Honors & Awards

  • Lifetime Fellow, American Society fo Cell Biology (2018)
  • Basil O'Connor Award, March of Dimes (1989)
  • Scholar in Biomedical Science, Lucille P. Markey Foundation (1985)
  • Presidential Young Investigator, National Science Foundation (1989)
  • Established Investigator, American Heart Association (1993)

Professional Education

  • A.B., Bowdoin College, Biochemistry (1976)
  • Ph.D., MIT, Biochemistry (1982)

Current Research and Scholarly Interests

The ER is the "port of entry" for proteins destined for the cell surface and beyond. The vast majority of proteins entering the secretory pathway are synthesized on ribosomes docked at ER translocons and are co-transationally translocated into the ER lumen. Proteins synthesized at the ER are subject to covalent modifications that include N- and O-glycosylation, disulfide bond formation, and in some cases, proline and lysine hydroxylation. Membrane proteins must be threaded co-translocationally into the lipid bilayer to become membrane-integrated, often with complex topologies and typically form hetero- or homo- oligomers. This highly complex "protein biogenesis" process is assisted by a diverse network of folding catalysts and protein-modifying enzymes and is scrutinized by molecular chaperones and other "quality control" factors which ensure that only correctly folded and assembled proteins exit the ER and proceed to distal compartments of the secretory pathway.

The Kopito laboratory seeks a molecular understanding of how cells maintain the fidelity of their proteomes. Unlike DNA, which can be repaired if damaged or incorrectly made, proteins cannot be mended. Instead, damaged or incorrectly synthesized proteins must be rapidly and efficiently destroyed lest they form toxic aggregates.

Our goal is to elucidate the functional networks that coordinate protein synthesis and quality control in the early secretory pathway. Currently the lab is focused on two specific systems: ERAD and ribosome UFMylation.

2023-24 Courses

Stanford Advisees

Graduate and Fellowship Programs

All Publications

  • Parallel CRISPR-Cas9 screens identify mechanisms of PLIN2 and lipid droplet regulation. Developmental cell Roberts, M. A., Deol, K. K., Mathiowetz, A. J., Lange, M., Leto, D. E., Stevenson, J., Hashemi, S. H., Morgens, D. W., Easter, E., Heydari, K., Nalls, M. A., Bassik, M. C., Kampmann, M., Kopito, R. R., Faghri, F., Olzmann, J. A. 2023


    Despite the key roles of perilipin-2 (PLIN2) in governing lipid droplet (LD) metabolism, the mechanisms that regulate PLIN2 levels remain incompletely understood. Here, we leverage a set of genome-edited human PLIN2 reporter cell lines in a series of CRISPR-Cas9 loss-of-function screens, identifying genetic modifiers that influence PLIN2 expression and post-translational stability under different metabolic conditions and in different cell types. These regulators include canonical genes that control lipid metabolism as well as genes involved in ubiquitination, transcription, and mitochondrial function. We further demonstrate a role for the E3 ligase MARCH6 in regulating triacylglycerol biosynthesis, thereby influencing LD abundance and PLIN2 stability. Finally, our CRISPR screens and several published screens provide the foundation for CRISPRlipid (http://crisprlipid.org), an online data commons for lipid-related functional genomics data. Our study identifies mechanisms of PLIN2 and LD regulation and provides an extensive resource for the exploration of LD biology and lipid metabolism.

    View details for DOI 10.1016/j.devcel.2023.07.001

    View details for PubMedID 37494933

  • RPL26/uL24 UFMylation is essential for ribosome-associated quality control at the endoplasmic reticulum. Proceedings of the National Academy of Sciences of the United States of America Scavone, F., Gumbin, S. C., Da Rosa, P. A., Kopito, R. R. 2023; 120 (16): e2220340120


    Ribosomes that stall while translating cytosolic proteins are incapacitated by incomplete nascent chains, termed "arrest peptides" (APs) that are destroyed by the ubiquitin proteasome system (UPS) via a process known as the ribosome-associated quality control (RQC) pathway. By contrast, APs on ribosomes that stall while translocating secretory proteins into the endoplasmic reticulum (ER-APs) are shielded from cytosol by the ER membrane and the tightly sealed ribosome-translocon junction (RTJ). How this junction is breached to enable access of cytosolic UPS machinery and 26S proteasomes to translocon- and ribosome-obstructing ER-APs is not known. Here, we show that UPS and RQC-dependent degradation of ER-APs strictly requires conjugation of the ubiquitin-like (Ubl) protein UFM1 to 60S ribosomal subunits at the RTJ. Therefore, UFMylation of translocon-bound 60S subunits modulates the RTJ to promote access of proteasomes and RQC machinery to ER-APs.

    View details for DOI 10.1073/pnas.2220340120

    View details for PubMedID 37036982

  • RPL26/uL24 UFMylation is essential for ribosome-associated quality control at the endoplasmic reticulum. bioRxiv : the preprint server for biology Scavone, F., Gumbin, S. C., DaRosa, P. A., Kopito, R. R. 2023


    Ribosomes that stall while translating cytosolic proteins are incapacitated by incomplete nascent chains, termed "arrest peptides" (APs) that are destroyed by the ubiquitin proteasome system (UPS) via a process known as the ribosome-associated quality control (RQC) pathway. By contrast, APs on ribosomes that stall while translocating secretory proteins into the endoplasmic reticulum (ER-APs) are shielded from cytosol by the ER membrane and the tightly sealed ribosome-translocon junction (RTJ). How this junction is breached to enable access of cytosolic UPS machinery and 26S proteasomes to translocon- and ribosome-obstructing ER-APs is not known. Here, we show that UPS and RQC-dependent degradation of ER-APs strictly requires conjugation of the ubiquitin-like (Ubl) protein UFM1 to 60S ribosomal subunits at the RTJ. Therefore, UFMylation of translocon-bound 60S subunits modulates the RTJ to promote access of proteasomes and RQC machinery to ER-APs.UFM1 is a ubiquitin-like protein that is selectively conjugated to the large (60S) subunit of ribosomes bound to the endoplasmic reticulum (ER), but the specific biological function of this modification is unclear. Here, we show that UFMylation facilitates proteasome-mediated degradation of arrest polypeptides (APs) which are generated following splitting of ribosomes that stall during co-translational translocation of secretory proteins into the ER. We propose that UFMylation weakens the tightly sealed ribosome-translocon junction, thereby allowing the cytosolic ubiquitin-proteasome and ribosome-associated quality control machineries to access ER-APs.

    View details for DOI 10.1101/2023.03.08.531792

    View details for PubMedID 36945571

    View details for PubMedCentralID PMC10028864

  • A non-canonical scaffold-type E3 ligase complex mediates protein UFMylation. The EMBO journal Peter, J. J., Magnussen, H. M., DaRosa, P. A., Millrine, D., Matthews, S. P., Lamoliatte, F., Sundaramoorthy, R., Kopito, R. R., Kulathu, Y. 2022: e111015


    Protein UFMylation, i.e., post-translational modification with ubiquitin-fold modifier 1 (UFM1), is essential for cellular and endoplasmic reticulum homeostasis. Despite its biological importance, we have a poor understanding of how UFM1 is conjugated onto substrates. Here, we use a rebuilding approach to define the minimal requirements of protein UFMylation. We find that the reported cognate E3 ligase UFL1 is inactive on its own and instead requires the adaptor protein UFBP1 to form an active E3 ligase complex. Structure predictions suggest the UFL1/UFBP1 complex to be made up of winged helix (WH) domain repeats. We show that UFL1/UFBP1 utilizes a scaffold-type E3 ligase mechanism that activates the UFM1-conjugating E2 enzyme, UFC1, for aminolysis. Further, we characterize a second adaptor protein CDK5RAP3 that binds to and forms an integral part of the ligase complex. Unexpectedly, we find that CDK5RAP3 inhibits UFL1/UFBP1 ligase activity invitro. Results from reconstituting ribosome UFMylation suggest that CDK5RAP3 functions as a substrate adaptor that directs UFMylation to the ribosomal protein RPL26. In summary, our reconstitution approach reveals the biochemical basis of UFMylation and regulatory principles of this atypical E3 ligase complex.

    View details for DOI 10.15252/embj.2022111015

    View details for PubMedID 36121123

  • PEX19 Coordinates Neutral Lipid Storage in Cells in a Peroxisome-Independent Fashion. Frontiers in cell and developmental biology Lyschik, S., Lauer, A. A., Roth, T., Janitschke, D., Hollander, M., Will, T., Hartmann, T., Kopito, R. R., Helms, V., Grimm, M. O., Schrul, B. 2022; 10: 859052


    Cellular lipid metabolism is tightly regulated and requires a sophisticated interplay of multiple subcellular organelles to adapt to changing nutrient supply. PEX19 was originally described as an essential peroxisome biogenesis factor that selectively targets membrane proteins to peroxisomes. Metabolic aberrations that were associated with compromised PEX19 functions, were solely attributed to the absence of peroxisomes, which is also considered the underlying cause for Zellweger Spectrum Disorders. More recently, however, it was shown that PEX19 also mediates the targeting of the VCP/P97-recuitment factor UBXD8 to the ER from where it partitions to lipid droplets (LDs) but the physiological consequences remained elusive. Here, we addressed the intriguing possibility that PEX19 coordinates the functions of the major cellular sites of lipid metabolism. We exploited the farnesylation of PEX19 and deciphered the organelle-specific functions of PEX19 using systems level approaches. Non-farnesylated PEX19 is sufficient to fully restore the metabolic activity of peroxisomes, while farnesylated PEX19 controls lipid metabolism by a peroxisome-independent mechanism that can be attributed to sorting a specific protein subset to LDs. In the absence of this PEX19-dependent LD proteome, cells accumulate excess triacylglycerols and fail to fully deplete their neutral lipid stores under catabolic conditions, highlighting a hitherto unrecognized function of PEX19 in controlling neutral lipid storage and LD dynamics.

    View details for DOI 10.3389/fcell.2022.859052

    View details for PubMedID 35557938

  • Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses. eLife Donnelly, K. M., DeLorenzo, O. R., Zaya, A. D., Pisano, G. E., Thu, W. M., Luo, L. n., Kopito, R. R., Panning Pearce, M. M. 2020; 9


    Emerging evidence supports the hypothesis that pathogenic protein aggregates associated with neurodegenerative diseases spread from cell to cell through the brain in a manner akin to infectious prions. Here, we show that mutant huntingtin (mHtt) aggregates associated with Huntington disease transfer anterogradely from presynaptic to postsynaptic neurons in the adult Drosophila olfactory system. Trans-synaptic transmission of mHtt aggregates is inversely correlated with neuronal activity and blocked by inhibiting caspases in presynaptic neurons, implicating synaptic dysfunction and cell death in aggregate spreading. Remarkably, mHtt aggregate transmission across synapses requires the glial scavenger receptor Draper and involves a transient visit to the glial cytoplasm, indicating that phagocytic glia act as obligatory intermediates in aggregate spreading between synaptically-connected neurons. These findings expand our understanding of phagocytic glia as double-edged players in neurodegeneration-by clearing neurotoxic protein aggregates, but also providing an opportunity for prion-like seeds to evade phagolysosomal degradation and propagate further in the brain.

    View details for DOI 10.7554/eLife.58499

    View details for PubMedID 32463364

  • Ribosomal protein RPL26 is the principal target of UFMylation. Proceedings of the National Academy of Sciences of the United States of America Walczak, C. P., Leto, D. E., Zhang, L., Riepe, C., Muller, R. Y., DaRosa, P. A., Ingolia, N. T., Elias, J. E., Kopito, R. R. 2019


    Ubiquitin fold modifier 1 (UFM1) is a small, metazoan-specific, ubiquitin-like protein modifier that is essential for embryonic development. Although loss-of-function mutations in UFM1 conjugation are linked to endoplasmic reticulum (ER) stress, neither the biological function nor the relevant cellular targets of this protein modifier are known. Here, we show that a largely uncharacterized ribosomal protein, RPL26, is the principal target of UFM1 conjugation. RPL26 UFMylation and de-UFMylation is catalyzed by enzyme complexes tethered to the cytoplasmic surface of the ER and UFMylated RPL26 is highly enriched on ER membrane-bound ribosomes and polysomes. Biochemical analysis and structural modeling establish that UFMylated RPL26 and the UFMylation machinery are in close proximity to the SEC61 translocon, suggesting that this modification plays a direct role in cotranslational protein translocation into the ER. These data suggest that UFMylation is a ribosomal modification specialized to facilitate metazoan-specific protein biogenesis at the ER.

    View details for PubMedID 30626644

  • Methods for genetic analysis of mammalian ER-associated degradation UBIQUITIN-DEPENDENT PROTEIN DEGRADATION Leto, D. E., Kopito, R. R., Hochstrasser, M. 2019; 619: 97–120
  • Methods for genetic analysis of mammalian ER-associated degradation. Methods in enzymology Leto, D. E., Kopito, R. R. 2019; 619: 97–120


    Identification and degradation of misfolded proteins by the ubiquitin-proteasome system (UPS) is crucial for maintaining proteostasis, but only a handful of UPS components have been linked to the recognition of specific substrates. Studies in Saccharomyces cerevisiae using systematic perturbation of nonessential genes have uncovered UPS components that recognize and ubiquitylate model substrates of the UPS; however, similar analyses in metazoans have been limited. In this chapter, we describe methods for using CRISPR/Cas9 technology combined with genome-wide high complexity single guide (sgRNA) libraries and a transcriptional shutoff strategy for phenotypic selection based on kinetic measurements of protein turnover to identify the genes required to degrade model clients of the mammalian ER-associated degradation system. We also discuss considerations for screen design, execution, and interpretation.

    View details for PubMedID 30910031

  • Acute unfolding of a single protein immediately stimulates recruitment of ubiquitin protein ligase E3C (UBE3C) to 26S proteasomes. The Journal of biological chemistry Gottlieb, C. D., Thompson, A. C., Ordureau, A. n., Harper, J. W., Kopito, R. R. 2019


    The intracellular accumulation of aggregated misfolded proteins is a cytopathological hallmark of neurodegenerative diseases. However, the functional relationship between protein misfolding and aggregation and the cellular proteostasis network that monitors and maintains proteome health are poorly understood. Previous studies have associated translational suppression and transcriptional remodeling with the appearance of protein aggregates, but whether these responses are induced by aggregates or their misfolded monomeric or oligomeric precursors remains unclear. Because aggregation in cells is rapid, non-linear, and asynchronous, it has not been possible to deconvolve these kinetically linked processes to determine the earliest cellular responses to misfolded proteins. Upon removal of the synthetic, biologically inert ligand shield-1 (S1), AgDD, an engineered variant FK506-binding protein (FKBP1A), rapidly (t1/2 ~5 min) unfolds and self-associates, forming detergent-insoluble, microscopic cytoplasmic aggregates. Using global diglycine-capture (K-GG) proteomics, we found here that this solubility transition is associated with immediate increases in ubiquitylation of AgDD itself, along with that of endogenous proteins that are components of the ribosome and the 26S proteasome. We also found that the earliest cellular responses to acute S1 removal include recruitment of ubiquitin protein ligase E3C (UBE3C) to the 26S proteasome and ubiquitylation of two key proteasomal ubiquitin receptors, 26S proteasome regulatory subunit RPN10 (RPN10) and Rpn13 homolog (RPN13 or ADRM1). We conclude that these proteasomal responses are due to AgDD protein misfolding and not to the presence of detergent-insoluble aggregates.

    View details for DOI 10.1074/jbc.RA119.009654

    View details for PubMedID 31375563

  • Genome-wide CRISPR Analysis Identifies Substrate-Specific Conjugation Modules in ER-Associated Degradation. Molecular cell Leto, D. E., Morgens, D. W., Zhang, L., Walczak, C. P., Elias, J. E., Bassik, M. C., Kopito, R. R. 2018


    The ubiquitin proteasome system (UPS) maintains the integrity of the proteome by selectively degrading misfolded or mis-assembled proteins, but the rules that govern how conformationally defective proteins in the secretory pathway are selected from the structurally and topologically diverse constellation of correctly folded membrane and secretory proteins for efficient degradation by cytosolic proteasomes is not well understood. Here, we combine parallel pooled genome-wide CRISPR-Cas9 forward genetic screening with a highly quantitative and sensitive protein turnover assay to discover a previously undescribed collaboration between membrane-embedded cytoplasmic ubiquitin E3 ligases to conjugate heterotypic branched or mixed ubiquitin (Ub) chains on substrates of endoplasmic-reticulum-associated degradation (ERAD). These findings demonstrate that parallel CRISPR analysis can be used to deconvolve highly complex cell biological processes and identify new biochemical pathways in protein quality control.

    View details for PubMedID 30581143

  • Proteomic analysis of monolayer-integrated proteins on lipid droplets identifies amphipathic interfacial alpha-helical membrane anchors. Proceedings of the National Academy of Sciences of the United States of America Pataki, C. I., Rodrigues, J., Zhang, L., Qian, J., Efron, B., Hastie, T., Elias, J. E., Levitt, M., Kopito, R. R. 2018


    Despite not spanning phospholipid bilayers, monotopic integral proteins (MIPs) play critical roles in organizing biochemical reactions on membrane surfaces. Defining the structural basis by which these proteins are anchored to membranes has been hampered by the paucity of unambiguously identified MIPs and a lack of computational tools that accurately distinguish monolayer-integrating motifs from bilayer-spanning transmembrane domains (TMDs). We used quantitative proteomics and statistical modeling to identify 87 high-confidence candidate MIPs in lipid droplets, including 21 proteins with predicted TMDs that cannot be accommodated in these monolayer-enveloped organelles. Systematic cysteine-scanning mutagenesis showed the predicted TMD of one candidate MIP, DHRS3, to be a partially buried amphipathic alpha-helix in both lipid droplet monolayers and the cytoplasmic leaflet of endoplasmic reticulum membrane bilayers. Coarse-grained molecular dynamics simulations support these observations, suggesting that this helix is most stable at the solvent-membrane interface. The simulations also predicted similar interfacial amphipathic helices when applied to seven additional MIPs from our dataset. Our findings suggest that interfacial helices may be a common motif by which MIPs are integrated into membranes, and provide high-throughput methods to identify and study MIPs.

    View details for PubMedID 30104359

  • Redundant and Antagonistic Roles of XTP3B and OS9 in Decoding Glycan and Non-glycan Degrons in ER-Associated Degradation MOLECULAR CELL van der Goot, A. T., Pearce, M. P., Leto, D. E., Shaler, T. A., Kopito, R. R. 2018; 70 (3): 516-+


    Glycoproteins engaged in unproductive folding in the ER are marked for degradation by a signal generated by progressive demannosylation of substrate N-glycans that is decoded by ER lectins, but how the two lectins, OS9 and XTP3B, contribute to non-glycosylated protein triage is unknown. We generated cell lines with homozygous deletions of both lectins individually and in combination. We found that OS9 and XTP3B redundantly promote glycoprotein degradation and stabilize the SEL1L/HRD1 dislocon complex, that XTP3B profoundly inhibits the degradation of non-glycosylated proteins, and that OS9 antagonizes this inhibition. The relative expression of OS9 and XTP3B and the distribution of glycan and non-glycan degrons within the same protein contribute to the fidelity and processivity of glycoprotein triage and, therefore, determine the fates of newly synthesized proteins in the early secretory pathway.

    View details for PubMedID 29706535

    View details for PubMedCentralID PMC5935522

  • Characterization of protein complexes of the endoplasmic reticulum-associated degradation E3 ubiquitin ligase Hrd1. journal of biological chemistry Hwang, J., Walczak, C. P., Shaler, T. A., Olzmann, J. A., Zhang, L., Elias, J. E., Kopito, R. R. 2017; 292 (22): 9104-9116


    Hrd1 is the core structural component of a large endoplasmic reticulum membrane-embedded protein complex that coordinates the destruction of folding-defective proteins in the early secretory pathway. Defining the composition, dynamics, and ultimately, the structure of the Hrd1 complex is a crucial step in understanding the molecular basis of glycoprotein quality control but has been hampered by the lack of suitable techniques to interrogate this complex under native conditions. In this study we used genome editing to generate clonal HEK293 (Hrd1.KI) cells harboring a homozygous insertion of a small tandem affinity tag knocked into the endogenous Hrd1 locus. We found that steady-state levels of tagged Hrd1 in these cells are indistinguishable from those of Hrd1 in unmodified cells and that the tagged variant is functional in supporting the degradation of well characterized luminal and membrane substrates. Analysis of detergent-solubilized Hrd1.KI cells indicates that the composition and stoichiometry of Hrd1 complexes are strongly influenced by Hrd1 expression levels. Analysis of affinity-captured Hrd1 complexes from these cells by size-exclusion chromatography, immunodepletion, and absolute quantification mass spectrometry identified two major high-molecular-mass complexes with distinct sets of interacting proteins and variable stoichiometries, suggesting a hitherto unrecognized heterogeneity in the functional units of Hrd1-mediated protein degradation.

    View details for DOI 10.1074/jbc.M117.785055

    View details for PubMedID 28411238

  • Prion-Like Characteristics of Polyglutamine-Containing Proteins. Cold Spring Harbor perspectives in medicine Pearce, M. M., Kopito, R. R. 2017


    Transmissible spongiform encephalopathies are infectious neurodegenerative diseases caused by the conversion of prion protein (PrP) into a self-replicating conformation that spreads via templated conversion of natively folded PrP molecules within or between cells. Recent studies provide compelling evidence that prion-like behavior is a general property of most protein aggregates associated with neurodegenerative diseases. Many of these disorders are associated with spontaneous protein aggregation, but genetic mutations can increase the aggregation propensity of specific proteins, including expansion of polyglutamine (polyQ) tracts, which is causative of nine inherited neurodegenerative diseases. Aggregates formed by polyQ-expanded huntingtin (Htt) in Huntington's disease can transfer between cells and seed the aggregation of cytoplasmic wild-type Htt in a prion-like manner. Additionally, prion-like properties of glutamine-rich proteins underlie nonpathological processes in yeast and higher eukaryotes. Here, we review current evidence supporting prion-like characteristics of polyQ and glutamine-rich proteins.

    View details for DOI 10.1101/cshperspect.a024257

    View details for PubMedID 28096245

  • Ubiquitin Accumulation on Disease Associated Protein Aggregates Is Correlated with Nuclear Ubiquitin Depletion, Histone De-Ubiquitination and Impaired DNA Damage Response PLOS ONE Ben Yehuda, A., Risheq, M., Novoplansky, O., Bersuker, K., Kopito, R. R., Goldberg, M., Brandeis, M. 2017; 12 (1)


    Deposition of ubiquitin conjugates on inclusion bodies composed of protein aggregates is a definitive cytopathological hallmark of neurodegenerative diseases. We show that accumulation of ubiquitin on polyQ IB, associated with Huntington's disease, is correlated with extensive depletion of nuclear ubiquitin and histone de-ubiquitination. Histone ubiquitination plays major roles in chromatin regulation and DNA repair. Accordingly, we observe that cells expressing IB fail to respond to radiomimetic DNA damage, to induce gamma-H2AX phosphorylation and to recruit 53BP1 to damaged foci. Interestingly ubiquitin depletion, histone de-ubiquitination and impaired DNA damage response are not restricted to PolyQ aggregates and are associated with artificial aggregating luciferase mutants. The longevity of brain neurons depends on their capacity to respond to and repair extensive ongoing DNA damage. Impaired DNA damage response, even modest one, could thus lead to premature neuron aging and mortality.

    View details for DOI 10.1371/journal.pone.0169054

    View details for Web of Science ID 000391621500030

    View details for PubMedID 28052107

    View details for PubMedCentralID PMC5215683

  • Protein misfolding in neurodegenerative diseases: implications and strategies. Translational neurodegeneration Sweeney, P., Park, H., Baumann, M., Dunlop, J., Frydman, J., Kopito, R., McCampbell, A., LeBlanc, G., Venkateswaran, A., Nurmi, A., Hodgson, R. 2017; 6: 6-?


    A hallmark of neurodegenerative proteinopathies is the formation of misfolded protein aggregates that cause cellular toxicity and contribute to cellular proteostatic collapse. Therapeutic options are currently being explored that target different steps in the production and processing of proteins implicated in neurodegenerative disease, including synthesis, chaperone-assisted folding and trafficking, and degradation via the proteasome and autophagy pathways. Other therapies, like mTOR inhibitors and activators of the heat shock response, can rebalance the entire proteostatic network. However, there are major challenges that impact the development of novel therapies, including incomplete knowledge of druggable disease targets and their mechanism of action as well as a lack of biomarkers to monitor disease progression and therapeutic response. A notable development is the creation of collaborative ecosystems that include patients, clinicians, basic and translational researchers, foundations and regulatory agencies to promote scientific rigor and clinical data to accelerate the development of therapies that prevent, reverse or delay the progression of neurodegenerative proteinopathies.

    View details for DOI 10.1186/s40035-017-0077-5

    View details for PubMedID 28293421

  • Ron R. Kopito: Unfolding the Secrets of Protein Aggregation. Trends in cell biology Kopito, R. R. 2016; 26 (8): 559-560

    View details for DOI 10.1016/j.tcb.2016.05.001

    View details for PubMedID 27238420

  • Peroxin-dependent targeting of a lipid-droplet-destined membrane protein to ER subdomains NATURE CELL BIOLOGY Schrul, B., Kopito, R. R. 2016; 18 (7): 740-?


    Lipid droplets (LDs) are endoplasmic reticulum (ER)-derived lipid storage organelles uniquely encapsulated by phospholipid monolayers. LD membrane proteins are embedded into the monolayer in a monotopic hairpin topology and are therefore likely to have requirements for their biogenesis distinct from those inserting as bitopic and polytopic proteins into phospholipid bilayers. UBXD8 belongs to a subfamily of hairpin proteins that localize to both the ER and LDs, and are initially inserted into the cytoplasmic leaflet of the ER bilayer before partitioning to the LD monolayer. The molecular machinery responsible for inserting hairpin proteins into membranes, however, is unknown. Here, we report that newly synthesized UBXD8 is post-translationally inserted into discrete ER subdomains by a mechanism requiring cytosolic PEX19 and membrane-integrated PEX3, proteins hitherto exclusively implicated in peroxisome biogenesis. Farnesylation of PEX19 uncouples ER/LD and peroxisome targeting, expanding the function of this peroxin to an ER-targeting pathway and suggesting a coordinated biogenesis of LDs and peroxisomes.

    View details for DOI 10.1038/ncb3373

    View details for Web of Science ID 000378840900007

    View details for PubMedID 27295553

  • Protein misfolding specifies recruitment to cytoplasmic inclusion bodies JOURNAL OF CELL BIOLOGY Bersuker, K., Brandeis, M., Kopito, R. R. 2016; 213 (2): 229-241


    Inclusion bodies (IBs) containing aggregated disease-associated proteins and polyubiquitin (poly-Ub) conjugates are universal histopathological features of neurodegenerative diseases. Ub has been proposed to target proteins to IBs for degradation via autophagy, but the mechanisms that govern recruitment of ubiquitylated proteins to IBs are not well understood. In this paper, we use conditionally destabilized reporters that undergo misfolding and ubiquitylation upon removal of a stabilizing ligand to examine the role of Ub conjugation in targeting proteins to IBs that are composed of an N-terminal fragment of mutant huntingtin, the causative protein of Huntington's disease. We show that reporters are excluded from IBs in the presence of the stabilizing ligand but are recruited to IBs after ligand washout. However, we find that Ub conjugation is not necessary to target reporters to IBs. We also report that forced Ub conjugation by the Ub fusion degradation pathway is not sufficient for recruitment to IBs. Finally, we find that reporters and Ub conjugates are stable at IBs. These data indicate that compromised folding states, rather than conjugation to Ub, can specify recruitment to IBs.

    View details for DOI 10.1083/jcb.201511024

    View details for Web of Science ID 000375513900013

    View details for PubMedID 27114501

  • Prion-like transmission of neuronal huntingtin aggregates to phagocytic glia in the Drosophila brain. Nature communications Pearce, M. M., Spartz, E. J., Hong, W., Luo, L., Kopito, R. R. 2015; 6: 6768-?


    The brain has a limited capacity to self-protect against protein aggregate-associated pathology, and mounting evidence supports a role for phagocytic glia in this process. We have established a Drosophila model to investigate the role of phagocytic glia in clearance of neuronal mutant huntingtin (Htt) aggregates associated with Huntington disease. We find that glia regulate steady-state numbers of Htt aggregates expressed in neurons through a clearance mechanism that requires the glial scavenger receptor Draper and downstream phagocytic engulfment machinery. Remarkably, some of these engulfed neuronal Htt aggregates effect prion-like conversion of soluble, wild-type Htt in the glial cytoplasm. We provide genetic evidence that this conversion depends strictly on the Draper signalling pathway, unveiling a previously unanticipated role for phagocytosis in transfer of pathogenic protein aggregates in an intact brain. These results suggest a potential mechanism by which phagocytic glia contribute to both protein aggregate-related neuroprotection and pathogenesis in neurodegenerative disease.

    View details for DOI 10.1038/ncomms7768

    View details for PubMedID 25866135

  • Prion-like transmission of neuronal huntingtin aggregates to phagocytic glia in the Drosophila brain. Nature communications Pearce, M. M., Spartz, E. J., Hong, W., Luo, L., Kopito, R. R. 2015; 6: 6768-?


    The brain has a limited capacity to self-protect against protein aggregate-associated pathology, and mounting evidence supports a role for phagocytic glia in this process. We have established a Drosophila model to investigate the role of phagocytic glia in clearance of neuronal mutant huntingtin (Htt) aggregates associated with Huntington disease. We find that glia regulate steady-state numbers of Htt aggregates expressed in neurons through a clearance mechanism that requires the glial scavenger receptor Draper and downstream phagocytic engulfment machinery. Remarkably, some of these engulfed neuronal Htt aggregates effect prion-like conversion of soluble, wild-type Htt in the glial cytoplasm. We provide genetic evidence that this conversion depends strictly on the Draper signalling pathway, unveiling a previously unanticipated role for phagocytosis in transfer of pathogenic protein aggregates in an intact brain. These results suggest a potential mechanism by which phagocytic glia contribute to both protein aggregate-related neuroprotection and pathogenesis in neurodegenerative disease.

    View details for DOI 10.1038/ncomms7768

    View details for PubMedID 25866135

  • Heat shock response activation exacerbates inclusion body formation in a cellular model of huntington disease. journal of biological chemistry Bersuker, K., Hipp, M. S., Calamini, B., Morimoto, R. I., Kopito, R. R. 2013; 288 (33): 23633-23638


    The cellular heat shock response (HSR) protects cells from toxicity associated with defective protein folding and this pathway is widely viewed as a potential pharmacological target to treat neurodegenerative diseases linked to protein aggregation. Here we show that the HSR is not activated by mutant huntingtin (htt) even in cells selected for the highest expression levels and for the presence of inclusion bodies containing aggregated protein. Surprisingly, HSR activation by HSF1 overexpression or by administration of a small molecule activator lowers the concentration threshold at which htt forms inclusion bodies (IB) in cells expressing aggregation-prone, polyglutamine expanded fragments of htt. These data suggest that the HSR does not mitigate IB formation.

    View details for DOI 10.1074/jbc.C113.481945

    View details for PubMedID 23839939

  • Spatial regulation of UBXD8 and p97/VCP controls ATGL-mediated lipid droplet turnover PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Olzmann, J. A., Richter, C. M., Kopito, R. R. 2013; 110 (4): 1345-1350


    UBXD8 is a membrane-embedded recruitment factor for the p97/VCP segregase that has been previously linked to endoplasmic reticulum (ER)-associated degradation and to the control of triacylglycerol synthesis in the ER. UBXD8 also has been identified as a component of cytoplasmic lipid droplets (LDs), but neither the mechanisms that control its trafficking between the ER and LDs nor its functions in the latter organelle have been investigated previously. Here we report that association of UBXD8 with the ER-resident rhomboid pseudoprotease UBAC2 specifically restricts trafficking of UBXD8 to LDs, and that the steady-state partitioning of UBXD8 between the ER and LDs can be experimentally manipulated by controlling the relative expression of these two proteins. We exploit this interaction to show that UBXD8-mediated recruitment of p97/VCP to LDs increases LD size by inhibiting the activity of adipose triglyceride lipase (ATGL), the rate-limiting enzyme in triacylglycerol hydrolysis. Our findings show that UBXD8 binds directly to ATGL and promotes dissociation of its endogenous coactivator, CGI-58. These data indicate that UBXD8 and p97/VCP play central integrative roles in cellular energy homeostasis.

    View details for DOI 10.1073/pnas.1213738110

    View details for Web of Science ID 000314453900046

    View details for PubMedID 23297223

    View details for PubMedCentralID PMC3557085

  • Simultaneous Measurement of Amyloid Fibril Formation by Dynamic Light Scattering and Fluorescence Reveals Complex Aggregation Kinetics PLOS ONE Streets, A. M., Sourigues, Y., Kopito, R. R., Melki, R., Quake, S. R. 2013; 8 (1)


    An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington's disease, in vitro. Huntington's disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer's and Parkinson's disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, β-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports β-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation.

    View details for DOI 10.1371/journal.pone.0054541

    View details for Web of Science ID 000313738900076

    View details for PubMedID 23349924

    View details for PubMedCentralID PMC3547910

  • Simultaneous measurement of amyloid fibril formation by dynamic light scattering and fluorescence reveals complex aggregation kinetics. PloS one Streets, A. M., Sourigues, Y., Kopito, R. R., Melki, R., Quake, S. R. 2013; 8 (1)


    An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington's disease, in vitro. Huntington's disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer's and Parkinson's disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, β-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports β-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation.

    View details for DOI 10.1371/journal.pone.0054541

    View details for PubMedID 23349924

    View details for PubMedCentralID PMC3547910

  • The Mammalian endoplasmic reticulum-associated degradation system. Cold Spring Harbor perspectives in biology Olzmann, J. A., Kopito, R. R., Christianson, J. C. 2013; 5 (9)


    The endoplasmic reticulum (ER) is the site of synthesis for nearly one-third of the eukaryotic proteome and is accordingly endowed with specialized machinery to ensure that proteins deployed to the distal secretory pathway are correctly folded and assembled into native oligomeric complexes. Proteins failing to meet this conformational standard are degraded by ER-associated degradation (ERAD), a complex process through which folding-defective proteins are selected and ultimately degraded by the ubiquitin-proteasome system. ERAD proceeds through four tightly coupled steps involving substrate selection, dislocation across the ER membrane, covalent conjugation with polyubiquitin, and proteasomal degradation. The ERAD machinery shows a modular organization with central ER membrane-embedded ubiquitin ligases linking components responsible for recognition in the ER lumen to the ubiquitin-proteasome system in the cytoplasm. The core ERAD machinery is highly conserved among eukaryotes and much of our basic understanding of ERAD organization has been derived from genetic and biochemical studies of yeast. In this article we discuss how the core ERAD machinery is organized in mammalian cells.

    View details for DOI 10.1101/cshperspect.a013185

    View details for PubMedID 23232094

  • Unassembled CD147 is an endogenous endoplasmic reticulum-associated degradation substrate MOLECULAR BIOLOGY OF THE CELL Tyler, R. E., Pearce, M. M., Shaler, T. A., Olzmann, J. A., Greenblatt, E. J., Kopito, R. R. 2012; 23 (24): 4668-4678


    Degradation of folding- or assembly-defective proteins by the endoplasmic reticulum-associated degradation (ERAD) ubiquitin ligase, Hrd1, is facilitated by a process that involves recognition of demannosylated N-glycans by the lectin OS-9/XTP3-B via the adaptor protein SEL1L. Most of our knowledge of the machinery that commits proteins to this fate in metazoans comes from studies of overexpressed mutant proteins in heterologous cells. In this study, we used mass spectrometry to identify core-glycoslyated CD147 (CD147(CG)) as an endogenous substrate of the ERAD system that accumulates in a complex with OS-9 following SEL1L depletion. CD147 is an obligatory assembly factor for monocarboxylate transporters. The majority of newly synthesized endogenous CD147(CG) was degraded by the proteasome in a Hrd1-dependent manner. CD147(CG) turnover was blocked by kifunensine, and interaction of OS-9 and XTP3-B with CD147(CG) was inhibited by mutations to conserved residues in their lectin domains. These data establish unassembled CD147(CG) as an endogenous, constitutive ERAD substrate of the OS-9/SEL1L/Hrd1 pathway.

    View details for DOI 10.1091/mbc.E12-06-0428

    View details for Web of Science ID 000314405100002

    View details for PubMedID 23097496

    View details for PubMedCentralID PMC3521676

  • ALIX Is a Lys63-Specific Polyubiquitin Binding Protein that Functions in Retrovirus Budding DEVELOPMENTAL CELL Dowlatshahi, D. P., Sandrin, V., Vivona, S., Shaler, T. A., Kaiser, S. E., Melandri, F., Sundquist, W. I., Kopito, R. R. 2012; 23 (6): 1247-1254


    The diversity of ubiquitin (Ub)-dependent signaling is attributed to the ability of this small protein to form different types of covalently linked polyUb chains and to the existence of Ub binding proteins that interpret this molecular syntax. We used affinity capture/mass spectrometry to identify ALIX, a component of the ESCRT pathway, as a Ub binding protein. We report that the V domain of ALIX binds directly and selectively to K63-linked polyUb chains, exhibiting a strong preference for chains composed of more than three Ub. Sequence analysis identified two potential Ub binding sites on a single α-helical surface within the coiled-coil region of the V domain. Mutation of these putative Ub binding sites inhibited polyUb binding to the isolated V domain in vitro and impaired budding of lentiviruses. These data reveal an important role for K63 polyUb binding by ALIX in retroviral release.

    View details for DOI 10.1016/j.devcel.2012.10.023

    View details for Web of Science ID 000312429200020

    View details for PubMedID 23201121

    View details for PubMedCentralID PMC3522770

  • Making the cut: intramembrane cleavage by a rhomboid protease promotes ERAD NATURE STRUCTURAL & MOLECULAR BIOLOGY Greenblatt, E. J., Olzmann, J. A., Kopito, R. R. 2012; 19 (10): 979-981


    Endoplasmic reticulum–associated degradation (ERAD) is a cellular protein quality-control process that disposes of proteasomal substrates from the early secretory pathway. Recent work shows that the endoplasmic reticulum–resident rhomboid protease RHBDL4 facilitates ERAD by recognizing and cleaving integral membrane substrates. The work indicates that intramembrane proteolysis may have a general role in the extraction of misfolded membrane proteins from the endoplasmic reticulum.

    View details for DOI 10.1038/nsmb.2398

    View details for Web of Science ID 000309591000004

    View details for PubMedID 23037595

  • Fibrillar Structure and Charge Determine the Interaction of Polyglutamine Protein Aggregates with the Cell Surface JOURNAL OF BIOLOGICAL CHEMISTRY Trevino, R. S., Lauckner, J. E., Sourigues, Y., Pearce, M. M., Bousset, L., Melki, R., Kopito, R. R. 2012; 287 (35): 29722-29728


    The pathogenesis of most neurodegenerative diseases, including transmissible diseases like prion encephalopathy, inherited disorders like Huntington disease, and sporadic diseases like Alzheimer and Parkinson diseases, is intimately linked to the formation of fibrillar protein aggregates. It is becoming increasingly appreciated that prion-like intercellular transmission of protein aggregates can contribute to the stereotypical spread of disease pathology within the brain, but the mechanisms underlying the binding and uptake of protein aggregates by mammalian cells are largely uninvestigated. We have investigated the properties of polyglutamine (polyQ) aggregates that endow them with the ability to bind to mammalian cells in culture and the properties of the cell surface that facilitate such uptake. Binding and internalization of polyQ aggregates are common features of mammalian cells and depend upon both trypsin-sensitive and trypsin-resistant saturable sites on the cell surface, suggesting the involvement of cell surface proteins in this process. polyQ aggregate binding depends upon the presence of a fibrillar amyloid-like structure and does not depend upon electrostatic interaction of fibrils with the cell surface. Sequences in the huntingtin protein that flank the amyloid-forming polyQ tract also influence the extent to which aggregates are able to bind to cell surfaces.

    View details for DOI 10.1074/jbc.M112.372474

    View details for Web of Science ID 000308286900048

    View details for PubMedID 22753412

    View details for PubMedCentralID PMC3436181

  • Indirect inhibition of 26S proteasome activity in a cellular model of Huntington's disease JOURNAL OF CELL BIOLOGY Hipp, M. S., Patel, C. N., Bersuker, K., Riley, B. E., Kaiser, S. E., Shaler, T. A., Brandeis, M., Kopito, R. R. 2012; 196 (5): 573-587


    Pathognomonic accumulation of ubiquitin (Ub) conjugates in human neurodegenerative diseases, such as Huntington's disease, suggests that highly aggregated proteins interfere with 26S proteasome activity. In this paper, we examine possible mechanisms by which an N-terminal fragment of mutant huntingtin (htt; N-htt) inhibits 26S function. We show that ubiquitinated N-htt-whether aggregated or not-did not choke or clog the proteasome. Both Ub-dependent and Ub-independent proteasome reporters accumulated when the concentration of mutant N-htt exceeded a solubility threshold, indicating that stabilization of 26S substrates is not linked to impaired Ub conjugation. Above this solubility threshold, mutant N-htt was rapidly recruited to cytoplasmic inclusions that were initially devoid of Ub. Although synthetically polyubiquitinated N-htt competed with other Ub conjugates for access to the proteasome, the vast majority of mutant N-htt in cells was not Ub conjugated. Our data confirm that proteasomes are not directly impaired by aggregated N-terminal fragments of htt; instead, our data suggest that Ub accumulation is linked to impaired function of the cellular proteostasis network.

    View details for DOI 10.1083/jcb.201110093

    View details for Web of Science ID 000301323100005

    View details for PubMedID 22371559

    View details for PubMedCentralID PMC3307690

  • Perturbation of the Hematopoietic System during Embryonic Liver Development Due to Disruption of Polyubiquitin Gene Ubc in Mice PLOS ONE Ryu, K., Park, H., Rossi, D. J., Weissman, I. L., Kopito, R. R. 2012; 7 (2)


    Disruption of the polyubiquitin gene Ubc leads to a defect in fetal liver development, which can be partially rescued by increasing the amount of ubiquitin. However, it is still not known why Ubc is required for fetal liver development and the nature of the defective cell types responsible for embryonic lethality have not been characterized. In this study, we assessed the cause of embryonic lethality with respect to the fetal liver hematopoietic system. We found that Ubc was highly expressed in the embryonic liver, and the proliferation capacity of fetal liver cells was reduced in Ubc(-/-) embryos. Specifically, Ubc was most highly expressed in hematopoietic cells, and the proliferation capacity of hematopoietic cells was significantly impaired in Ubc(-/-) embryos. While hematopoietic cell and hematopoietic stem cell (HSC) frequency was maintained in Ubc(-/-) embryos, the absolute number of these cells was diminished because of reduced total liver cell number in Ubc(-/-) embryos. Transplantations of fetal liver cells into lethally irradiated recipient mice by non-competitive and competitive reconstitution methods indicated that disruption of Ubc does not significantly impair the intrinsic function of fetal liver HSCs. These findings suggest that disruption of Ubc reduces the absolute number of HSCs in embryonic livers, but has no significant effect on the autonomous function of HSCs. Thus, the lethality of Ubc(-/-) embryos is not the result of intrinsic HSC failure.

    View details for DOI 10.1371/journal.pone.0032956

    View details for Web of Science ID 000303003500114

    View details for PubMedID 22393459

  • Live-cell imaging of ubiquitin-proteasome system function. Methods in molecular biology (Clifton, N.J.) Hipp, M. S., Bersuker, K., Kopito, R. R. 2012; 832: 463-472


    The role of the ubiquitin-proteasome system (UPS) in maintaining protein homeostasis has generated a demand for assays that quantify UPS function in the presence of chemical and protein UPS inhibitors. Here, we describe protocols that measure changes in UPS reporter levels in response to changes in the expression level, localization, or aggregation state of a second protein. We utilize cell lines stably expressing fluorescent UPS substrates that are transfected with a second protein tagged with a compatible fluorophore. We describe protocols to correlate levels of UPS substrates with changes in the levels or properties of the transfected protein.

    View details for DOI 10.1007/978-1-61779-474-2_33

    View details for PubMedID 22350906

  • Defining human ERAD networks through an integrative mapping strategy NATURE CELL BIOLOGY Christianson, J. C., Olzmann, J. A., Shaler, T. A., Sowa, M. E., Bennett, E. J., Richter, C. M., Tyler, R. E., Greenblatt, E. J., Harper, J. W., Kopito, R. R. 2012; 14 (1): 93-U176

    View details for DOI 10.1038/ncb2383

    View details for Web of Science ID 000298421100015

  • Defining human ERAD networks through an integrative mapping strategy. Nature cell biology Christianson, J. C., Olzmann, J. A., Shaler, T. A., Sowa, M. E., Bennett, E. J., Richter, C. M., Tyler, R. E., Greenblatt, E. J., Harper, J. W., Kopito, R. R. 2012; 14 (1): 93-105


    Proteins that fail to correctly fold or assemble into oligomeric complexes in the endoplasmic reticulum (ER) are degraded by a ubiquitin- and proteasome-dependent process known as ER-associated degradation (ERAD). Although many individual components of the ERAD system have been identified, how these proteins are organized into a functional network that coordinates recognition, ubiquitylation and dislocation of substrates across the ER membrane is not well understood. We have investigated the functional organization of the mammalian ERAD system using a systems-level strategy that integrates proteomics, functional genomics and the transcriptional response to ER stress. This analysis supports an adaptive organization for the mammalian ERAD machinery and reveals a number of metazoan-specific genes not previously linked to ERAD.

    View details for DOI 10.1038/ncb2383

    View details for PubMedID 22119785

    View details for PubMedCentralID PMC3250479

  • Derlin-1 is a rhomboid pseudoprotease required for the dislocation of mutant alpha-1 antitrypsin from the endoplasmic reticulum NATURE STRUCTURAL & MOLECULAR BIOLOGY Greenblatt, E. J., Olzmann, J. A., Kopito, R. R. 2011; 18 (10): 1147-U115


    The degradation of misfolded secretory proteins is ultimately mediated by the ubiquitin-proteasome system in the cytoplasm, therefore endoplasmic reticulum-associated degradation (ERAD) substrates must be dislocated across the ER membrane through a process driven by the AAA ATPase p97/VCP. Derlins recruit p97/VCP and have been proposed to be part of the dislocation machinery. Here we report that Derlins are inactive members of the rhomboid family of intramembrane proteases and bind p97/VCP through C-terminal SHP boxes. Human Derlin-1 harboring mutations within the rhomboid domain stabilized mutant α-1 antitrypsin (NHK) at the cytosolic face of the ER membrane without disrupting the p97/VCP interaction. We propose that substrate interaction and p97/VCP recruitment are separate functions that are essential for dislocation and can be assigned respectively to the rhomboid domain and the C terminus of Derlin-1. These data suggest that intramembrane proteolysis and protein dislocation share unexpected mechanistic features.

    View details for DOI 10.1038/nsmb.2111

    View details for Web of Science ID 000295931400009

    View details for PubMedID 21909096

    View details for PubMedCentralID PMC3196324

  • Lipid Droplet Formation Is Dispensable for Endoplasmic Reticulum-associated Degradation JOURNAL OF BIOLOGICAL CHEMISTRY Olzmann, J. A., Kopito, R. R. 2011; 286 (32): 27872-27874


    Proteins that fail to fold or assemble in the endoplasmic reticulum (ER) are destroyed by cytoplasmic proteasomes through a process known as ER-associated degradation. Substrates of this pathway are initially sequestered within the ER lumen and must therefore be dislocated across the ER membrane to be degraded. It has been proposed that generation of bicellar structures during lipid droplet formation may provide an "escape hatch" through which misfolded proteins, toxins, and viruses can exit the ER. We have directly tested this hypothesis by exploiting yeast strains defective in lipid droplet formation. Our data demonstrate that lipid droplet formation is dispensable for the dislocation of a plant toxin and the degradation of both soluble and integral membrane glycoproteins.

    View details for DOI 10.1074/jbc.C111.266452

    View details for Web of Science ID 000293557800005

    View details for PubMedID 21693705

    View details for PubMedCentralID PMC3151032

  • Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools NATURE METHODS Kaiser, S. E., Riley, B. E., Shaler, T. A., Trevino, R. S., Becker, C. H., Schulman, H., Kopito, R. R. 2011; 8 (8): 691-U129


    The protein ubiquitin is an important post-translational modifier that regulates a wide variety of biological processes. In cells, ubiquitin is apportioned among distinct pools, which include a variety of free and conjugated species. Although maintenance of a dynamic and complex equilibrium among ubiquitin pools is crucial for cell survival, the tools necessary to quantify each cellular ubiquitin pool have been limited. We have developed a quantitative mass spectrometry approach to measure cellular concentrations of ubiquitin species using isotope-labeled protein standards and applied it to characterize ubiquitin pools in cells and tissues. Our method is convenient, adaptable and should be a valuable tool to facilitate our understanding of this important signaling molecule.

    View details for DOI 10.1038/NMETH.1649

    View details for Web of Science ID 000293220600027

    View details for PubMedID 21743460

    View details for PubMedCentralID PMC3196335

  • Altered Testicular Gene Expression Patterns in Mice Lacking the Polyubiquitin Gene Ubb MOLECULAR REPRODUCTION AND DEVELOPMENT Sinnar, S. A., Small, C. L., Evanoff, R. M., Reinholdt, L. G., Griswold, M. D., Kopito, R. R., Ryu, K. 2011; 78 (6): 415-425


    Ubiquitin (Ub) is an essential protein found in all eukaryotic cells and plays important roles in a variety of cellular functions including germ cell development. We have previously reported that targeted disruption of the polyubiquitin gene Ubb results in male and female infertility in Ubb(-/-) mice, with germ cells arrested at meiotic prophase I. Although reduced Ub levels in germ cells are believed to be responsible for the fertility defect in Ubb(-/-) mice, it is still unclear how reduced Ub levels result in sterility. Here we describe the results of a microarray analysis of the murine testicular transcriptome, which demonstrates dramatically altered gene expression patterns in Ubb(-/-) mice, possibly related to reduced levels of histone 2A (H2A) ubiquitylation. We find that large numbers of genes related to fertility, metabolism, transcription, and the ubiquitin-proteasome system (UPS) are misregulated in Ubb(-/-) mice. Such wide-ranging alterations in gene expression suggest that loss of the Ubb gene does not mimic a single-gene defect phenotype, but instead may affect gene expression more globally. These dramatic changes in gene expression could, at least in part, contribute to the complex fertility and metabolic phenotypes seen in these mice.

    View details for DOI 10.1002/mrd.21318

    View details for Web of Science ID 000292500700005

    View details for PubMedID 21542049

    View details for PubMedCentralID PMC3133727

  • Autophagy inhibition engages Nrf2-p62 Ub-associated signaling AUTOPHAGY Riley, B. E., Kaiser, S. E., Kopito, R. R. 2011; 7 (3): 338-340

    View details for DOI 10.4161/auto.7.3.14780

    View details for Web of Science ID 000287849800014

    View details for PubMedID 21252622

    View details for PubMedCentralID PMC3359479

  • Ubiquitin accumulation in autophagy-deficient mice is dependent on the Nrf2-mediated stress response pathway: a potential role for protein aggregation in autophagic substrate selection JOURNAL OF CELL BIOLOGY Riley, B. E., Kaiser, S. E., Shaler, T. A., Ng, A. C., Hara, T., Hipp, M. S., Lage, K., Xavier, R. J., Ryu, K. Y., Taguchi, K., Yamamoto, M., Tanaka, K., Mizushima, N., Komatsu, M., Kopito, R. R. 2010; 191 (3): 537-552


    Genetic ablation of autophagy in mice leads to liver and brain degeneration accompanied by the appearance of ubiquitin (Ub) inclusions, which has been considered to support the hypothesis that ubiquitination serves as a cis-acting signal for selective autophagy. We show that tissue-specific disruption of the essential autophagy genes Atg5 and Atg7 leads to the accumulation of all detectable Ub-Ub topologies, arguing against the hypothesis that any particular Ub linkage serves as a specific autophagy signal. The increase in Ub conjugates in Atg7(-/-) liver and brain is completely suppressed by simultaneous knockout of either p62 or Nrf2. We exploit a novel assay for selective autophagy in cell culture, which shows that inactivation of Atg5 leads to the selective accumulation of aggregation-prone proteins, and this does not correlate with an increase in substrate ubiquitination. We propose that protein oligomerization drives autophagic substrate selection and that the accumulation of poly-Ub chains in autophagy-deficient circumstances is an indirect consequence of activation of Nrf2-dependent stress response pathways.

    View details for DOI 10.1083/jcb.201005012

    View details for PubMedID 21041446

  • Loss of polyubiquitin gene Ubb leads to metabolic and sleep abnormalities in mice NEUROPATHOLOGY AND APPLIED NEUROBIOLOGY Ryu, K., Fujiki, N., Kazantzis, M., Garza, J. C., Bouley, D. M., Stahl, A., Lu, X., Nishino, S., Kopito, R. R. 2010; 36 (4): 285-299


    Ubiquitin performs essential roles in a myriad of signalling pathways required for cellular function and survival. Recently, we reported that disruption of the stress-inducible ubiquitin-encoding gene Ubb reduces ubiquitin content in the hypothalamus and leads to adult-onset obesity coupled with a loss of arcuate nucleus neurones and disrupted energy homeostasis in mice. Neuropeptides expressed in the hypothalamus control both metabolic and sleep behaviours. In order to demonstrate that the loss of Ubb results in broad hypothalamic abnormalities, we attempted to determine whether metabolic and sleep behaviours were altered in Ubb knockout mice.Metabolic rate and energy expenditure were measured in a metabolic chamber, and sleep stage was monitored via electroencephalographic/electromyographic recording. The presence of neurodegeneration and increased reactive gliosis in the hypothalamus were also evaluated.We found that Ubb disruption leads to early-onset reduced activity and metabolic rate. Additionally, we have demonstrated that sleep behaviour is altered and sleep homeostasis is disrupted in Ubb knockout mice. These early metabolic and sleep abnormalities are accompanied by persistent reactive gliosis and the loss of arcuate nucleus neurones, but are independent of neurodegeneration in the lateral hypothalamus.Ubb knockout mice exhibit phenotypes consistent with hypothalamic dysfunction. Our data also indicate that Ubb is essential for the maintenance of the ubiquitin levels required for proper regulation of metabolic and sleep behaviours in mice.

    View details for DOI 10.1111/j.1365-2990.2009.01057.x

    View details for Web of Science ID 000277712100003

    View details for PubMedID 20002312

  • Prion-like transmission of protein aggregates in neurodegenerative diseases NATURE REVIEWS MOLECULAR CELL BIOLOGY Brundin, P., Melki, R., Kopito, R. 2010; 11 (4): 301-307


    Neurodegenerative diseases are commonly associated with the accumulation of intracellular or extracellular protein aggregates. Recent studies suggest that these aggregates are capable of crossing cellular membranes and can directly contribute to the propagation of neurodegenerative disease pathogenesis. We propose that, once initiated, neuropathological changes might spread in a 'prion-like' manner and that disease progression is associated with the intercellular transfer of pathogenic proteins. The transfer of naked infectious particles between cells could therefore be a target for new disease-modifying therapies.

    View details for DOI 10.1038/nrm2873

    View details for Web of Science ID 000275850400014

    View details for PubMedID 20308987

  • SPFH1 and SPFH2 mediate the ubiquitination and degradation of inositol 1,4,5-trisphosphate receptors in muscarinic receptor-expressing HeLa cells BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH Wang, Y., Pearce, M. M., Sliter, D. A., Olzmann, J. A., Christianson, J. C., Kopito, R. R., Boeckmann, S., Gagen, C., Leichner, G. S., Roitelman, J., Wojcikiewicz, R. J. 2009; 1793 (11): 1710-1718


    Inositol 1,4,5-trisphosphate (IP(3)) receptors are endoplasmic reticulum (ER) membrane calcium channels that, upon activation, become substrates for the ER-associated degradation (ERAD) pathway. While it is clear that IP(3) receptors are polyubiquitinated and are transferred to the proteasome by a p97-based complex, currently very little is known about the proteins that initially select activated IP(3) receptors for ERAD. Here, we have transfected HeLa cells to stably express m3 muscarinic receptors to allow for the study of IP(3) receptor ERAD in this cell type, and show that IP(3) receptors are polyubiquitinated and then degraded by the proteasome in response to carbachol, a muscarinic agonist. In seeking to identify proteins that mediate IP(3) receptor ERAD we found that both SPFH1 and SPFH2 (also known as erlin 1 and erlin 2), which exist as a hetero-oligomeric complex, rapidly associate with IP(3) receptors in a manner that precedes polyubiquitination and the association of p97. Suppression of SPFH1 and SPFH2 expression by RNA interference markedly inhibited carbachol-induced IP(3) receptor polyubiquitination and degradation, but did not affect carbachol-induced calcium mobilization or IkappaBalpha processing, indicating that the SPFH1/2 complex is a key player in IP(3) receptor ERAD, acting at a step after IP(3) receptor activation, but prior to IP(3) receptor polyubiquitination. Suppression of SPFH1 and SPFH2 expression had only slight effects on the turnover of some exogenous model ERAD substrates, and had no effect on sterol-induced ERAD of endogenous 3-hydroxy-3-methylglutaryl-CoA reductase. Overall, these studies show that m3 receptor-expressing HeLa cells are a valuable system for studying IP(3) receptor ERAD, and suggest that the SPFH1/2 complex is a factor that selectively mediates the ERAD of activated IP(3) receptors.

    View details for DOI 10.1016/j.bbamcr.2009.09.004

    View details for Web of Science ID 000271552500009

    View details for PubMedID 19751772

    View details for PubMedCentralID PMC2764810

  • The polyubiquitin Ubc gene modulates histone H2A monoubiquitylation in the R6/2 mouse model of Huntington's disease JOURNAL OF CELLULAR AND MOLECULAR MEDICINE Bett, J. S., Benn, C. L., Ryu, K., Kopito, R. R., Bates, G. P. 2009; 13 (8B): 2645-2657


    Huntington's disease (HD) is an inherited neurodegenerative disease caused by the expansion of a polyglutamine tract in the protein huntingtin (htt). HD brains are characterized by the presence of ubiquitin-positive neuronal inclusion bodies, suggesting that disturbances in the distribution of cellular ubiquitin may contribute to disease pathology. The fact that several neurodegenerative diseases are caused by mutations in ubiquitin-processing enzymes and that the polyubiquitin genes are required for resistance to cellular stress led us to investigate the effect of perturbing the ubiquitin system in HD. We crossed R6/2 transgenic HD mice with heterozygous polyubiquitin Ubc knockout mice (Ubc+/-) and assessed the effect on the R6/2 neurological phenotype. Although the R6/2 phenotype was largely unaffected, surprisingly we observed some subtle improvements in various behavioural activities correlating with heterozygous Ubc knockout. Interestingly, immunoblot analysis revealed that the levels of monoubiquitylated histone H2A (uH2A), a modification associated with gene repression, were significantly increased in the brains of R6/2 mice. Furthermore, the reduction of Ubc expression in R6/2; Ubc+/- mice largely prevented this increase in uH2A levels. However, we were not able to show by the use of a limited number of quantitative RT-PCR assays that changes in the amount of uH2A in the R6/2-Ubc mice had an effect on disease-associated transcriptional abnormalities. These results suggest that the expression of aggregation-prone mutant htt causes disturbances to the ubiquitin system, which may contribute to disease due to the diverse and important roles of ubiquitin.

    View details for DOI 10.1111/j.1582-4934.2008.00543.x

    View details for Web of Science ID 000272190800089

    View details for PubMedID 19602042

  • Cytoplasmic penetration and persistent infection of mammalian cells by polyglutamine aggregates NATURE CELL BIOLOGY Ren, P., Lauckner, J. E., Kachirskaia, I., Heuser, J. E., Melki, R., Kopito, R. R. 2009; 11 (2): 219-U232


    Sequence-specific nucleated protein aggregation is closely linked to the pathogenesis of most neurodegenerative diseases and constitutes the molecular basis of prion formation. Here we report that fibrillar polyglutamine peptide aggregates can be internalized by mammalian cells in culture where they gain access to the cytosolic compartment and become co-sequestered in aggresomes together with components of the ubiquitin-proteasome system and cytoplasmic chaperones. Remarkably, these internalized fibrillar aggregates are able to selectively recruit soluble cytoplasmic proteins with which they share homologous but not heterologous amyloidogenic sequences, and to confer a heritable phenotype on cells expressing the homologous amyloidogenic protein from a chromosomal locus.

    View details for DOI 10.1038/ncb1830

    View details for Web of Science ID 000263285500021

    View details for PubMedID 19151706

    View details for PubMedCentralID PMC2757079

  • Misfolded proteins partition between two distinct quality control compartments NATURE Kaganovich, D., Kopito, R., Frydman, J. 2008; 454 (7208): 1088-U36


    The accumulation of misfolded proteins in intracellular amyloid inclusions, typical of many neurodegenerative disorders including Huntington's and prion disease, is thought to occur after failure of the cellular protein quality control mechanisms. Here we examine the formation of misfolded protein inclusions in the eukaryotic cytosol of yeast and mammalian cell culture models. We identify two intracellular compartments for the sequestration of misfolded cytosolic proteins. Partition of quality control substrates to either compartment seems to depend on their ubiquitination status and aggregation state. Soluble ubiquitinated misfolded proteins accumulate in a juxtanuclear compartment where proteasomes are concentrated. In contrast, terminally aggregated proteins are sequestered in a perivacuolar inclusion. Notably, disease-associated Huntingtin and prion proteins are preferentially directed to the perivacuolar compartment. Enhancing ubiquitination of a prion protein suffices to promote its delivery to the juxtanuclear inclusion. Our findings provide a framework for understanding the preferential accumulation of amyloidogenic proteins in inclusions linked to human disease.

    View details for DOI 10.1038/nature07195

    View details for Web of Science ID 000258719600031

    View details for PubMedID 18756251

    View details for PubMedCentralID PMC2746971

  • Hypothalamic neurodegeneration and adult-onset obesity in mice lacking the Ubb polyubiquitin gene PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ryu, K., Garza, J. C., Lu, X., Barsh, G. S., Kopito, R. R. 2008; 105 (10): 4016-4021


    Nearly all neurodegenerative diseases are associated with abnormal accumulation of ubiquitin (Ub) conjugates within neuronal inclusion bodies. To directly test the hypothesis that depletion of cellular Ub is sufficient to cause neurodegeneration, we have disrupted Ubb, one of four genes that supply Ub in the mouse. Here, we report that loss of Ubb led to a progressive degenerative disorder affecting neurons within the arcuate nucleus of the hypothalamus. This neurodegenerative cytopathology was accompanied by impaired hypothalamic control of energy balance and adult-onset obesity. Ubb was highly expressed in vulnerable hypothalamic neurons and total Ub levels were selectively reduced in the hypothalamus of Ubb-null mice. These findings demonstrate that maintenance of adequate supplies of cellular Ub is essential for neuronal survival and establish that decreased Ub availability is sufficient to cause neuronal dysfunction and death.

    View details for DOI 10.1073/pnas.0800096105

    View details for Web of Science ID 000253930600064

    View details for PubMedID 18299572

    View details for PubMedCentralID PMC2268782

  • OS-9 and GRP94 deliver mutant alpha 1-antitrypsin to the Hrd1-SEL1L ubiquitin ligase complex for ERAD NATURE CELL BIOLOGY Christianson, J. C., Shaler, T. A., Tyler, R. E., Kopito, R. R. 2008; 10 (3): 272-U13


    Terminally misfolded or unassembled proteins in the early secretory pathway are degraded by a ubiquitin- and proteasome-dependent process known as ER-associated degradation (ERAD). How substrates of this pathway are recognized within the ER and delivered to the cytoplasmic ubiquitin-conjugating machinery is unknown. We report here that OS-9 and XTP3-B/Erlectin are ER-resident glycoproteins that bind to ERAD substrates and, through the SEL1L adaptor, to the ER-membrane-embedded ubiquitin ligase Hrd1. Both proteins contain conserved mannose 6-phosphate receptor homology (MRH) domains, which are required for interaction with SEL1L, but not with substrate. OS-9 associates with the ER chaperone GRP94 which, together with Hrd1 and SEL1L, is required for the degradation of an ERAD substrate, mutant alpha(1)-antitrypsin. These data suggest that XTP3-B and OS-9 are components of distinct, partially redundant, quality control surveillance pathways that coordinate protein folding with membrane dislocation and ubiquitin conjugation in mammalian cells.

    View details for DOI 10.1038/ncb1689

    View details for Web of Science ID 000253778300009

    View details for PubMedID 18264092

    View details for PubMedCentralID PMC2757077

  • The mouse polyubiquitin gene Ubb is essential for meiotic progression MOLECULAR AND CELLULAR BIOLOGY Ryu, K., Sinnar, S. A., Reinholdt, L. G., Vaccari, S., Hall, S., Garcia, M. A., Zaitseva, T. S., Bouley, D. M., Boekelheide, K., Handel, M. A., Conti, M., Kopito, R. R. 2008; 28 (3): 1136-1146


    Ubiquitin is encoded in mice by two polyubiquitin genes, Ubb and Ubc, that are considered to be stress inducible and two constitutively expressed monoubiquitin (Uba) genes. Here we report that targeted disruption of Ubb results in male and female infertility due to failure of germ cells to progress through meiosis I and hypogonadism. In the absence of Ubb, spermatocytes and oocytes arrest during meiotic prophase, before metaphase of the first meiotic division. Although cellular ubiquitin levels are believed to be maintained by a combination of functional redundancy among the four ubiquitin genes, stress inducibility of the two polyubiquitin genes, and ubiquitin recycling by proteasome-associated isopeptidases, our results indicate that ubiquitin is required for and consumed during meiotic progression. The striking similarity of the meiotic phenotype in Ubb(-/-) germ cells to the sporulation defect in fission yeast (Schizosaccharomyces pombe) lacking a polyubiquitin gene suggests that a meiotic role of the polyubiquitin gene has been conserved throughout eukaryotic evolution.

    View details for DOI 10.1128/MCB.01566-07

    View details for Web of Science ID 000252606100024

    View details for PubMedID 18070917

    View details for PubMedCentralID PMC2223379

  • Global changes to the ubiquitin system in Huntington's disease NATURE Bennett, E. J., Shaler, T. A., Woodman, B., Ryu, K., Zaitseva, T. S., Becker, C. H., Bates, G. P., Schulman, H., Kopito, R. R. 2007; 448 (7154): 704-U11


    Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by expansion of CAG triplet repeats in the huntingtin (HTT) gene (also called HD) and characterized by accumulation of aggregated fragments of polyglutamine-expanded HTT protein in affected neurons. Abnormal enrichment of HD inclusion bodies with ubiquitin, a diagnostic characteristic of HD and many other neurodegenerative disorders including Alzheimer's and Parkinson's diseases, has suggested that dysfunction in ubiquitin metabolism may contribute to the pathogenesis of these diseases. Because modification of proteins with polyubiquitin chains regulates many essential cellular processes including protein degradation, cell cycle, transcription, DNA repair and membrane trafficking, disrupted ubiquitin signalling is likely to have broad consequences for neuronal function and survival. Although ubiquitin-dependent protein degradation is impaired in cell-culture models of HD and of other neurodegenerative diseases, it has not been possible to evaluate the function of the ubiquitin-proteasome system (UPS) in HD patients or in animal models of the disease, and a functional role for UPS impairment in neurodegenerative disease pathogenesis remains controversial. Here we exploit a mass-spectrometry-based method to quantify polyubiquitin chains and demonstrate that the abundance of these chains is a faithful endogenous biomarker of UPS function. Lys 48-linked polyubiquitin chains accumulate early in pathogenesis in brains from the R6/2 transgenic mouse model of HD, from a knock-in model of HD and from human HD patients, establishing that UPS dysfunction is a consistent feature of HD pathology. Lys 63- and Lys 11-linked polyubiquitin chains, which are not typically associated with proteasomal targeting, also accumulate in the R6/2 mouse brain. Thus, HD is linked to global changes in the ubiquitin system to a much greater extent than previously recognized.

    View details for DOI 10.1038/nature06022

    View details for Web of Science ID 000248598000047

    View details for PubMedID 17687326

  • The mouse polyubiquitin gene UbC is essential for fetal liver development, cell-cycle progression and stress tolerance EMBO JOURNAL Ryu, K., Maehr, R., Gilchrist, C. A., Long, M. A., Bouley, D. M., Mueller, B., Ploegh, H. L., Kopito, R. R. 2007; 26 (11): 2693-2706


    UbC is one of two stress-inducible polyubiquitin genes in mammals and is thought to supplement the constitutive UbA genes in maintaining cellular ubiquitin (Ub) levels during episodes of cellular stress. We have generated mice harboring a targeted disruption of the UbC gene. UbC(-/-) embryos die between embryonic days 12.5 and 14.5 in utero, most likely owing to a severe defect in liver cell proliferation. Mouse embryonic fibroblasts from UbC(-/-) embryos exhibit reduced growth rates, premature senescence, increased apoptosis and delayed cell-cycle progression, with slightly, but significantly, decreased steady-state Ub levels. UbC(-/-) fibroblasts are hypersensitive to proteasome inhibitors and heat shock, and unable to adequately increase Ub levels in response to these cellular stresses. Most, but not all of the UbC(-/-) phenotypes can be rescued by providing additional Ub from a poly hemagglutinin-tagged Ub minigene expressed from the Hprt locus. We propose that UbC is regulated by a process that senses Ub pool dynamics. These data establish that UbC constitutes an essential source of Ub during cell proliferation and stress that cannot be compensated by other Ub genes.

    View details for DOI 10.1038/sj.emboj.7601722

    View details for Web of Science ID 000247084100008

    View details for PubMedID 17491588

    View details for PubMedCentralID PMC1888680

  • Impaired post-translational folding of familial ALS-linked Cu, Zn superoxide dismutase mutants EMBO JOURNAL Bruns, C. K., Kopito, R. R. 2007; 26 (3): 855-866


    Over 110 structurally diverse missense mutations in the superoxide dismutase (SOD1) gene have been linked to the pathogenesis of familial amyotrophic lateral sclerosis (FALS), yet the mechanism by which these lead to cytotoxicity still remains unknown. We have synthesized wild-type and mutant SOD1 in synchronized cell-free reticulocyte extracts replete with the full complement of molecular chaperones and folding facilitators that are normally required to fold this metalloenzyme. Here, we report that, despite being a small, single-domain protein, human SOD1 folds post-translationally to a hyperstable native-like conformation without a requirement for ATP-dependent molecular chaperones. SOD1 folding requires tight Zn but not Cu binding and proceeds through at least three kinetically and biochemically distinct states. We find that all 11 FALS-associated SOD1 mutants examined using this system delay the kinetics of folding, but do not necessarily preclude the formation of native-like states. These data suggest a model whereby impaired post-translational folding increases the population of on- and off-pathway folding intermediates that could provide an important source of proto-toxic protein, and suggest a unifying mechanism for SOD1-linked FALS pathogenesis.

    View details for DOI 10.1038/sj.emboj.7601528

    View details for Web of Science ID 000244082500022

    View details for PubMedID 17255946

    View details for PubMedCentralID PMC1794386

  • Cellular mechanisms of protein quality control. Rinsho shinkeigaku = Clinical neurology Bennett, E. J., Shaler, T., Gonzalez-Zulueta, M., Schulman, H. F., Iwata, A., Riley, B. E., Johnston, J. A., Bucci, M., Nukina, N., Ellerby, L., Kopito, R. R. 2006; 46 (11): 805-?

    View details for PubMedID 17432184

  • Cellular mechanisms of protein quality control Bennett, E. J., Shaler, T., Gonzalez-Zulueta, M., Schulman, H. F., Iwata, A., Riley, B. E., Johnston, J. A., Bucci, M., Nukina, N., Ellerby, L., Kopito, R. R. LANDES BIOSCIENCE. 2006: 344–44
  • Ubiquitin-specific protease 2 as a tool for quantification of total ubiquitin levels in biological specimens ANALYTICAL BIOCHEMISTRY Ryu, K., Baker, R. T., Kopito, R. R. 2006; 353 (1): 153-155

    View details for DOI 10.1016/j.ab.2006.03.038

    View details for Web of Science ID 000237856700024

    View details for PubMedID 16643835

  • Central pore residues mediate the p97/VCP activity required for ERAD MOLECULAR CELL DeLaBarre, B., Christianson, J. C., Kopito, R. R., Brunger, A. T. 2006; 22 (4): 451-462


    The AAA-ATPase p97/VCP facilitates protein dislocation during endoplasmic reticulum-associated degradation (ERAD). To understand how p97/VCP accomplishes dislocation, a series of point mutants was made to disrupt distinguishing structural features of its central pore. Mutants were evaluated in vitro for ATPase activity in the presence and absence of synaptotagmin I (SytI) and in vivo for ability to process the ERAD substrate TCRalpha. Synaptotagmin induces a 4-fold increase in the ATPase activity of wild-type p97/VCP (p97/VCP(wt)), but not in mutants that showed an ERAD impairment. Mass spectrometry of crosslinked synaptotagmin . p97/VCP revealed interactions near Trp551 and Phe552. Additionally, His317, Arg586, and Arg599 were found to be essential for substrate interaction and ERAD. Except His317, which serves as an interaction nexus, these residues all lie on prominent loops within the D2 pore. These data support a model of substrate dislocation facilitated by interactions with p97/VCP's D2 pore.

    View details for DOI 10.1016/j.molcel.2006.03.036

    View details for Web of Science ID 000237813300007

    View details for PubMedID 16713576

  • Intersecting pathways to neurodegeneration in Parkinson's disease: Effects of the pesticide rotenone on DJ-1, alpha-synuclein, and the ubiquitin-proteasome system NEUROBIOLOGY OF DISEASE Betarbet, R., Canet-Aviles, R. A., Sherer, T. B., Mastroberardino, P. G., McLendon, C., Kim, J. H., Lund, S., Na, H. M., Taylor, G., Bence, N. F., Kopito, R., Seo, B. B., Yagi, T., Klinefelter, G., Cookson, M. R., Greenamyre, J. T. 2006; 22 (2): 404-420


    Sporadic Parkinson's disease (PD) is most likely caused by a combination of environmental exposures and genetic susceptibilities, although there are rare monogenic forms of the disease. Mitochondrial impairment at complex I, oxidative stress, alpha-synuclein aggregation, and dysfunctional protein degradation, have been implicated in PD pathogenesis, but how they are related to each other is unclear. To further evaluated PD pathogenesis here, we used in vivo and in vitro models of chronic low-grade complex I inhibition with the pesticide rotenone. Chronic rotenone exposure in vivo caused oxidative modification of DJ-1, accumulation of alpha-synuclein, and proteasomal impairment. Interestingly, the effects become more regionally restricted such that systemic complex I inhibition eventually results in highly selective degeneration of the nigrostriatal pathway. DJ-1 modifications, alpha-synuclein accumulation, and proteasomal dysfunction were also seen in vitro and these effects could be prevented with alpha-tocopherol. Thus, chronic exposure to a pesticide and mitochondrial toxin brings into play three systems, DJ-1, alpha-synuclein, and the ubiquitin-proteasome system, and implies that mitochondrial dysfunction and oxidative stress link environmental and genetic forms of the disease.

    View details for DOI 10.1016/j.nbd.2005.12.003

    View details for Web of Science ID 000237378000020

    View details for PubMedID 16439141

  • HDAC6 and microtubules are required for autophagic degradation of aggregated Huntingtin JOURNAL OF BIOLOGICAL CHEMISTRY Iwata, A., Riley, B. E., Johnston, J. A., Kopito, R. R. 2005; 280 (48): 40282-40292


    CNS neurons are endowed with the ability to recover from cytotoxic insults associated with the accumulation of proteinaceous polyglutamine aggregates via a process that appears to involve capture and degradation of aggregates by autophagy. The ubiquitin-proteasome system protects cells against proteotoxicity by degrading soluble monomeric misfolded aggregation-prone proteins but is ineffective against, and impaired by, non-native protein oligomers. Here we show that autophagy is induced in response to impaired ubiquitin proteasome system activity. We show that ATG proteins, molecular determinants of autophagic vacuole formation, and lysosomes are recruited to pericentriolar cytoplasmic inclusion bodies by a process requiring an intact microtubule cytoskeleton and the cytoplasmic deacetylase HDAC6. These data suggest that HDAC6-dependent retrograde transport on microtubules is used by cells to increase the efficiency and selectivity of autophagic degradation.

    View details for DOI 10.1074/jbc.M508786200

    View details for Web of Science ID 000233461300070

    View details for PubMedID 16192271

  • Increased susceptibility of cytoplasmic over nuclear polyglutamine aggregates to autophagic degradation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Iwata, A., Christianson, J. C., Bucci, M., Ellerby, L. M., Nukina, N., Forno, L. S., Kopito, R. R. 2005; 102 (37): 13135-13140


    CNS neurons are endowed with the ability to recover from cytotoxic insults associated with the accumulation of proteinaceous aggregates in mouse models of polyglutamine disease, but the cellular mechanism underlying this phenomenon is unknown. Here, we show that autophagy is essential for the elimination of aggregated forms of mutant huntingtin and ataxin-1 from the cytoplasmic but not nuclear compartments. Human orthologs of yeast autophagy genes, molecular determinants of autophagic vacuole formation, are recruited to cytoplasmic but not nuclear inclusion bodies in vitro and in vivo. These data indicate that autophagy is a critical component of the cellular clearance of toxic protein aggregates and may help to explain why protein aggregates are more toxic when directed to the nucleus.

    View details for DOI 10.1073/pnas.0505801102

    View details for Web of Science ID 000231916300025

    View details for PubMedID 16141322

    View details for PubMedCentralID PMC1201602

  • Formation of morphologically similar globular aggregates from diverse aggregation-prone proteins in mammalian cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mukai, H., Isagawa, T., Goyama, E., Tanaka, S., Bence, N. F., Tamura, A., Ono, Y., Kopito, R. R. 2005; 102 (31): 10887-10892


    Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine repeat expansion in the first exon of the huntingtin (Htt) protein. N-terminal Htt peptides with polyglutamine tracts in the pathological range (51-122 glutamines) form high-molecular-weight protein aggregates with fibrillar morphology in vitro, and they form discrete inclusion bodies in a cell-culture model. However, in some studies, formation of discrete Htt inclusions does not correlate well with cell death. We coexpressed N-terminal Htt fragments containing 91 glutamines fused to different affinity tags in HEK293 cells, and we isolated small aggregates by double sequential-affinity chromatography to assure the isolation of multimeric molecules. Transmission electron microscopy and atomic force microscopy revealed the isolated aggregates as globules or clusters of globules 4-50 nm in diameter without any detectable fibrillar species. Because small nonfibrillar oligomers, not mature fibrils, recently have been suggested to be the principal cytotoxic species in neurodegenerative disease, these Htt globular aggregates formed in cells may represent the pathogenic form of mutant Htt.

    View details for DOI 10.1073/pnas.0409283102

    View details for Web of Science ID 000231102400029

    View details for PubMedID 16040812

  • Subversion of cellular autophagosomal machinery by RNA viruses PLOS BIOLOGY Jackson, W. T., Giddings, T. H., Taylor, M. P., Mulinyawe, S., RABINOVITCH, M., Kopito, R. R., Kirkegaard, K. 2005; 3 (5): 861-871


    Infection of human cells with poliovirus induces the proliferation of double-membraned cytoplasmic vesicles whose surfaces are used as the sites of viral RNA replication and whose origin is unknown. Here, we show that several hallmarks of cellular autophagosomes can be identified in poliovirus-induced vesicles, including colocalization of LAMP1 and LC3, the human homolog of Saccharomyces cerevisiae Atg8p, and staining with the fluorophore monodansylcadaverine followed by fixation. Colocalization of LC3 and LAMP1 was observed early in the poliovirus replicative cycle, in cells infected with rhinoviruses 2 and 14, and in cells that express poliovirus proteins 2BC and 3A, known to be sufficient to induce double-membraned vesicles. Stimulation of autophagy increased poliovirus yield, and inhibition of the autophagosomal pathway by 3-methyladenine or by RNA interference against mRNAs that encode two different proteins known to be required for autophagy decreased poliovirus yield. We propose that, for poliovirus and rhinovirus, components of the cellular machinery of autophagosome formation are subverted to promote viral replication. Although autophagy can serve in the innate immune response to microorganisms, our findings are inconsistent with a role for the induced autophagosome-like structures in clearance of poliovirus. Instead, we argue that these double-membraned structures provide membranous supports for viral RNA replication complexes, possibly enabling the nonlytic release of cytoplasmic contents, including progeny virions, from infected cells.

    View details for DOI 10.1371/journal.pbio.0030156

    View details for Web of Science ID 000229125400014

    View details for PubMedID 15884975

    View details for PubMedCentralID PMC1084330

  • Global impairment of the ubiquitin-proteasome system by nuclear or cytoplasmic protein aggregates precedes inclusion body formation MOLECULAR CELL Bennett, E. J., Bence, N. F., Jayakumar, R., Kopito, R. R. 2005; 17 (3): 351-365


    The highly conserved ubiquitin-proteasome system (UPS) controls the stability of most nuclear and cytoplasmic proteins and is therefore essential for virtually all aspects of cellular function. We have previously shown that the UPS is impaired in the presence of aggregated proteins that become deposited into cytoplasmic inclusion bodies (IBs). Here, we report that production of protein aggregates specifically targeted to either the nucleus or cytosol leads to global impairment of UPS function in both cellular compartments and is independent of sequestration of aggregates into IBs. The observation of severe UPS impairment in compartments lacking detectable aggregates or aggregation-prone protein, together with the lack of interference of protein aggregates on 26S proteasome function in vitro, suggests that UPS impairment is unlikely to be a consequence of direct choking of proteasomes by protein aggregates. These data suggest a common proteotoxic mechanism for nuclear and cytoplasmic protein aggregates in the pathogenesis of neurodegenerative disease.

    View details for DOI 10.1016/j.molcel.2004.12.021

    View details for Web of Science ID 000226905200005

    View details for PubMedID 15694337

  • Effect of ubiquitin expression on neuropathogenesis in a mouse model of familial amyotrophic lateral sclerosis NEUROPATHOLOGY AND APPLIED NEUROBIOLOGY Gilchrist, C. A., Gray, D. A., Stieber, A., GONATAS, N. K., Kopito, R. R. 2005; 31 (1): 20-33


    The ubiquitin-proteasome system (UPS) is a central component in the cellular defence against potentially toxic protein aggregates. UPS dysfunction is linked to the pathogenesis of both sporadic and inherited neurodegenerative diseases, including dominantly inherited familial amyotrophic lateral sclerosis (fALS). To investigate the role of the UPS in fALS pathogenesis, transgenic mice expressing mutant G9 3A Cu,Zn superoxide dismutase (SOD1) were crossed with transgenic mice expressing epitope tagged, wild-type or dominant-negative mutant ubiquitin (Ub(K48R)). RNase protection assays were used to confirm expression of the Ub transgenes in spinal cord and ubiquitin transgene levels were estimated to account for 9-12% of total ubiquitin. Mice expressing the G9 3A transgene exhibited neurological symptoms and histopathological changes typical of this model irrespective of ubiquitin transgene status. Impaired rotarod performance was observed in all G9 3A transgenics by 7 weeks of age irrespective of ubiquitin genotype. The presence of wild-type or mutant ubiquitin transgenes resulted in a small but significant delay in the onset of clinical symptoms and mild acceleration of disease progression, without influencing overall survival. These data suggest that relatively small changes in ubiquitin expression can influence the development of neurodegenerative disease and are consistent with a neuroprotective role for the UPS.

    View details for DOI 10.1111/j.1365-2990.2004.00604.x

    View details for Web of Science ID 000225949100003

    View details for PubMedID 15634228

  • Suppression of wild-type rhodopsin maturation by mutants linked to autosomal dominant retinitis pigmentosa JOURNAL OF BIOLOGICAL CHEMISTRY Rajan, R. S., Kopito, R. R. 2005; 280 (2): 1284-1291


    Autosomal dominant retinitis pigmentosa (ADRP) has been linked to mutations in the gene encoding rhodopsin. Most RP-linked rhodopsin mutants are unable to fold correctly in the endoplasmic reticulum, are degraded by the ubiquitin proteasome system, and are highly prone to forming detergent-insoluble high molecular weight aggregates. Here we have reported that coexpression of folding-deficient, but not folding-proficient, ADRP-linked rhodopsin mutants impairs delivery of the wild-type protein to the plasma membrane. Fluorescence resonance energy transfer and co-precipitation studies revealed that mutant and wild-type rhodopsins form a high molecular weight, detergent-insoluble complex in which the two proteins are in close (<70 A) proximity. Co-expression of ARDP-linked rhodopsin folding-deficient mutants resulted in enhanced proteasome-mediated degradation and steady-state ubiquitination of the wild-type protein. These data suggested a dominant negative effect on conformational maturation that may underlie the dominant inheritance of ARDP.

    View details for DOI 10.1074/jbc.M406448200

    View details for Web of Science ID 000226195200053

    View details for PubMedID 15509574

  • Application and analysis of the GFP(u) family of ubiquitin-proteasome system reporters UBIQUITIN AND PROTEIN DEGRADATION, PT B Bence, N. F., Bennett, E. J., Kopito, R. R. 2005; 399: 481-490


    The relevance of the ubiquitin proteasome system (UPS) to disease and fundamental cellular processes has generated a demand for methods to monitor the activity of this system in living cells and organisms. Here we describe the GFP(u) family of UPS reporters. These reporters are constitutively degraded, ubiquitin-dependent proteasome substrates that can be used to monitor UPS function in the living cell. The GFP(u) reporter family consists of three variants that can report on global, nuclear, and cytoplasmic UPS function. This article focuses on the properties and design of these reporters and highlights appropriate techniques and applications for their use.

    View details for DOI 10.1016/50076-6879(05)99033-2

    View details for Web of Science ID 000233597300033

    View details for PubMedID 16338377

  • The missing linker: An unexpected role for a histone deacetylase MOLECULAR CELL Kopito, R. R. 2003; 12 (6): 1349-1351


    Molecular motors are the long-haul carriers of eukaryotic cells, moving cargos bidirectionally along microtubule tracks. In the December 12th issue of Cell, report that HDAC6, a tubulin deacetylase, functions as an adaptor that links cargos of aggregated protein to the minus end-directed motor, cytoplasmic dynein.

    View details for Web of Science ID 000187511600005

    View details for PubMedID 14690590

  • Immunoglobulin light chains dictate vesicular transport-dependent and -independent routes for IgM degradation by the ubiquitin-proteasome pathway JOURNAL OF BIOLOGICAL CHEMISTRY Elkabetz, Y., Kerem, A., Tencer, L., Winitz, D., Kopito, R. R., Bar-Nun, S. 2003; 278 (21): 18922-18929


    Degradation of IgM mu heavy chains in light chain-negative pre-B cells is independent of vesicular transport, as is evident by its insensitivity to brefeldin A or cell permeabilization. Conversely, by the same criteria, degradation of the secretory mu heavy chain in light chain-expressing B cells depends on vesicular transport. To investigate whether the presence of conventional light chains or the developmental stage of the B-lymphocytes dictates the degradative route taken by mu, we express in 70Z/3 pre-B cells either lambda ectopically or kappa by lipopolysaccharides-stimulated differentiation into B cells and show their assembly with mu heavy chains. The resulting sensitivity of mu degradation to brefeldin A and cell permeabilization demonstrates that conventional light chains, a hallmark of B cell differentiation, are necessary and sufficient to divert mu from a vesicular transport-independent to a vesicular transport-dependent degradative route. Although both routes converge at the ubiquitin-proteasome degradation pathway, only in light chain-expressing cells is vesicular transport a prerequisite for mu ubiquitination.

    View details for DOI 10.1074/jbc.M.08730200

    View details for Web of Science ID 000182932200028

    View details for PubMedID 12754269

  • Recognition of a single transmembrane degron by sequential quality control checkpoints MOLECULAR BIOLOGY OF THE CELL Fayadat, L., Kopito, R. R. 2003; 14 (3): 1268-1278


    To understand the relationship between conformational maturation and quality control-mediated proteolysis in the secretory pathway, we engineered the well-characterized degron from the alpha-subunit of the T-cell antigen receptor (TCRalpha) into the alpha-helical transmembrane domain of homotrimeric type I integral membrane protein, influenza hemagglutinin (HA). Although the membrane degron does not appear to interfere with acquisition of native secondary structure, as assessed by the formation of native intrachain disulfide bonds, only approximately 50% of nascent mutant HA chains (HA(++)) become membrane-integrated and acquire complex N-linked glycans indicative of transit to a post-ER compartment. The remaining approximately 50% of nascent HA(++) chains fail to integrate into the lipid bilayer and are subject to proteasome-dependent degradation. Site-specific cleavage by extracellular trypsin and reactivity with conformation-specific monoclonal antibodies indicate that membrane-integrated HA(++) molecules are able to mature to the plasma membrane with a conformation indistinguishable from that of HA(wt). These apparently native HA(++) molecules are, nevertheless, rapidly degraded by a process that is insensitive to proteasome inhibitors but blocked by lysosomotropic amines. These data suggest the existence in the secretory pathway of at least two sequential quality control checkpoints that recognize the same transmembrane degron, thereby ensuring the fidelity of protein deployment to the plasma membrane.

    View details for DOI 10.1091/mbc.E02-06-0363

    View details for Web of Science ID 000181578700034

    View details for PubMedID 12631739

    View details for PubMedCentralID PMC151595

  • Cystic fibrosis: premature degradation of mutant proteins as a molecular disease mechanism. Methods in molecular biology (Clifton, N.J.) Gelman, M. S., Kopito, R. R. 2003; 232: 27-37

    View details for PubMedID 12840537

  • Rescuing protein conformation: prospects for pharmacological therapy in cystic fibrosis JOURNAL OF CLINICAL INVESTIGATION Gelman, M. S., Kopito, R. R. 2002; 110 (11): 1591-1597

    View details for DOI 10.1172/JCI200216786

    View details for Web of Science ID 000179706500003

    View details for PubMedID 12464661

  • A rhodopsin mutant linked to autosomal dominant retinitis pigmentosa is prone to aggregate and interacts with the ubiquitin proteasome system JOURNAL OF BIOLOGICAL CHEMISTRY Illing, M. E., Rajan, R. S., Bence, N. F., Kopito, R. R. 2002; 277 (37): 34150-34160


    The inherited retinal degenerations are typified by retinitis pigmentosa (RP), a heterogeneous group of inherited disorders that causes the destruction of photoreceptor cells, the retinal pigmented epithelium, and choroid. This group of blinding conditions affects over 1.5 million persons worldwide. Approximately 30-40% of human autosomal dominant (AD) RP is caused by dominantly inherited missense mutations in the rhodopsin gene. Here we show that P23H, the most frequent RP mutation in American patients, renders rhodopsin extremely prone to form high molecular weight oligomeric species in the cytoplasm of transfected cells. Aggregated P23H accumulates in aggresomes, which are pericentriolar inclusion bodies that require an intact microtubule cytoskeleton to form. Using fluorescence resonance energy transfer (FRET), we observe that P23H aggregates in the cytoplasm even at extremely low expression levels. Our data show that the P23H mutation destabilizes the protein and targets it for degradation by the ubiquitin proteasome system. P23H is stabilized by proteasome inhibitors and by co-expression of a dominant negative form of ubiquitin. We show that expression of P23H, but not wild-type rhodopsin, results in a generalized impairment of the ubiquitin proteasome system, suggesting a mechanism for photoreceptor degeneration that links RP to a broad class of neurodegenerative diseases.

    View details for DOI 10.1074/jbc.M204955200

    View details for Web of Science ID 000177959100082

    View details for PubMedID 12091393

  • Cytoplasmic dynein/dynactin mediates the assembly of aggresomes CELL MOTILITY AND THE CYTOSKELETON Johnston, J. A., Illing, M. E., Kopito, R. R. 2002; 53 (1): 26-38


    Aggresomes are pericentrosomal cytoplasmic structures into which aggregated, ubiquitinated, misfolded proteins are sequestered. Misfolded proteins accumulate in aggresomes when the capacity of the intracellular protein degradation machinery is exceeded. Previously, we demonstrated that an intact microtubule cytoskeleton is required for the aggresome formation [Johnston et al., 1998: J. Cell Biol. 143:1883-1898]. In this study, we have investigated the involvement of microtubules (MT) and MT motors in this process. Induction of aggresomes containing misfolded DeltaF508 CFTR is accompanied by a redistribution of the retrograde motor cytoplasmic dynein that colocalizes with aggresomal markers. Coexpression of the p50 (dynamitin) subunit of the dynein/dynactin complex prevents the formation of aggresomes, even in the presence of proteasome inhibitors. Using in vitro microtubule binding assays in conjunction with immunogold electron microscopy, our data demonstrate that misfolded DeltaF508 CFTR associate with microtubules. We conclude that cytoplasmic dynein/dynactin is responsible for the directed transport of misfolded protein into aggresomes. The implications of these findings with respect to the pathogenesis of neurodegenerative disease are discussed.

    View details for DOI 10.1002/cm.10057

    View details for Web of Science ID 000177870800002

    View details for PubMedID 12211113

  • A principal role for the proteasome in endoplasmic reticulum-associated degradation of misfolded intracellular cystic fibrosis transmembrane conductance regulator JOURNAL OF BIOLOGICAL CHEMISTRY Gelman, M. S., Kannegaard, E. S., Kopito, R. R. 2002; 277 (14): 11709-11714


    Endoplasmic reticulum-associated degradation of misfolded cystic fibrosis transmembrane conductance regulator (CFTR) protein is known to involve the ubiquitin-proteasome system. In addition, an ATP-independent proteolytic system has been suggested to operate in parallel with this pathway and become up-regulated when proteasomes are inhibited (Jensen, T. J., Loo, M. A., Pind, S., Williams, D. B., Goldberg, A. L., and Riordan, J. R. (1995) Cell 83, 129-135). In this study, we use two independent techniques, pulse-chase labeling and a noninvasive fluorescence cell-based assay, to investigate the proteolytic pathways underlying the degradation of misfolded CFTR. Here we report that only inhibitors of the proteasome have a significant effect on preventing the degradation of CFTR, whereas cell-permeable inhibitors of lysosomal degradation, autophagy, and several classes of protease had no measurable effect on CFTR degradation, when used either alone or in combination with the specific proteasome inhibitor carbobenzoxy-l-leucyl-leucyl-l-leucinal (MG132). Our results suggest that ubiquitin-proteasome-mediated degradation is the dominant pathway for disposal of misfolded CFTR in mammalian cells and provide new mechanistic insight into endoplasmic reticulum-associated degradation.

    View details for DOI 10.1074/jbc.M111958200

    View details for Web of Science ID 000174846400015

    View details for PubMedID 11812794

  • Cysteine residues in the nucleotide binding domains regulate the conductance state of CFTR channels BIOPHYSICAL JOURNAL Harrington, M. A., Kopito, R. R. 2002; 82 (3): 1278-1292


    Gating of cystic fibrosis transmembrane conductance regulator (CFTR) channels requires intermolecular or interdomain interactions, but the exact nature and physiological significance of those interactions remains uncertain. Subconductance states of the channel may result from alterations in interactions among domains, and studying mutant channels enriched for a single conductance type may elucidate those interactions. Analysis of CFTR channels in inside-out patches revealed that mutation of cysteine residues in NBD1 and NBD2 affects the frequency of channel opening to the full-size versus a 3-pS subconductance. Mutating cysteines in NBD1 resulted in channels that open almost exclusively to the 3-pS subconductance, while mutations of cysteines in NBD2 decreased the frequency of subconductance openings. Wild-type channels open to both size conductances and make fast transitions between them within a single open burst. Full-size and subconductance openings of both mutant and wild-type channels are similarly activated by ATP and phosphorylation. However, the different size conductances open very differently in the presence of a nonhydrolyzable ATP analog, with subconductance openings significantly shortened by ATPgammaS, while full-size channels are locked open. In wild-type channels, reducing conditions increase the frequency and decrease the open time of subconductance channels, while oxidizing conditions decrease the frequency of subconductance openings. In contrast, in the cysteine mutants studied, altering redox potential has little effect on gating of the subconductance.

    View details for Web of Science ID 000174170700015

    View details for PubMedID 11867445

  • Specificity in intracellular protein aggregation and inclusion body formation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rajan, R. S., Illing, M. E., Bence, N. F., Kopito, R. R. 2001; 98 (23): 13060-13065


    Protein aggregation is widely considered to be a nonspecific coalescence of misfolded proteins, driven by interactions between solvent-exposed hydrophobic surfaces that are normally buried within a protein's interior. Accordingly, abnormal interactions between misfolded proteins with normal cellular constituents has been proposed to underlie the toxicity associated with protein aggregates in many neurodegenerative disorders. Here we have used fluorescence resonance energy transfer and deconvolution microscopy to investigate the degree to which unrelated misfolded proteins expressed in the same cells coaggregate with one another. Our data reveal that in cells, protein aggregation exhibits exquisite specificity even among extremely hydrophobic substrates expressed at very high levels.

    View details for Web of Science ID 000172076800043

    View details for PubMedID 11687604

  • Impairment of the ubiquitin-proteasome system by protein aggregation SCIENCE Bence, N. F., Sampat, R. M., Kopito, R. R. 2001; 292 (5521): 1552-1555


    Intracellular deposition of aggregated and ubiquitylated proteins is a prominent cytopathological feature of most neurodegenerative disorders. Whether protein aggregates themselves are pathogenic or are the consequence of an underlying molecular lesion is unclear. Here, we report that protein aggregation directly impaired the function of the ubiquitin-proteasome system. Transient expression of two unrelated aggregation-prone proteins, a huntingtin fragment containing a pathogenic polyglutamine repeat and a folding mutant of cystic fibrosis transmembrane conductance regulator, caused nearly complete inhibition of the ubiquitin-proteasome system. Because of the central role of ubiquitin-dependent proteolysis in regulating fundamental cellular events such as cell division and apoptosis, our data suggest a potential mechanism linking protein aggregation to cellular disregulation and cell death.

    View details for Web of Science ID 000168963800047

    View details for PubMedID 11375494

  • Aggresomes, inclusion bodies and protein aggregation TRENDS IN CELL BIOLOGY Kopito, R. R. 2000; 10 (12): 524-530


    Intracellular and extracellular accumulation of aggregated protein are linked to many diseases, including ageing-related neurodegeneration and systemic amyloidosis. Cells avoid accumulating potentially toxic aggregates by mechanisms including the suppression of aggregate formation by molecular chaperones and the degradation of misfolded proteins by proteasomes. Once formed, aggregates tend to be refractory to proteolysis and to accumulate in inclusion bodies. This accumulation has been assumed to be a diffusion-limited process, but recent studies suggest that, in animal cells, aggregated proteins are specifically delivered to inclusion bodies by dynein-dependent retrograde transport on microtubules. This microtubule-dependent inclusion body is called an aggresome.

    View details for Web of Science ID 000165657700003

    View details for PubMedID 11121744

  • Formation of high molecular weight complexes of mutant Cu,Zn-superoxide dismutase in a mouse model for familial amyotrophic lateral sclerosis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Johnston, J. A., Dalton, M. J., Gurney, M. E., Kopito, R. R. 2000; 97 (23): 12571-12576


    Deposition of aggregated protein into neurofilament-rich cytoplasmic inclusion bodies is a common cytopathological feature of neurodegenerative disease. How-or indeed whether-protein aggregation and inclusion body formation cause neurotoxicity are presently unknown. Here, we show that the capacity of superoxide dismutase (SOD) to aggregate into biochemically distinct, high molecular weight, insoluble protein complexes (IPCs) is a gain of function associated with mutations linked to autosomal dominant familial amyotrophic lateral sclerosis. SOD IPCs are detectable in spinal cord extracts from transgenic mice expressing mutant SOD several months before inclusion bodies and motor neuron pathology are apparent. Sequestration of mutant SOD into cytoplasmic inclusion bodies resembling aggresomes requires retrograde transport on microtubules. These data indicate that aggregation and inclusion body formation are mechanistically and temporally distinct processes.

    View details for Web of Science ID 000165225800042

    View details for PubMedID 11050163

  • Conformational disease NATURE CELL BIOLOGY Kopito, R. R., Ron, D. 2000; 2 (11): E207-E209


    A large and diverse number of diseases are now recognized as 'conformational diseases', caused by adoption of non-native protein conformations that lead to aggregation. The recent conference, 'Alpha1-antitrypsin deficiency and other conformational diseases', held in Airlie, Virginia, USA (27-30 June, 2000) focused on some of the common pathways by which cells protect themselves from toxicity associated with protein misfolding and aggregation.

    View details for Web of Science ID 000165207500008

    View details for PubMedID 11056553

  • Aggresomes and Russell bodies - Symptoms of cellular indigestion? EMBO REPORTS Kopito, R. R., Sitia, R. 2000; 1 (3): 225-231


    All cells are equipped with a proteolytic apparatus that eliminates damaged, misfolded and incorrectly assembled proteins. The principal engine of cytoplasmic proteolysis, the 26S proteasome, requires that substrates be unfolded to gain access to the active site; consequently, it is relatively ineffective at degrading aggregated proteins. Cellular indigestion occurs when the production of aggregation-prone proteins exceeds the cell's (or organelle's) capacity to eliminate them. Cellular pathways that resolve this indigestion exist, but appear to have limited capacities. Russell bodies and aggresomes are manifestations of cellular indigestion in the endoplasmic reticulum and cytoplasmic compartments, respectively, and are often associated with disease.

    View details for DOI 10.1093/embo-reports/kvd052

    View details for Web of Science ID 000165766200011

    View details for PubMedID 11256604

    View details for PubMedCentralID PMC1083726

  • The role of multiubiquitination in dislocation and degradation of the alpha subunit of the T cell antigen receptor JOURNAL OF BIOLOGICAL CHEMISTRY Yu, H., Kopito, R. R. 1999; 274 (52): 36852-36858


    Unassembled alpha subunits of the T cell receptor (TCRalpha) are degraded by proteasomes following their dislocation from the endoplasmic reticulum membrane. We previously demonstrated that a variant of TCRalpha lacking lysines (KalphaR) is degraded by this pathway with kinetics indistinguishable from those of the wild type protein (Yu, H., Kaung, G., Kobayashi, S., and Kopito, R. R. (1997) J. Biol. Chem. 272, 20800-20804), demonstrating that ubiquitination on lysines is not required for TCRalpha degradation by the proteasome. Here, we show that dislocation and degradation of TCRalpha and KalphaR are suppressed by dominant negative ubiquitin coexpression and by mutations in the ubiquitin activating enzyme, indicating that their degradation requires a functional ubiquitin pathway. A cytoplasmic TCRalpha variant that mimics a dislocated degradation intermediate was degraded 5 times more rapidly than full-length TCRalpha, suggesting that dislocation from the endoplasmic reticulum membrane is the rate-limiting step in TCRalpha degradation. We conclude that ubiquitination is required both for dislocation and for targeting TCRalpha chains to the proteasome.

    View details for Web of Science ID 000084382700007

    View details for PubMedID 10601236

  • Redox reagents and divalent cations alter the kinetics of cystic fibrosis transmembrane conductance regulator channel gating JOURNAL OF BIOLOGICAL CHEMISTRY Harrington, M. A., Gunderson, K. L., Kopito, R. R. 1999; 274 (39): 27536-27544


    Gating of the cystic fibrosis Cl(-) channel requires hydrolysis of ATP by its nucleotide binding folds, but how this process controls the kinetics of channel gating is poorly understood. In the present work we show that the kinetics of channel gating and presumably the rate of ATP hydrolysis depends on the species of divalent cation present and the oxidation state of the protein. With Ca(2+) as the dominant divalent cation instead of Mg(2+), the open burst duration of the channel is increased approximately 20-fold, and this change is reversible upon washout of Ca(2+). In contrast, "soft" divalent cations such as Cd(2+) interact covalently with cystic fibrosis transmembrane conductance regulator (CFTR). These metals decrease both opening and closing rates of the channel, and the effects are not reversed by washout. Oxidation of CFTR channels with a variety of oxidants resulted in a similar slowing of channel gating. In contrast, reducing agents had the opposite effect, increasing both opening and closing rates of the channel. In cell-attached patches, CFTR channels exhibit both oxidized and reduced types of gating, raising the possibility that regulation of the redox state of the channel may be a physiological mode of control of CFTR channel activity.

    View details for Web of Science ID 000082739100028

    View details for PubMedID 10488089

  • Aggresomes: A cellular response to misfolded proteins Johnston, J. A., Ward, C. L., Kopito, R. R. FEDERATION AMER SOC EXP BIOL. 1999: A1520–A1520
  • Biosynthesis and degradation of CFTR. Physiological reviews Kopito, R. R. 1999; 79 (1): S167-73


    Biosynthesis and Degradation of CFTR. Physiol. Rev. 79, Suppl.: S167-S173, 1999. - Many of the mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that cause cystic fibrosis interfere with the folding and biosynthetic processing of nascent CFTR molecules in the endoplasmic reticulum. Mutations in the cytoplasmic nucleotide binding domains, including the common allele DeltaF508, decrease the efficiency of CFTR folding, reduce the probability of its dissociation from molecular chaperones, and largely prevent its maturation through the secretory pathway to the plasma membrane. These mutant CFTR molecules are rapidly degraded by cytoplasmic proteasomes by a process that requires covalent modification by multiubiquitination. The effects of temperature and chemical chaperones on the intracellular processing of mutant CFTR molecules suggest that strategies aimed at increasing the folding yield of this protein in vivo may eventually lead to the development of novel therapies for cystic fibrosis.

    View details for PubMedID 9922380

  • Aggresomes: A cellular response to misfolded proteins JOURNAL OF CELL BIOLOGY Johnston, J. A., Ward, C. L., Kopito, R. R. 1998; 143 (7): 1883-1898


    Intracellular deposition of misfolded protein aggregates into ubiquitin-rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Why these aggregates form despite the existence of cellular machinery to recognize and degrade misfolded protein and how they are delivered to cytoplasmic inclusions are not known. We have investigated the intracellular fate of cystic fibrosis transmembrane conductance regulator (CFTR), an inefficiently folded integral membrane protein which is degraded by the cytoplasmic ubiquitin-proteasome pathway. Overexpression or inhibition of proteasome activity in transfected human embryonic kidney or Chinese hamster ovary cells led to the accumulation of stable, high molecular weight, detergent-insoluble, multiubiquitinated forms of CFTR. Using immunofluorescence and transmission electron microscopy with immunogold labeling, we demonstrate that undegraded CFTR molecules accumulate at a distinct pericentriolar structure which we have termed the aggresome. Aggresome formation is accompanied by redistribution of the intermediate filament protein vimentin to form a cage surrounding a pericentriolar core of aggregated, ubiquitinated protein. Disruption of microtubules blocks the formation of aggresomes. Similarly, inhibition of proteasome function also prevented the degradation of unassembled presenilin-1 molecules leading to their aggregation and deposition in aggresomes. These data lead us to propose that aggresome formation is a general response of cells which occurs when the capacity of the proteasome is exceeded by the production of aggregation-prone misfolded proteins.

    View details for Web of Science ID 000077900300010

    View details for PubMedID 9864362

  • Aggresomes: A cellular response to misfolded proteins Johnson, J. A., Ward, C. W., Kopito, R. R. AMER SOC CELL BIOLOGY. 1998: 260A–260A
  • Indirect requirement for multi-ubiquitination in TCR alpha degradation Yu, H., Kopito, R. AMER SOC CELL BIOLOGY. 1998: 459A–459A
  • Cytosolic pH regulates G(Cl) through control of phosphorylation states of CFTR AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Reddy, M. M., Kopito, R. R., Quinton, P. M. 1998; 275 (4): C1040-C1047


    Our objective in this study was to determine the effect of changes in luminal and cytoplasmic pH on cystic fibrosis transmembrane regulator (CFTR) Cl- conductance (GCl). We monitored CFTR GCl in the apical membranes of sweat ducts as reflected by Cl- diffusion potentials (VCl) and transepithelial conductance (GCl). We found that luminal pH (5.0-8.5) had little effect on the cAMP/ATP-activated CFTR GCl, showing that CFTR GCl is maintained over a broad range of extracellular pH in which it functions physiologically. However, we found that phosphorylation activation of CFTR GCl is sensitive to intracellular pH. That is, in the presence of cAMP and ATP [adenosine 5'-O-(3-thiotriphosphate)], CFTR could be phosphorylated at physiological pH (6.8) but not at low pH (approximately 5.5). On the other hand, basic pH prevented endogenous phosphatase(s) from dephosphorylating CFTR. After phosphorylation of CFTR with cAMP and ATP, CFTR GCl is normally deactivated within 1 min after cAMP is removed, even in the presence of 5 mM ATP. This deactivation was due to an increase in endogenous phosphatase activity relative to kinase activity, since it was reversed by the reapplication of ATP and cAMP. However, increasing cytoplasmic pH significantly delayed the deactivation of CFTR GCl in a dose-dependent manner, indicating inhibition of dephosphorylation. We conclude that CFTR GCl may be regulated via shifts in cytoplasmic pH that mediate reciprocal control of endogenous kinase and phosphatase activities. Luminal pH probably has little direct effect on these mechanisms. This regulation of CFTR may be important in shifting electrolyte transport in the duct from conductive to nonconductive modes.

    View details for Web of Science ID 000076267000016

    View details for PubMedID 9755057

  • Topology of the region surrounding Glu(681) of human AE1 protein, the erythrocyte anion exchanger JOURNAL OF BIOLOGICAL CHEMISTRY Tang, X. B., Fujinaga, J., Kopito, R., Casey, J. R. 1998; 273 (35): 22545-22553


    AE1 protein transports Cl- and HCO3- across the erythrocyte membrane by an electroneutral exchange mechanism. Glu681 of human AE1 may form part of the anion translocation apparatus and the permeability barrier. We have therefore studied the structure of the sequence surrounding Glu681, using scanning cysteine mutagenesis. Residues of the Ser643 (adjacent to the glycosylation site) to Ser690 region of cysteineless mutant (AE1C-) were replaced individually with cysteine. The ability of mutants to mediate Cl-/HCO3- exchange in transfected HEK293 cells revealed that extracellular mutants, W648C, I650C, P652C, L655C, and F659C have an important role in transport. By contrast, only transmembrane mutation E681C fully blocked anion exchange activity. The topology of the region was investigated by comparing cysteine labeling with the membrane-permeant cysteine-directed reagent 3-(N-maleimidylpropionyl)biocytin, with or without prior labeling with membrane-impermeant lucifer yellow iodoacetamide (LYIA). Two regions readily label with 3-(N-maleimidylpropionyl)biocytin (Ser643-Met663 and Ile684-Ser690). We propose that poorly labeled Met664-Gln683 corresponds to transmembrane segment 8 of AE1. Regions Ser643-Met663 and Ile684-Ser690 localize, respectively, to extracellular and intracellular sites on the basis of accessibility to LYIA. On the basis of LYIA accessibility, we propose that the Arg656-Met663 region forms a "vestibule" that leads anions to the transport channel. Glu681 is located 3 amino acids from the C terminus of transmembrane segment 8, which places the membrane permeability barrier within 5 A of the intracellular surface of the membrane.

    View details for Web of Science ID 000075616600057

    View details for PubMedID 9712881

  • Cotranslational ubiquitination of cystic fibrosis transmembrane conductance regulator in vitro JOURNAL OF BIOLOGICAL CHEMISTRY Sato, S., Ward, C. L., Kopito, R. R. 1998; 273 (13): 7189-7192


    Ubiquitination is a covalent protein modification that can target proteins in eukaryotic cells for degradation by the 26 S proteasome. Substrates for this degradation pathway include abnormal proteins that arise from misfolding and/or mutation. How and when the ubiquitination machinery recognizes misfolded proteins and targets them for degradation remains largely unknown. We have previously shown that cystic fibrosis transmembrane conductance regulator (CFTR), is rapidly degraded in a ubiquitin-dependent fashion, without any detectable lag following its synthesis (Ward, C. L., and Kopito, R. R. (1994) J. Biol. Chem. 269, 25710-25718), suggesting that ubiquitination and protein synthesis may be temporally linked. In the present study, we have investigated the timing of CFTR ubiquitination relative to its translation in reticulocyte lysates containing 125I-ubiquitin. In synchronized, proteasome-inhibited lysates, translation of full-length CFTR chains was completed in approximately 30 min, whereas modification of CFTR with [125I]ubiquitin was evident by 20 min, indicating that ubiquitination precedes the completion of full-length polypeptide chains. Moreover, ubiquitin was also found to be transferred to nascent CFTR chains while attached to ribosomes. Together, these data establish that ubiquitination, which is widely assumed to be a post-translational event, can occur cotranslationally and suggest a role for ubiquitination early in protein biosynthesis.

    View details for Web of Science ID 000072738500004

    View details for PubMedID 9516408

  • A cluster of cytoplasmic histidine residues specifies pH dependence of the AE2 plasma membrane anion exchanger. Cell Kobayashi, S., Kopito, R. R. 1997; 90 (6): following 1159-?

    View details for PubMedID 9340197

  • Cytosolic degradation of T-cell receptor alpha chains by the proteasome JOURNAL OF BIOLOGICAL CHEMISTRY Yu, H., Kaung, G., Kobayashi, S., Kopito, R. R. 1997; 272 (33): 20800-20804


    The T-cell antigen receptor (TCR) is an hetero-oligomeric membrane complex composed of at least seven transmembrane polypeptide chains that has served as a model for the assembly and degradation of integral membrane proteins in the endoplasmic reticulum (ER). Unassembled TCRalpha chains fail to mature to the Golgi apparatus and are rapidly degraded by a non-lysosomal "ER degradation" pathway that has been proposed to be autonomous to the ER. In these studies we show that the degradation of core-glycosylated TCRalpha is blocked by N-acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN) and lactacystin, implicating the proteasome in ER degradation. Either acute or chronic treatment of TCRalpha-transfected cells with proteasome inhibitors cause the core-glycosylated TCRalpha chains to progressively shift to an approximately 28-kDa form that lacks N-linked oligosaccharides and the N-terminal signal peptide. The susceptibility of this 28-kDa species to extravesicular protease indicates that it is not protected by the ER membrane and, hence, cytoplasmic. These data suggest a model in which TCRalpha chains that are translocated across the membrane, core-glycosylated, but fail to assemble are dislocated back to the cytoplasm for degradation by cytoplasmic proteasomes. Our data also suggest that covalent modification of TCRalpha with ubiquitin is not required for its degradation.

    View details for Web of Science ID A1997XR22100074

    View details for PubMedID 9252404

  • Cystic fibrosis transmembrane conductance regulator and adenosine triphosphate - Response SCIENCE Reddy, M. M., Quinton, P. M., HAWS, C., Wine, J. J., Grygorczyk, R., Tabcharani, J. A., Hanrahan, J. W., Gunderson, K. L., Kopito, R. R. 1997; 275 (5304): 1325-1325
  • ER quality control: The cytoplasmic connection CELL Kopito, R. R. 1997; 88 (4): 427-430

    View details for Web of Science ID A1997WJ69100001

    View details for PubMedID 9038332

  • Determination of topology and pore-lining residues in the transmembrane segment 8 region of AE1, the human erythrocyte anion exchange protein Casey, J. R., Tang, X. B., Kopito, R. R. CELL PRESS. 1997: TU346–TU346
  • A cluster of cytoplasmic histidine residues specifies pH dependence of the AE2 plasma membrane anion exchanger CELL Sekler, I., Kobayashi, S., Kopito, R. R. 1996; 86 (6): 929-935


    Intracellular pH is maintained by a dynamic equilibrium balancing the opposing forces of proton loading and proton extrusion. By providing an efflux pathway for base, anion exchangers constitute a key component of the plasma membrane proton-loading machinery. The data in this paper identify a histidine-rich sequence within the cytoplasmic domain of the nonerythroid anion exchanger, AE2, that serves as an intracellular pH "sensor" that modulates anion exchange activity within the physiological range of cytoplasmic pH. These data reveal an interaction between the two major domains of the anion exchanger and suggest a novel substrate feedback mechanism by which intracellular protons directly control the activity of an acid-loading plasma membrane ion transporter.

    View details for Web of Science ID A1996VJ44300011

    View details for PubMedID 8808628

  • Immunocytochemical localization of vacuolar H+-ATPase and Cl--HCO3- anion exchanger (Erythrocyte band-3 protein) in avian osteoclasts: Effect of calcium-deficient diet on polar expression of the H+-ATPase pump CALCIFIED TISSUE INTERNATIONAL Bastani, B., Ross, F. P., Kopito, R. R., Gluck, S. L. 1996; 58 (5): 332-336


    Osteoclasts attach to the bone surface and resorb bone by secreting protons into an isolated subosteoclastic compartment. Previous studies have shown the presence of a vacuolar type H+-ATPase, and a functional Cl--HCO3- anion exchanger in the osteoclast. In the present studies, using a monoclonal antibody to the 31-kDa subunit of H+-ATPase and a rabbit antiserum to the erythrocyte band-3 protein (Cl--HCO3- anion exchanger) we have immunocytochemically localized the respective pumps in bone sections obtained from chickens fed a normal or a calcium-deficient diet for 4 weeks. Our results indicate that although H+-ATPase is either evenly distributed throughout the osteoclast or is more polarized at its ruffled membrane juxtaposed to the bone surface, the band-3 protein immunoreactivity is always localized to the plasma membrane which is not attached to the bone surface (basolateral membrane). Four weeks of a calcium-deficient diet resulted in a significant increase in the percentage of osteoclasts that were polarized for the H+-ATPase pump at their ruffled membrane, and a trend toward increased total number of osteoclasts, although the latter did not reach statistical significance (P = 0.09). These changes were not accompanied by a significant increase in the intensity of staining for H+-ATPase. Band-3 protein immunoreactivity was always prominent, limited to the basolateral membrane, and did not alter with calcium-deficient diet or with changes in the degree of H+-ATPase polarization.

    View details for Web of Science ID A1996UJ24700006

    View details for PubMedID 8661962

  • Failure of the cystic fibrosis transmembrane conductance regulator to conduct ATP SCIENCE Reddy, M. M., Quinton, P. M., HAWS, C., Wine, J. J., Grygorczyk, R., Tabcharani, J. A., Hanrahan, J. W., Gunderson, K. L., Kopito, R. R. 1996; 271 (5257): 1876-1879


    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel regulated by protein kinase A and adenosine triphosphate (ATP). Loss of CFTR-mediated chloride ion conductance from the apical plasma membrane of epithelial cells is a primary physiological lesion in cystic fibrosis. CFTR has also been suggested to function an an ATP channel, although the size of the ATP anion is much larger than the estimated size of the CFTR pore. ATP was not conducted through CFTR in intact organs, polarized human lung cell lines, stably transfected mammalian cell lines, or planar lipid bilayers reconstituted with CFTR protein. These findings suggest that ATP permeation through the CFTR is unlikely to contribute to the normal function of CFTR or to the pathogenesis of cystic fibrosis.

    View details for Web of Science ID A1996UC77800050

    View details for PubMedID 8596959

  • Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation JOURNAL OF BIOLOGICAL CHEMISTRY Sato, S., Ward, C. L., Krouse, M. E., Wine, J. J., Kopito, R. R. 1996; 271 (2): 635-638


    The common delta F508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) interferes with the biosynthetic folding of nascent CFTR polypeptides, leading to their retention and rapid degradation in an intracellular compartment proximal to the Golgi apparatus. Neither the pathway by which wild-type CFTR folds nor the mechanism by which the Phe508 deletion interferes with this process is well understood. We have investigated the effect of glycerol, a polyhydric alcohol known to stabilize protein conformation, on the folding of CFTR and delta F508 in vivo. Incubation of transient and stable delta F508 transfectants with 10% glycerol induced a significant accumulation of delta F508 protein bearing complex N-linked oligosaccharides, indicative of their transit to a compartment distal to the endoplasmic reticulum (ER). This accumulation was accompanied by an increase in mean whole cell cAMP activated chloride conductance, suggesting that the glycerol-rescued delta F508 polypeptides form functional plasma membrane CFTR channels. These effects were dose- and time-dependent and fully reversible. Glycerol treatment also stabilized immature (core-glycosylated) delta F508 and CFTR molecules that are normally degraded rapidly. These effects of glycerol were not due to a general disruption of ER quality control processes but appeared to correlate with the degree of temperature sensitivity of specific CFTR mutations. These data suggest a model in which glycerol serves to stabilize an otherwise unstable intermediate in CFTR biosynthesis, maintaining it in a conformation that is competent for folding and subsequent release from the ER quality control apparatus.

    View details for Web of Science ID A1996TP88900009

    View details for PubMedID 8557666



    Most cases of cystic fibrosis are caused by mutations that interfere with the biosynthetic folding of the cystic fibrosis transmembrane conductance regulator (CFTR), leading to the rapid degradation of CFTR molecules that have not matured beyond the endoplasmic reticulum (ER). The mechanism by which integral membrane proteins including CFTR are recognized and targeted for ER degradation and the proteolytic machinery involved in this process are not well understood. We show here that the degradation of both wild-type and mutant CFTR is inhibited by two potent proteasome inhibitors that induce the accumulation of polyubiquitinated forms of immature CFTR. CFTR degradation was also inhibited by coexpression of a dominant negative ubiquitin mutant and in cells bearing a temperature-sensitive mutation in the ubiquitin-activating enzyme, confirming that ubiquitination is required for rapid CFTR degradation.

    View details for Web of Science ID A1995RY58300016

    View details for PubMedID 7553863



    CFTR is a member of the traffic ATPase superfamily and a Cl- ion channel that appears to require ATP hydrolysis for gating. Analysis of single CFTR Cl- channels reconstituted into planar lipid bilayers revealed the presence of two open conductance states that are connected to each other and to the closed state by an asymmetric cycle of gating events. We show here that the transition between the two open conductance states is directly coupled to ATP hydrolysis by one of the consensus nucleotide-binding folds, designated NBF2. Moreover, the transition between the closed state and one of the open states is linked to the binding of ATP. This analysis permits real-time visualization of conformational changes associated with a single cycle of ATP hydrolysis by a single protein molecule and suggests a model describing a role for ATP in CFTR gating.

    View details for Web of Science ID A1995RL76000010

    View details for PubMedID 7543023



    Mutant (delta F508) and wild-type cystic fibrosis transmembrane conductance regulator (CFTR) were synthesized initially as an approximately 140-kDa core-glycosylated precursor, which, in the case of wild-type CFTR, was chased to an approximately 160 kDa form bearing complex oligosaccharides. Mutant CFTR disappeared from the detergent-soluble cell extract with rapid (t1/2 = 27 min) kinetics. Only approximately 25% of the initially synthesized wild-type 140-kDa CFTR precursor was detected as mature protein; the remaining approximately 75% decayed with kinetics (t1/2 = 33 min) indistinguishable from those of the mutant. Rapid degradation kinetics and inefficient processing of wild-type CFTR were also observed in the colonic carcinoma lines HT29 and T84 and in stably transfected C127 cells, which express 5-50 times lower levels of CFTR. These results suggest that inefficient processing and rapid degradation of wild-type CFTR precursor are an intrinsic property of CFTR in these diverse cell types and are not an artifact of overexpression. Degradation of wild-type and mutant 140-kDa CFTR began without significant lag following synthesis. These data suggest that wild-type and delta F508 CFTR differ in the efficiency of folding of the core-glycosylated primary translation product.

    View details for Web of Science ID A1994PQ49100072

    View details for PubMedID 7523390



    Plasma membrane anion exchangers constitute a multigene family that contributes to the regulation of intracellular pH and chloride concentration in many cell types. We have characterized two polypeptide isoforms of the AE3 gene that are expressed in the rat retina. Using antipeptide antibodies specific for defined NH2-terminal and COOH-terminal epitopes, we have identified a 165 kDa polypeptide whose expression is restricted to the primary glial cell type of the retina, the Müller cell, and a 125 kDa polypeptide that is expressed in horizontal neurons. Expression of the Müller cell isoform exhibits a polarized distribution and is highest in basal endfoot processes. These AE3 isoforms exhibit a distinct developmental expression pattern in postnatal rat retina. The neuronal isoform is undetectable in neonatal retina until postnatal day 10-15, correlating strongly with the onset of retinal function.

    View details for Web of Science ID A1994PL02800046

    View details for PubMedID 7931579



    We studied Cl-/HCO3- exchange function in acutely dissociated single hippocampal neurons from adult and fetal day 18 rat using a fluorescent intracellular pH (pHi) indicator dye. The presence of Cl-/HCO3- exchange activity was assayed by observing the elevation in pHi upon acute reversal of the Cl- gradient. Resting intracellular pH in acutely dissociated neurons of both adult and fetal tissue was significantly higher than that of cultured fetal hippocampal neurons (day 10-12 in culture). Acute removal of extracellular Cl- caused a rapid and reversible increase in pHi by 0.25 pH units in adult neurons but had virtually no effect in similarly dissociated fetal neurons. Cl-/HCO3- exchange activity was also undetectable in fetal cultured hippocampal neurons. The mRNA for the anion exchanger AE3 is expressed abundantly in adult rodent neurons. AE3 is a potential candidate molecule for the observed Cl-/HCO3- exchange activity. In situ hybridization was used to monitor expression of the AE3 gene in these two age groups. We found that both adult and fetal neurons express AE3 mRNA. These results indicate that AE3 may not function as a Cl-/HCO3- exchanger in fetal neurons, in contrast to its possible role in the adult brain.

    View details for Web of Science ID A1993LJ04400040

    View details for PubMedID 8348323



    The human erythrocyte anion-exchange protein (band 3 or AE1) was cloned from a fetal liver cDNA library. Three overlapping clones, encompassing 3637 nucleotides, were analyzed in detail. These encode a 911-amino acid protein (Mr 101,791) and detect a single 4.7-kilobase species in human reticulocyte RNA. The corresponding gene is located on chromosome 17. The protein is similar in structure to other anion exchangers and is divided into three regions: a hydrophilic, cytoplasmic domain that interacts with a variety of membrane and cytoplasmic proteins (residues 1-403); a hydrophobic, transmembrane domain that forms the anion antiporter (residues 404-882); and an acidic, C-terminal domain of unknown function (residues 883-911). The N-terminal domain contains several conserved sections (e.g., residues 57-86, 102-164, 219-347, and 375-403), some of which may contribute to binding sites for ankyrin, protein 4.1, or protein 4.2. The membrane domain is highly conserved with the exception of a single segment (residues 543-567) that contains several sites for cleavage of the protein by extracellular proteases. Based on hydropathy analyses and the wealth of available topographical and functional data, a model is proposed in which the protein crosses the membrane 14 times.

    View details for Web of Science ID A1989CC53300005

    View details for PubMedID 2594752

  • IDENTIFICATION OF A 185-KDA BAND 3-RELATED POLYPEPTIDE IN OXYNTIC CELLS AMERICAN JOURNAL OF PHYSIOLOGY Thomas, H. A., Machen, T. E., Smolka, A., Baron, R., Kopito, R. R. 1989; 257 (3): C537-C544


    Polyclonal antibodies to the purified mouse erythrocyte anion exchange protein (band 3) and to a conserved COOH-terminal peptide of mouse band 3 (alpha-Ct) recognized a single major 185-kDa polypeptide in immunoblots of a membrane fraction prepared from rabbit gastric glands. Competition studies revealed that the epitopes shared between the rabbit gastric 185-kDa antigen and the approximately 100-kDa mouse erythrocyte band 3 protein are restricted to the COOH-terminal domain of band 3, which is known to contain the catalytic site for anion exchange activity. Immunofluorescence microscopy was used to demonstrate that this band 3-related polypeptide is associated with the plasma membrane in a subpopulation of gastric gland cells composed exclusively of oxyntic cells, as judged by the coincidence of immunofluorescence with alpha-Ct and with a monoclonal antibody to the gastric H+-K+-ATPase. This alpha-Ct-reactive antigen was further localized to the cytoplasmic face of the basolateral membrane of oxyntic cells, which correlates well with the physiologically determined site of anion exchange activity. These data demonstrate the presence in gastric oxyntic cells of a novel member of the family of proteins related to the erythrocyte anion exchanger. The possibility that the 185-kDa polypeptide is an anion exchanger is discussed.

    View details for Web of Science ID A1989AQ38700016

    View details for PubMedID 2551174



    Band 3 multigene family consists of several distinct but structurally related polypeptides which are probably involved in the transport of anions across the plasma membrane of both erythrocytes and nonerythroid cells. A novel member of this family of polypeptides that resides in the Golgi complex was identified with antibodies to Band 3. The Golgi antigen had a larger molecular size and was antigenically distinct from Band 3 in the amino-terminal domain. It was expressed most prominently in cells that secrete large amounts of sulfated proteins and proteoglycans. This polypeptide may participate in sulfate transport across Golgi membranes.

    View details for Web of Science ID A1988R135900035

    View details for PubMedID 2461589



    Anion exchange is a nearly ubiquitous cellular transport function which contributes to the regulation of cell pH and of cell volume. However, the only plasma membrane anion exchanger of known identity and sequence is erythroid band 3. Both hybridization and immunologic data support the presence of band 3-related mRNAs and proteins in nonerythroid tissues. We have used low stringency hybridization with the murine band 3 cDNA to clone a band 3-related cDNA from murine kidney and from 70Z/3 pre-B cells. The cDNA encodes a band 3-related protein (B3RP) of 1237 amino acids, with a predicted mass of 137 kDa. The carboxyl-terminal hydrophobic domain of B3RP has an amino acid sequence 67% identical to that of band 3, with a very similar predicted secondary structure. The amino-terminal hydrophilic domain of B3RP has two sections. The section adjacent to the putative membrane-associated segment is 33% identical in amino acid sequence to the amino-terminal, cytoplasmic domain of band 3. The other, far amino-terminal section of B3RP has no correspondent in the band 3 sequence. B3RP mRNA is present in a variety of epithelial and other tissues and probably encodes an anion exchange protein of wide distribution.

    View details for Web of Science ID A1988Q882600085

    View details for PubMedID 3182834

  • Structure and tissue-specific expression of the mouse anion-exchanger gene in erythroid and renal cells. Society of General Physiologists series Kopito, R. R., Andersson, M. M., Herzlinger, D. A., Al-Awqati, Q., Lodish, H. F. 1988; 43: 151-161

    View details for PubMedID 3077542

  • A molecular biological approach to the study of anion transport. Kidney international. Supplement Alper, S. L., Kopito, R. R., Lodish, H. F. 1987; 23: S117-33

    View details for PubMedID 3326961



    Primer extension and nuclease protection analysis were used to map the 5' end of mRNA transcripts of the single gene encoding the mouse erythroid anion-exchange protein, band 3. RNA from induced Friend murine erythroleukemia cells contains transcripts whose 5' ends are heterogeneous but that map to five clustered sites between 146 and 189 nucleotides upstream of the initiator AUG codon. The steady-state level of band 3 mRNA increases markedly following dimethyl sulfoxide-induced differentiation, but the pattern of transcription initiation does not vary. mRNA from anemic and normal mouse spleen exhibits the same pattern, with the addition of another transcript whose 5' end maps to position -260. In contrast, mRNA from mouse kidney has a single predominant transcript, mapping to -260. These data establish that the band 3 gene is expressed in kidney as well as in erythroid cells and suggest the presence of tissue-specific alternate promoter elements within the first exon of the band 3 gene. The nucleotide sequence of 1.7 kilobases of genomic DNA 5' to the first intron of the single-copy mouse band 3 gene, although moderately (G + C)-rich, has no "TATA" or "CAAT" boxes or other homologies with globin or other eukaryotic polymerase II promoter regions.

    View details for Web of Science ID A1987K504900040

    View details for PubMedID 3478687

  • STRUCTURE AND ORGANIZATION OF THE MURINE BAND-3 GENE JOURNAL OF BIOLOGICAL CHEMISTRY Kopito, R. R., Andersson, M., Lodish, H. F. 1987; 262 (17): 8035-8040


    The Band 3 protein mediates the reversible exchange of chloride and bicarbonate anions across the plasma membrane of erythrocytes, and probably, certain epithelial cells. It also serves to anchor the spectrin cytoskeleton to the plasma membrane via its association with ankyrin. We have isolated and largely sequenced the 17-kilobase murine Band 3 gene. We show that this gene is present in a single copy in the mouse genome and have identified and mapped the 19 intervening sequences. The locations of the intron/exon junctions in the Band 3 mRNA correlate with predicted structural features of the erythrocyte Band 3 protein structure and membrane topology. One of the introns within this gene contains a single copy of a murine Alu-type high dispersed sequence, in addition to several unusual tandemly repeated sequences.

    View details for Web of Science ID A1987H684000017

    View details for PubMedID 3036795

  • RADIOENZYMATIC ASSAY OF PLASMA MEVALONATE METHODS IN ENZYMOLOGY Parker, T. S., Kopito, R. R., Brunengraber, H. 1985; 110: 58-71

    View details for Web of Science ID A1985AFZ4900007

    View details for PubMedID 2991705



    A full-length clone encoding the mouse erythrocyte anion exchange protein, band 3, has been isolated from a cDNA library using an antibody against the mature erythrocyte protein. The complete nucleotide sequence has been determined. Substantial homology is evident between the deduced murine amino acid sequence and published sequences of fragments of human band 3 protein. The amino-terminal 420 and the carboxy-terminal 32 residues constitute polar, soluble domains, while the intervening 475 amino acids are likely to be intimately associated with the lipid bilayer. Hydrophobic analysis of this sequence, together with structural studies on the human protein, suggests the possibility of at least 12 membrane spans, predicting that both the amino- and carboxy-termini are intracellular.

    View details for Web of Science ID A1985ARJ3400001

    View details for PubMedID 3840489



    The amino-acid sequence of murine band 3, deduced from the nucleotide sequence of a complementary DNA clone, confirms that this integral membrane glycoprotein is composed of two major structural domains which correlate with its dual functions as the anchor for the erythrocyte cytoskeleton and as a plasma membrane anion antiporter. This latter activity resides within a highly hydrophobic domain that crosses the plasma membrane at least 12 times.

    View details for Web of Science ID A1985AMJ4900073

    View details for PubMedID 2410791



    The shunt pathway of mevalonate metabolism (Edmond, J., and Popják, G. (1974) J. Biol. Chem. 249, 66-71) has been studied in isolated kidneys from rats perfused with physiological concentrations of variously labeled [14C]- and [3H]mevalonates. The rate of operation of the shunt pathway was quantified by the production of either 14CO2 or 3H2O from the tracers. The measured rates of 14CO2 production from [14C] mevalonate were converted to rates of mitochondrial acetyl-CoA production by methods which take into account underestimations of metabolic rates derived from 14CO2 production. We have shown that the sex difference in renal shunting of mevalonate (Wiley, M. H., Howton, M. M., and Siperstein, M. D. (1979) J. Biol. Chem. 254, 837-842) occurs at physiological levels of substrate. The shunt pathway diverts up to 17% of the flux of mevalonate entering the cholesterol synthesis pathway in the kidney. It may, therefore, play a role in the long term regulation of cholesterol synthesis in this organ, as had been hypothesized by Edmond and Popják.

    View details for Web of Science ID A1984RY41400058

    View details for PubMedID 6706942

  • THE SHUNT PATHWAY OF MEVALONATE METABOLISM IN THE ISOLATED PERFUSED-RAT-LIVER JOURNAL OF BIOLOGICAL CHEMISTRY WEINSTOCK, S. B., Kopito, R. R., Endemann, G., Tomera, J. F., Marinier, E., Murray, D. M., Brunengraber, H. 1984; 259 (14): 8939-8944


    The shunt pathway of mevalonate metabolism (Edmond, J., and Popják, G. (1974) J. Biol. Chem. 249, 66-71) has been studied in isolated livers from fed rats perfused with physiological concentrations of variously labeled [14C]mevalonates. The measured rates of 14CO2 production were converted to rates of mitochondrial acetyl-CoA production from mevalonate by methods which take into account underestimations of metabolic rates derived from 14CO2 production. Our data confirm that the shunt pathway leads to mitochondrial acetyl-CoA. The apparent negligible rate of mevalonate shunting in liver, previously reported by others, stems from the very low contribution (congruent to 0.1%) of plasma mevalonate to total mevalonate metabolism in the liver. This contribution was assessed from the relative incorporations of 3H2O and [5-14C]mevalonate into sterols. In livers from fed rats, the shunt diverts about 5% of the production of mevalonate. The total rate of mevalonate shunting in the liver is about 200 times greater than in two kidneys. The liver is therefore the main site of mevalonate shunting in the rat.

    View details for Web of Science ID A1984TB56300044

    View details for PubMedID 6430893



    A procedure is described to convert rates of (14)CO(2) production into rates of mitochondrial acetyl-CoA production from a (14)C-labelled substrate. The principle is illustrated in perfused rat liver and kidney by the differential yield of (14)CO(2) from 4-methyl-2-oxo[1-(14)C]valerate and 4-methyl-2-oxo[2-(14)C]valerate.

    View details for Web of Science ID A1983PY70400032

    View details for PubMedID 6405741

  • METABOLISM OF PLASMA MEVALONATE IN RATS AND HUMANS JOURNAL OF LIPID RESEARCH Kopito, R. R., WEINSTOCK, S. B., Freed, L. E., Murray, D. M., Brunengraber, H. 1982; 23 (4): 577-583


    A circadian rhythm in plasma mevalonate was identified in human subjects. This variation, over a 5-fold range, is paralleled by a rhythm in urinary excretion. No such diurnal change in plasma mevalonate was observed in schedule-fed, light-cycled rats, despite the presence of a pronounced rhythm in liver HMG-Coa reductase and sterol synthesis. A linear correlation was found between liver HMG-CoA reductase activity and the rate of hepatic sterol synthesis. Sterol synthesis accounted for 59% of the HMG-CoA reductase activity. A 4-fold increase in plasma mevalonate following bilateral nephrectomy did not feed back on liver HMG-CoA reductase. Turnover rates for circulating R- and S-mevalonate were determined by the kinetics of tritiated tracers. S-Mevalonate exhibited first-order kinetics with a T 1/2 of 19 to 23 min, while R-mevalonate kinetics could be resolved into two phases with half-lives of 9 and 42 min. The renal uptake of circulating mevalonate was measured by the initial rate of increase in plasma mevalonate immediately following bilateral nephrectomy; this was confirmed by determination of the renal arterio-venous difference. This value ranges between 500 and 600 pmol/min for a 250-g rat.

    View details for Web of Science ID A1982NQ91200007

    View details for PubMedID 7097123



    The urinary excretion and the incorporation into lipids of R[3-14C]mevalonate was investigated in isolated rat kidneys perfused with physiological concentration sof the substrate (80-500 pmol/ml). The clearance of R[3-14C]mevalonate and of the unnatural enantiomer S[5-14C]mevalonate were compared to the glomerular filtration ratea measured by the clearance of inulin. Evidence is presented that half of R-mevalonate filtered in the glomerulus is reabsorbed in the tubule whereas S-mevalonate is not reabsorbed. The kidney tubule appears to discriminate between the R and S forms of the mevalonate salt. Urinary excretion and incorporation into lipids accounted for 22% and 46%, respectively, of the uptake of R[3-14C]mevalonate from the perfusate. The label of R[3-14C]mevalonate recovered in lipids was distributed among saponifiable (15%), digitonin-precipitable sterols (18%) and squalene + prenols (67%). Sterol synthesis in the kidney appears to be controlled, at least in part, by the level of circulatinga R-mevalonate.

    View details for Web of Science ID A1981MB98400004

    View details for PubMedID 7276751



    (R)-Mevalonate was identified by radioenzymatic assay in human and rat urines. This was confirmed by spectrophotometric enzymatic assay of an ether extract of human urine. The average excretion rate of (R)-mevalonate in humans was 1.7 mumol/24 hr which corresponds to 29% of the glomerular filtration rate. In anesthetized rats the average rate of (R)-mevalonate excretion was 350 pmol/min, corresponding to 44% of the glomerular filtration rate. These rates were not affected by the sex of the subjects or animals. After bilateral nephrectomy, the concentration of (R)-mevalonate in rat serum increased within 2 hr to a new level that was 5 times that of sham-operated controls. Nephrectomized rats showed a decrease in their tolerance to an intravenous load of mevalonate. These data show that (R)-mevalonate is normally excreted in the urine of humans and rats. Urinary excretion of (R)-mevalonate should therefore be taken into account in in vivo studies on the metabolism of this compound.

    View details for Web of Science ID A1980KN96400024

    View details for PubMedID 6255462



    Elevation of serum-bound sialic acid concentration in different disease states fails to correlate significantly with suppressive serum actions in mixed allogeneic lymphocyte cultures or phyto-haemagglutinin cultures. Heat-decomplemented serum from patients with abnormal levels of bound sialic acid was added to parallel cultures containing similar blood lymphocyte populations derived from normal humans. Wide fluctuations of the rate of incorporation of tritiated thymidine into nucleoprotein indicated presence of suppressive elements other than sialoprotein in the added serum components. Serum with rising sialyl concentration derived from patients with cancer showed slight tendency to augment mixed lymphocyte and phytohaemagglutinin responses. The findings suggest that the previously documented nonspecific suppressive action of serum sialoprotein on human host lymphoblastic response to neuraminidase-treated cancer cells represents a mechanism unique to that culture system rather than a manifestation of a general immunoregulatory function of serum sialoprotein.

    View details for Web of Science ID A1976CA27700006

    View details for PubMedID 133776