How does a cell decide what to do with a misfolded protein? My work to date has focused on protein quality control in eukaryotic cells, and specifically on the interplay between two key circuits: molecular chaperones and ubiquitin systems. During my PhD with Paul Workman (Institute of Cancer Research, UK), I characterized the mechanisms of ubiquitin-mediated degradation in the Hsp90 cancer proteome. My current work has identified that distinct chaperone and ubiquitin systems collaborate to clear nuclear versus cytoplasmic misfolded proteins in yeast.
By using an integrated approach, including use of super-resolution fluorescence microscopy and state-of-the-art proteomics, together with more conventional biochemical and cell biological techniques, my ultimate goal is to determine how protein quality control circuits influence, and are influenced by, specific ubiquitin linkages—and how this changes during aging and disease. Given that changing capacity of protein quality control is a major hallmark of aging and disease, understanding how ubiquitin linkage-specific protein quality control circuits are rewired during these processes will likely have important consequences for disease pathology and the development of novel therapies.
Basic Life Science Research Associate, Biology
Honors & Awards
Poster Prize, Bay Area Aging Meeting (2018)
Long-Term Fellowship, Human Frontiers Science Program (2015-2018)
Research & Travel Award, Cancer Research UK (2014)
Doctor of Philosophy, Institute of Cancer Research, University of London, Cancer Pharmacology (2012)
Bachelor of Arts, University of Cambridge, Natural Sciences (Biochemistry) (2007)
Current Research and Scholarly Interests
Protein misfolding in the cell creates toxic species linked to an array of diseases. Protective cellular protein quality control (PQC) mechanisms evolved to selectively recognize misfolded proteins and limit their toxic effects. Molecular chaperones recognize misfolded proteins, while the ubiquitin-proteasome system (UPS) promotes their clearance through the attachment of ubiquitin chains. We previously identified a PQC pathway for spatial sequestration and clearance of misfolded proteins, conserved from yeast to humans, that is amplified when the UPS is impaired. However, the identity of the E3 ubiquitin ligases involved in this pathway—and how they interact with the chaperone machinery—is unresolved. Starting with a fluorescence microscopy-based genetic screen in yeast, we show that distinct chaperone and ubiquitination circuitries cooperate in PQC of soluble misfolded proteins in the cytoplasm and nucleus. In contrast with the canonical model where Lys48-linked ubiquitin chains are sufficient for proteasomal targeting, we found that cytoplasmic misfolded proteins requires tagging with mixed ubiquitin chains that contain both Lys11 and Lys48 linkages to be degraded. Each type of linkage-specific ubiquitination requires a distinct combination of ubiquitin ligases and chaperones. Strikingly, unlike cytoplasmic PQC, proteasomal degradation of nuclear misfolded proteins only requires Lys48 ubiquitin linkages and is independent of Lys11-specific circuits. We conclude that cytoplasmic and nuclear PQC involve combinatorial recognition by defined sets of cooperating systems. The distinct PQC requirements reveal underlying differences in nuclear and cytoplasmic proteome management, with important implications for our understanding of a wide range of diseases.
Methods for measuring misfolded protein clearance in the budding yeast Saccharomyces cerevisiae.
Methods in enzymology
2019; 619: 27–45
Protein misfolding in the cell is linked to an array of diseases, including cancers, cardiovascular disease, type II diabetes, and numerous neurodegenerative disorders. Therefore, investigating cellular pathways by which misfolded proteins are trafficked and cleared ("protein quality control") is of both mechanistic and therapeutic importance. The clearance of most misfolded proteins involves the covalent attachment of one or more ubiquitin molecules; however, the precise fate of the ubiquitinated protein varies greatly, depending on the linkages present in the ubiquitin chain. Here, we discuss approaches for quantifying linkage-specific ubiquitination and clearance of misfolded proteins in the budding yeast Saccharomyces cerevisiae-a model organism used extensively for interrogation of protein quality control pathways, but which presents its own unique challenges for cell and molecular biology experiments. We present a fluorescence microscopy-based assay for monitoring the clearance of misfolded protein puncta, a cycloheximide-chase assay for calculating misfolded protein half-life, and two antibody-based methods for quantifying specific ubiquitin linkages on tagged misfolded proteins, including a 96-well plate-based ELISA. We hope these methods will be of use to the protein quality control, protein degradation, and ubiquitin biology communities.
View details for PubMedID 30910025
Distinct proteostasis circuits cooperate in nuclear and cytoplasmic protein quality control.
2018; 563 (7731): 407–11
Protein misfolding is linked to a wide array of human disorders, including Alzheimer's disease, Parkinson's disease and type II diabetes1,2. Protective cellular protein quality control (PQC) mechanisms have evolved to selectively recognize misfolded proteins and limit their toxic effects3-9, thus contributing to the maintenance of the proteome (proteostasis). Here we examine how molecular chaperones and the ubiquitin-proteasome system cooperate to recognize and promote the clearance of soluble misfolded proteins. Using a panel of PQC substrates with distinct characteristics and localizations, we define distinct chaperone and ubiquitination circuitries that execute quality control in the cytoplasm and nucleus. In the cytoplasm, proteasomal degradation of misfolded proteins requires tagging with mixed lysine 48 (K48)- and lysine 11 (K11)-linked ubiquitin chains. A distinct combination of E3 ubiquitin ligases and specific chaperones is required to achieve each type of linkage-specific ubiquitination. In the nucleus, however, proteasomal degradation of misfolded proteins requires only K48-linked ubiquitin chains, and is thus independent of K11-specific ligases and chaperones. The distinct ubiquitin codes for nuclear and cytoplasmic PQC appear to be linked to the function of the ubiquilin protein Dsk2, which is specifically required to clear nuclear misfolded proteins. Our work defines the principles of cytoplasmic and nuclear PQC as distinct, involving combinatorial recognition by defined sets of cooperating chaperones and E3 ligases. A better understanding of how these organelle-specific PQC requirements implement proteome integrity has implications for our understanding of diseases linked to impaired protein clearance and proteostasis dysfunction.
View details for PubMedID 30429547
Mechanisms and Functions of Spatial Protein Quality Control.
Annual review of biochemistry
A healthy proteome is essential for cell survival. Protein misfolding is linked to a rapidly expanding list of human diseases, ranging from neurodegenerative diseases to aging and cancer. Many of these diseases are characterized by the accumulation of misfolded proteins in intra- and extracellular inclusions, such as amyloid plaques. The clear link between protein misfolding and disease highlights the need to better understand the elaborate machinery that manages proteome homeostasis, or proteostasis, in the cell. Proteostasis depends on a network of molecular chaperones and clearance pathways involved in the recognition, refolding, and/or clearance of aberrant proteins. Recent studies reveal that an integral part of the cellular management of misfolded proteins is their spatial sequestration into several defined compartments. Here, we review the properties, function, and formation of these compartments. Spatial sequestration plays a central role in protein quality control and cellular fitness and represents a critical link to the pathogenesis of protein aggregation-linked diseases.
View details for DOI 10.1146/annurev-biochem-060815-014616
View details for PubMedID 28489421
- Phosphoproteomic-based identification of CDK8/CDK19 substrates in colorectal cancer AMER ASSOC CANCER RESEARCH. 2016
- Proteomic analysis of ubiquitination identifies the interplay between HSP90 inhibition and CUL5 in the control of autophagy AMER ASSOC CANCER RESEARCH. 2016
A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease.
Nature chemical biology
2015; 11 (12): 973-980
There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small-molecule inhibitor of the WNT pathway discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex-associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a type 1 binding mode involving insertion of the CDK8 C terminus into the ligand binding site. In contrast to type II inhibitors of CDK8 and CDK19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogs alter WNT pathway-regulated gene expression and other on-target effects of modulating CDK8 and CDK19, including expression of genes regulated by STAT1. Consistent with this, we find that phosphorylation of STAT1(SER727) is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors.
View details for DOI 10.1038/nchembio.1952
View details for PubMedID 26502155
View details for PubMedCentralID PMC4677459
E3 ubiquitin ligase Cullin-5 modulates multiple molecular and cellular responses to heat shock protein 90 inhibition in human cancer cells
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2014; 111 (18): 6834-6839
The molecular chaperone heat shock protein 90 (HSP90) is required for the activity and stability of its client proteins. Pharmacologic inhibition of HSP90 leads to the ubiquitin-mediated degradation of clients, particularly activated or mutant oncogenic protein kinases. Client ubiquitination occurs via the action of one or more E3 ubiquitin ligases. We sought to identify the role of Cullin-RING family E3 ubiquitin ligases in the cellular response to HSP90 inhibition. Through a focused siRNA screen of 28 Cullin-RING ligase family members, we found that CUL5 and RBX2 were required for degradation of several HSP90 clients upon treatment of human cancer cells with the clinical HSP90 inhibitor 17-AAG. Surprisingly, silencing Cullin-5 (CUL5) also delayed the earlier loss of HSP90 client protein activity at the same time as delaying cochaperone dissociation from inhibited HSP90-client complexes. Expression of a dominant-negative CUL5 showed that NEDD8 conjugation of CUL5 is required for client degradation but not for loss of client activity or recruitment of clients and HSP90 to CUL5. Silencing CUL5 reduced cellular sensitivity to three distinct HSP90 inhibitors, across four cancer types driven by different protein kinases. Our results reveal the importance of CUL5 in multiple aspects of the cellular response to HSP90 inhibition.
View details for DOI 10.1073/pnas.1322412111
View details for Web of Science ID 000335477300073
View details for PubMedID 24760825
View details for PubMedCentralID PMC4020071
ATP-competitive inhibitors block protein kinase recruitment to the Hsp90-Cdc37 system
NATURE CHEMICAL BIOLOGY
2013; 9 (5): 307-?
Protein kinase clients are recruited to the Hsp90 molecular chaperone system via Cdc37, which simultaneously binds Hsp90 and kinases and regulates the Hsp90 chaperone cycle. Pharmacological inhibition of Hsp90 in vivo results in degradation of kinase clients, with a therapeutic effect in dependent tumors. We show here that Cdc37 directly antagonizes ATP binding to client kinases, suggesting a role for the Hsp90-Cdc37 complex in controlling kinase activity. Unexpectedly, we find that Cdc37 binding to protein kinases is itself antagonized by ATP-competitive kinase inhibitors, including vemurafenib and lapatinib. In cancer cells, these inhibitors deprive oncogenic kinases such as B-Raf and ErbB2 of access to the Hsp90-Cdc37 complex, leading to their degradation. Our results suggest that at least part of the efficacy of ATP-competitive inhibitors of Hsp90-dependent kinases in tumor cells may be due to targeted chaperone deprivation.
View details for DOI 10.1038/NCHEMBIO.1212
View details for Web of Science ID 000317727600007
View details for PubMedID 23502424
- MOLECULAR BIOLOGY Choose your protein partners NATURE 2012; 490 (7420): 351-352
The expanding proteome of the molecular chaperone HSP90
2012; 11 (7): 1301-1308
The molecular chaperone HSP90 maintains the activity and stability of a diverse set of "client" proteins that play key roles in normal and disease biology. Around 20 HSP90 inhibitors that deplete the oncogenic clientele have entered clinical trials for cancer. However, the full extent of the HSP90-dependent proteome, which encompasses not only clients but also proteins modulated by downstream transcriptional responses, is still incompletely characterized and poorly understood. Earlier large-scale efforts to define the HSP90 proteome have been valuable but are incomplete because of limited technical sensitivity. Here we discuss previous large-scale surveys of proteome perturbations induced by HSP90 inhibitors in light of a significant new study using state-of-the-art SILAC technology combined with more sensitive high-resolution mass spectrometry (MS) that extends the catalog of proteomic changes in inhibitor-treated cancer cells. Among wide-ranging changes, major functional responses include downregulation of protein kinase activity and the DNA damage response alongside upregulation of the protein degradation machinery. Despite this improved proteomic coverage, there was surprisingly little overlap with previous studies. This may be due in part to technical issues but is likely also due to the variability of the HSP90 proteome with the inhibitor conditions used, the cancer cell type and the genetic status of client proteins. We suggest future proteomic studies to address these factors, to help distinguish client protein components from indirect transcriptional components and to address other key questions in fundamental and translational HSP90 research. Such studies should also reveal new biomarkers for patient selection and novel targets for therapeutic intervention.
View details for DOI 10.4161/cc.11.7.19722
View details for Web of Science ID 000302285300013
View details for PubMedID 22421145
View details for PubMedCentralID PMC3350876