Ph.D., Stanford University, Chemical and Systems Biology (2008)
M.S., Stanford University, Computer Science (2000)
B.S., University of California, San Diego, Computer Science (1998)
Current Research and Scholarly Interests
The Brandman Lab studies how cells sense and respond to stress. We employ an integrated set of techniques including single cell analysis, mathematical modeling, genomics, structural studies, and in vitro assays.
- Biochemistry Mini-Course
BIOC 202 (Aut)
Independent Studies (8)
- Directed Reading in Biochemistry
BIOC 299 (Aut, Win, Spr, Sum)
- Directed Reading in Biophysics
BIOPHYS 399 (Aut, Win, Spr, Sum)
- Graduate Research
BIOPHYS 300 (Aut, Win, Spr, Sum)
- Graduate Research and Special Advanced Work
BIOC 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
BIOC 370 (Aut, Win, Spr, Sum)
- Out-of-Department Graduate Research
BIO 300X (Aut, Win, Spr, Sum)
- The Teaching of Biochemistry
BIOC 221 (Aut, Win, Spr, Sum)
- Undergraduate Research
BIOC 199 (Aut, Win, Spr, Sum)
- Directed Reading in Biochemistry
Prior Year Courses
- Biochemistry Mini-Course
BIOC 202 (Aut)
- Biochemistry Mini-Course
BIOC 202 (Aut)
- Proteostatis: guarding the proteome in health and disease
BIOS 287 (Win)
- Biochemistry Mini-Course
Opi1-mediated transcriptional modulation orchestrates genotoxic stress response in budding yeast.
In budding yeast, the transcriptional repressor Opi1 regulates phospholipid biosynthesis by repressing expression of genes containing inositol-sensitive upstream activation sequences (UASINO). Upon genotoxic stress, cells activate the DNA Damage Response (DDR) to coordinate a complex network of signaling pathways aimed at preserving genomic integrity. Here, we reveal that Opi1 is important to modulate transcription in response to genotoxic stress. We find that cells lacking Opi1 exhibit hypersensitivity to genotoxins, along with a delayed G1 to S-phase transition and decreased gamma-H2A levels. Transcriptome analysis using RNA-seq reveals that Opi1 plays a central role in modulating essential biological processes during MMS-associated stress, including repression of phospholipid biosynthesis and transduction of mating signaling. Moreover, Opi1 induces sulfate assimilation and amino acid metabolic processes, such as arginine and histidine biosynthesis and glycine catabolism. Furthermore, we observe increased mitochondrial DNA instability in opi1Δ cells upon MMS treatment. Notably, we show that constitutive activation of the transcription factors Ino2-Ino4 is responsible for genotoxin sensitivity in Opi1-deficient cells, and the production of inositol pyrophosphates by Kcs1 counteracts Opi1 function specifically during MMS-induced stress. Overall, our findings highlight Opi1 as a critical sensor of genotoxic stress in budding yeast, orchestrating gene expression to facilitate appropriate stress responses.
View details for DOI 10.1093/genetics/iyad130
View details for PubMedID 37440469
Oxaliplatin disrupts nucleolar function through biophysical disintegration.
2022; 41 (6): 111629
Platinum (Pt) compounds such as oxaliplatin are among the most commonly prescribed anti-cancer drugs. Despite their considerable clinical impact, the molecular basis of platinum cytotoxicity and cancer specificity remain unclear. Here we show that oxaliplatin, a backbone for the treatment of colorectal cancer, causes liquid-liquid demixing of nucleoli at clinically relevant concentrations. Our data suggest that this biophysical defect leads to cell-cycle arrest, shutdown of Pol I-mediated transcription, and ultimately cell death. We propose that instead of targeting a single molecule, oxaliplatin preferentially partitions into nucleoli, where it modifies nucleolar RNA and proteins. This mechanism provides a general approach for drugging the increasing number of cellular processes linked to biomolecular condensates.
View details for DOI 10.1016/j.celrep.2022.111629
View details for PubMedID 36351392
ReporterSeq reveals genome-wide dynamic modulators of the heat shock response across diverse stressors.
Understanding cellular stress response pathways is challenging because of the complexity of regulatory mechanisms and response dynamics, which can vary with both time and the type of stress. We developed a reverse genetic method called ReporterSeq to comprehensively identify genes regulating a stress-induced transcription factor under multiple conditions in a time-resolved manner. ReporterSeq links RNA-encoded barcode levels to pathway-specific output under genetic perturbations, allowing pooled pathway activity measurements via DNA sequencing alone and without cell enrichment or single-cell isolation. We used ReporterSeq to identify regulators of the heat shock response (HSR), a conserved, poorly understood transcriptional program that protects cells from proteotoxicity and is misregulated in disease. Genome-wide HSR regulation in budding yeast was assessed across 15 stress conditions, uncovering novel stress-specific, time-specific, and constitutive regulators. ReporterSeq can assess the genetic regulators of any transcriptional pathway with the scale of pooled genetic screens and the precision of pathway-specific readouts.
View details for DOI 10.7554/eLife.57376
View details for PubMedID 34223816
Protein products of nonstop mRNA disrupt nucleolar homeostasis.
Cell stress & chaperones
Stalled mRNA translation results in the production of incompletely synthesized proteins that are targeted for degradation by ribosome-associated quality control (RQC). Here we investigated the fate of defective proteins translated from stall-inducing, nonstop mRNA that escape ubiquitylation by the RQC protein LTN1. We found that nonstop protein products accumulated in nucleoli and this localization was driven by polylysine tracts produced by translation of the poly(A) tails of nonstop mRNA. Nucleolar sequestration increased the solubility of invading proteins but disrupted nucleoli, altering their dynamics, morphology, and resistance to stress in cell culture and intact flies. Our work elucidates how stalled translation may affect distal cellular processes and may inform studies on the pathology of diseases caused by failures in RQC and characterized by nucleolar stress.
View details for DOI 10.1007/s12192-021-01200-w
View details for PubMedID 33619693
Primordial Protein Tails.
2021; 81 (1): 6–7
C-terminal tailing is an ancient and conserved form of peptide synthesis that protects cells from incomplete and potentially toxic translation products. Filbeck et al. (2020) and Crowe-McAuliffe et al. (2020) use structural, genetic, and biochemical approaches to elucidate the mechanisms driving C-terminal tailing.
View details for DOI 10.1016/j.molcel.2020.12.032
View details for PubMedID 33417855
Sis1 delivers the State of the Union.
The Journal of cell biology
2021; 220 (1)
The heat shock response (HSR) is a gene expression program that protects cells from heat and proteotoxic stressors. In this issue, Feder et al. (2020. J. Cell Biol.https://doi.org/10.1083/jcb.202005165) show that subcellular relocalization of the cochaperone Sis1 drives the HSR by de-suppressing the transcription factor Hsf1.
View details for DOI 10.1083/jcb.202011093
View details for PubMedID 33332552
Adaptability of the ubiquitin-proteasome system to proteolytic and folding stressors.
The Journal of cell biology
2021; 220 (3)
Aging, disease, and environmental stressors are associated with failures in the ubiquitin-proteasome system (UPS), yet a quantitative understanding of how stressors affect the proteome and how the UPS responds is lacking. Here we assessed UPS performance and adaptability in yeast under stressors using quantitative measurements of misfolded substrate stability and stress-dependent UPS regulation by the transcription factor Rpn4. We found that impairing degradation rates (proteolytic stress) and generating misfolded proteins (folding stress) elicited distinct effects on the proteome and on UPS adaptation. Folding stressors stabilized proteins via aggregation rather than overburdening the proteasome, as occurred under proteolytic stress. Still, the UPS productively adapted to both stressors using separate mechanisms: proteolytic stressors caused Rpn4 stabilization while folding stressors increased RPN4 transcription. In some cases, adaptation completely prevented loss of UPS substrate degradation. Our work reveals the distinct effects of proteotoxic stressors and the versatility of cells in adapting the UPS.
View details for DOI 10.1083/jcb.201912041
View details for PubMedID 33382395
Cellular Control of Viscosity Counters Changes in Temperature and Energy Availability.
Cellular functioning requires the orchestration of thousands of molecular interactions in time and space. Yet most molecules in a cell move by diffusion, which is sensitive to external factors like temperature. How cells sustain complex, diffusion-based systems across wide temperature ranges is unknown. Here, we uncover a mechanism by which budding yeast modulate viscosity in response to temperature and energy availability. This "viscoadaptation" uses regulated synthesis of glycogen and trehalose to vary the viscosity of the cytosol. Viscoadaptation functions as a stress response and a homeostatic mechanism, allowing cells to maintain invariant diffusion across a 20°C temperature range. Perturbations to viscoadaptation affect solubility and phase separation, suggesting that viscoadaptation may have implications for multiple biophysical processes in the cell. Conditions that lower ATP trigger viscoadaptation, linking energy availability to rate regulation of diffusion-controlled processes. Viscoadaptation reveals viscosity to be a tunable property for regulating diffusion-controlled processes in a changing environment.
View details for DOI 10.1016/j.cell.2020.10.017
View details for PubMedID 33157040
Detection and Degradation of Stalled Nascent Chains via Ribosome-Associated Quality Control.
Annual review of biochemistry
2020; 89: 417–42
Stalled protein synthesis produces defective nascent chains that can harm cells. In response, cells degrade these nascent chains via a process called ribosome-associated quality control (RQC). Here, we review the irregularities in the translation process that cause ribosomes to stall as well as how cells use RQC to detect stalled ribosomes, ubiquitylate their tethered nascent chains, and deliver the ubiquitylated nascent chains to the proteasome. We additionally summarize how cells respond to RQC failure.
View details for DOI 10.1146/annurev-biochem-013118-110729
View details for PubMedID 32569528
CAT Tails Drive Degradation of Stalled Polypeptides on and off the Ribosome
CELL PRESS. 2020: 181A
View details for Web of Science ID 000513023201157
Viscoadaptation Controls Diffusion and Intracellular Reaction Rates in Response to Heat and Energy Availability
CELL PRESS. 2020: 134A
View details for Web of Science ID 000513023200651
Aggregation of CAT tails blocks their degradation and causes proteotoxicity in S. cerevisiae.
2020; 15 (1): e0227841
The Ribosome-associated Quality Control (RQC) pathway co-translationally marks incomplete polypeptides from stalled translation with two signals that trigger their proteasome-mediated degradation. The E3 ligase Ltn1 adds ubiquitin and Rqc2 directs the large ribosomal subunit to append carboxy-terminal alanine and threonine residues (CAT tails). When excessive amounts of incomplete polypeptides evade Ltn1, CAT-tailed proteins accumulate and can self-associate into aggregates. CAT tail aggregation has been hypothesized to either protect cells by sequestering potentially toxic incomplete polypeptides or harm cells by disrupting protein homeostasis. To distinguish between these possibilities, we modulated CAT tail aggregation in Saccharomyces cerevisiae with genetic and chemical tools to analyze CAT tails in aggregated and un-aggregated states. We found that enhancing CAT tail aggregation induces proteotoxic stress and antagonizes degradation of CAT-tailed proteins, while inhibiting aggregation reverses these effects. Our findings suggest that CAT tail aggregation harms RQC-compromised cells and that preventing aggregation can mitigate this toxicity.
View details for DOI 10.1371/journal.pone.0227841
View details for PubMedID 31945107
MISTERMINATE Mechanistically Links Mitochondrial Dysfunction with Proteostasis Failure.
Mitochondrial dysfunction and proteostasis failure frequently coexist as hallmarks of neurodegenerative disease. How these pathologies are related is notwell understood. Here, we describe a phenomenon termed MISTERMINATE (mitochondrial-stress-induced translational termination impairment and protein carboxyl terminal extension), which mechanistically links mitochondrial dysfunction with proteostasis failure. We show that mitochondrial dysfunction impairs translational termination of nuclear-encoded mitochondrial mRNAs, including complex-I 30kD subunit (C-I30) mRNA, occurring on the mitochondrial surface in Drosophila and mammalian cells. Ribosomes stalled at the normal stop codon continue to add to the C terminus of C-I30 certain amino acids non-coded by mRNA template. C-terminally extended C-I30 is toxic when assembled into C-I and forms aggregates in the cytosol. Enhancing co-translational quality control prevents C-I30 C-terminal extension and rescues mitochondrial and neuromuscular degeneration in a Parkinson's disease model. These findings emphasize theimportance of efficient translation termination and reveal unexpected link between mitochondrial health and proteome homeostasis mediated by MISTERMINATE.
View details for DOI 10.1016/j.molcel.2019.06.031
View details for PubMedID 31378462
CAT tails drive degradation of stalled polypeptides on and off the ribosome.
Nature structural & molecular biology
Stalled translation produces incomplete, ribosome-tethered polypeptides that the ribosome-associated quality control (RQC) pathway targets for degradation via the E3 ubiquitin ligase Ltn1. During this process, the protein Rqc2 and the large ribosomal subunit elongate stalled polypeptides with carboxy-terminal alanine and threonine residues (CAT tails). Failure to degrade CAT-tailed proteins disrupts global protein homeostasis, as CAT-tailed proteins can aggregate and sequester chaperones. Why cells employ such a potentially toxic process during RQC is unclear. Here, we developed quantitative techniques to assess how CAT tails affect stalled polypeptide degradation in Saccharomyces cerevisiae. We found that CAT tails enhance the efficiency of Ltn1 in targeting structured polypeptides, which are otherwise poor Ltn1 substrates. If Ltn1 fails to ubiquitylate those stalled polypeptides or becomes limiting, CAT tails act as degrons, marking proteins for proteasomal degradation off the ribosome. Thus, CAT tails functionalize the carboxytermini of stalled polypeptides to drive their degradation on and off the ribosome.
View details for DOI 10.1038/s41594-019-0230-1
View details for PubMedID 31133701
- Finding the Right Finish Line in Eukaryotic Transcription. Biochemistry 2019
- Quantification of Hsp90 availability reveals differential coupling to the heat shock response JOURNAL OF CELL BIOLOGY 2018; 217 (11): 3809-3816
Quantification of Hsp90 availability reveals differential coupling to the heat shock response.
The Journal of cell biology
The heat shock response (HSR) is a protective gene expression program that is activated by conditions that cause proteotoxic stress. While it has been suggested that the availability of free chaperones regulates the HSR, chaperone availability and the HSR have never been precisely quantified in tandem under stress conditions. Thus, how the availability of chaperones changes in stress conditions and the extent to which these changes drive the HSR are unknown. In this study, we quantified Hsp90 chaperone availability and the HSR under multiple stressors. We show that Hsp90-dependent and -independent pathways both regulate the HSR, and the contribution of each pathway varies greatly depending on the stressor. Moreover, stressors that regulate the HSR independently of Hsp90 availability do so through the Hsp70 chaperone. Thus, the HSR responds to diverse defects in protein quality by monitoring the state of multiple chaperone systems independently.
View details for PubMedID 30131327
Asc1, Hel2, and Slh1 couple translation arrest to nascent chain degradation.
RNA (New York, N.Y.)
Premature arrest of protein synthesis within the open reading frame elicits a protective response that degrades the incomplete nascent chain. In this response, arrested 80S ribosomes are split into their large and small subunits, allowing assembly of the ribosome quality control complex (RQC), which targets nascent chains for degradation. How the cell recognizes arrested nascent chains among the vast pool of actively translating polypeptides is poorly understood. We systematically examined translation arrest and modification of nascent chains in Saccharomyces cerevisiae to characterize the steps that couple arrest to RQC targeting. We focused our analysis on two poorly understood 80S ribosome-binding proteins previously implicated in the response to failed translation, Asc1 and Hel2, as well as a new component of the pathway, Slh1, that we identified here. We found that premature arrest at ribosome stalling sequences still occurred robustly in the absence of Asc1, Hel2, and Slh1. However, these three factors were required for the RQC to modify the nascent chain. We propose that Asc1, Hel2, and Slh1 target arresting ribosomes and that this targeting event is a precondition for the RQC to engage the incomplete nascent chain and facilitate its degradation.
View details for DOI 10.1261/rna.060897.117
View details for PubMedID 28223409
View details for PubMedCentralID PMC5393187
Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains
FEDERATION AMER SOC EXP BIOL. 2016
View details for Web of Science ID 000406444002561
Ribosome-associated protein quality control.
Nature structural & molecular biology
2016; 23 (1): 7-15
Protein synthesis by the ribosome can fail for numerous reasons including faulty mRNA, insufficient availability of charged tRNAs and genetic errors. All organisms have evolved mechanisms to recognize stalled ribosomes and initiate pathways for recycling, quality control and stress signaling. Here we review the discovery and molecular dissection of the eukaryotic ribosome-associated quality-control pathway for degradation of nascent polypeptides arising from interrupted translation.
View details for DOI 10.1038/nsmb.3147
View details for PubMedID 26733220
Protein synthesis. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains.
2015; 347 (6217): 75-78
In Eukarya, stalled translation induces 40S dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here we report cryo-electron microscopy structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S subunit at sites exposed after 40S dissociation, placing the Ltn1p RING (Really Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged tRNA to the A site and directs the elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which a protein--not an mRNA--determines tRNA recruitment and the tagging of nascent chains with carboxy-terminal Ala and Thr extensions ("CAT tails").
View details for DOI 10.1126/science.1259724
View details for PubMedID 25554787
A Ribosome-Bound Quality Control Complex Triggers Degradation of Nascent Peptides and Signals Translation Stress
2012; 151 (5): 1042-1054
The conserved transcriptional regulator heat shock factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol. We surveyed Hsf1 activity in a genome-wide loss-of-function library in Saccaromyces cerevisiae as well as ~78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC, and Hsf1 senses an RQC-mediated translation-stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.
View details for DOI 10.1016/j.cell.2012.10.044
View details for Web of Science ID 000311423500017
View details for PubMedID 23178123
STIMulating Calcium Entry at ER-Plasma Membrane Junctions
CELL PRESS. 2009: 193A
View details for Web of Science ID 000426353300013
Feedback loops shape cellular signals in space and time
2008; 322 (5900): 390-395
Positive and negative feedback loops are common regulatory elements in biological signaling systems. We discuss core feedback motifs that have distinct roles in shaping signaling responses in space and time. We also discuss approaches to experimentally investigate feedback loops in signaling systems.
View details for DOI 10.1126/science.1160617
View details for Web of Science ID 000260094500033
View details for PubMedID 18927383
View details for PubMedCentralID PMC2680159
STIM2 is a feedback regulator that stabilizes basal cytosolic and endoplasmic reticulum Ca2+ levels
2007; 131 (7): 1327-1339
Deviations in basal Ca2+ levels interfere with receptor-mediated Ca2+ signaling as well as endoplasmic reticulum (ER) and mitochondrial function. While defective basal Ca2+ regulation has been linked to various diseases, the regulatory mechanism that controls basal Ca2+ is poorly understood. Here we performed an siRNA screen of the human signaling proteome to identify regulators of basal Ca2+ concentration and found STIM2 as the strongest positive regulator. In contrast to STIM1, a recently discovered signal transducer that triggers Ca2+ influx in response to receptor-mediated depletion of ER Ca2+ stores, STIM2 activated Ca2+ influx upon smaller decreases in ER Ca2+. STIM2, like STIM1, caused Ca2+ influx via activation of the plasma membrane Ca2+ channel Orai1. Our study places STIM2 at the center of a feedback module that keeps basal cytosolic and ER Ca2+ concentrations within tight limits.
View details for DOI 10.1016/j.cell.2007.11.039
View details for Web of Science ID 000252217200021
View details for PubMedID 18160041
View details for PubMedCentralID PMC2680164
Interlinked fast and slow positive feedback loops drive reliable cell decisions
2005; 310 (5747): 496-498
Positive feedback is a ubiquitous signal transduction motif that allows systems to convert graded inputs into decisive, all-or-none outputs. Here we investigate why the positive feedback switches that regulate polarization of budding yeast, calcium signaling, Xenopus oocyte maturation, and various other processes use multiple interlinked loops rather than single positive feedback loops. Mathematical simulations revealed that linking fast and slow positive feedback loops creates a "dual-time" switch that is both rapidly inducible and resistant to noise in the upstream signaling system.
View details for DOI 10.1126/science.1113834
View details for Web of Science ID 000232786000048
View details for PubMedID 16239477
View details for PubMedCentralID PMC3175767
Protein evolution in the context of Drosophila development
JOURNAL OF MOLECULAR EVOLUTION
2005; 60 (6): 774-U42
The tempo at which a protein evolves depends not only on the rate at which mutations arise but also on the selective effects that those mutations have at the organismal level. It is intuitive that proteins functioning during different stages of development may be predisposed to having mutations of different selective effects. For example, it has been hypothesized that changes to proteins expressed during early development should have larger phenotypic consequences because later stages depend on them. Conversely, changes to proteins expressed much later in development should have smaller consequences at the organismal level. Here we assess whether proteins expressed at different times during Drosophila development vary systematically in their rates of evolution. We find that proteins expressed early in development and particularly during mid-late embryonic development evolve unusually slowly. In addition, proteins expressed in adult males show an elevated evolutionary rate. These two trends are independent of each other and cannot be explained by peculiar rates of mutation or levels of codon bias. Moreover, the observed patterns appear to hold across several functional classes of genes, although the exact developmental time of the slowest protein evolution differs among each class. We discuss our results in connection with data on the evolution of development.
View details for DOI 10.1007/s00239-004-0241-2
View details for Web of Science ID 000230077700008
View details for PubMedID 15909223