Heather Baker
Doctor of Philosophy in Biochemistry and Molecular Biology (PhD)
Research Topic
Identifying key E3 ubiquitin ligases and co-factors that target disease-associated mutant cytosolic proteins for degradation
Thibault Mayor
Professor
Research Classification
Research Interests
Aging
Cell Biology
Neurodegenerative diseases
Proteasome
Protein Degradation
Protein Folding
Proteomics
Proteostasis
Ubiquitin
Yeast Genetics
Relevant Thesis-Based Degree Programs
Research Methodology
Mass Spectrometey
Cell Biology
biochemistry
Recruitment
Master's students
Doctoral students
Postdoctoral Fellows
Any time / year round
The main lab focus is protein quality control and how misfolded proteins are triaged within the cell. In the next years, we want to establish several new projects and here are some examples:
- Identification of protein quality control pathways mediating rare genetic diseases in human.
- Reconstitution of the quality control machinery in vitro to better understand the molecular mechanism.
- Characterization of the Nedd4/Rsp5 quality control pathway in mammalian cells.
- Development of new proteomics approaches to determine client repertoires of chaperone proteins.
- Characterization of the formation of heat stress granules.
- Characterization of changes in the protein homeostasis network upon aging
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Doctoral Student Supervision
Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Age drives changes in the composition of detergent-insoluble protein aggregates in brain tissue (2024)
Aging and protein aggregation diseases are inextricably linked. During aging, cellular protein quality control declines which results in diminished protein homeostasis (proteostasis). In model organisms, such as C. elegans, proteostatic decline with age has been linked to the onset of aggregation of proteins in wild-type animals. However, this correlation has not been extensively characterized in aging mammals. To investigate the potentially aggregated portion of the proteome, detergent-insoluble fractionation was used to isolate proteins that become insoluble with age in mouse brain tissue. Quantitative mass spectrometry was used to identify 171 proteins enriched in the detergent-insoluble fraction of older mice. This data was then compared with published datasets using detergent-insoluble fractionation with mouse brain tissue derived from various ages and disease models. Comparison of protein features across the datasets revealed that features associated with proteins found in the pellet fraction differ depending on the ages of the mice. In general, insoluble proteins from young mice were more likely to be RNA-binding, more disordered and more likely to be found in membraneless organelles. These traits become less prominent with age within the combined dataset, as proteins with more structure enter the pellet fraction. These findings suggested that there are likely alterations in the folding of the highly structured proteins that are affected by aging that cause them to become insoluble. Replication of these experiments in the cortex and hippocampus yielded detergent-insoluble proteins with similar features. In addition, consistent enrichment of subunits of the extracellular C1q complex was also observed in both brain regions along with an increase in immunoglobulin signal indicating that increased inflammation may contribute to the insolubility of certain proteins. To further investigate possible structural changes in the soluble portion of the proteome, limited proteolysis was used. These altered proteins tend to have more charged residues and annotated phosphorylation sites and are enriched for synaptic proteins. The complementary approaches employed in this study revealed minute alterations of the proteome that may help to better explain the slow decay of proteostasis observed upon aging.
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To aggregate or not to aggregate: a study of protein macro-assemblies and aggregation-prone polypeptides in Saccharomyces Cerevisiae (2022)
Protein aggregation is often a hallmark of protein homeostasis disruption in the cell. Accumulation of cytotoxic protein species has been associated to many diseases. However, protein aggregation has also been shown to be beneficial and provide biological functions in other cases. With a fine line between a functional and harmful outcome, protein aggregation is highly regulated and under constant monitoring of the protein homeostasis network. In this thesis, we combined cell fractionation with mass spectrometry to identify proteins aggregates and to delineate features associated with them.To identify protein aggregates in unstressed cells, we established a quantitative mass spectrometry approach that combines SILAC labeling and centrifugation. We found that these proteins are longer and lower abundant proteins with fewer hydrophobic residues and more low complexity and disordered regions. We also found features that suggest these proteins have more potential interaction partners and are involved in function macro assemblies.We next examined which yeast proteins aggregate under stress condition by using heat shock. We found that proteins that aggregate under heat-shock were enriched with known stress granule (SG) proteins. There proteins are also abundant and enriched in intrinsically disordered regions (IDRs). We found that several IDRs were sufficient for localization to SG. Lastly, we showed that the IDR of the Ubp3 was critical for yeast SG formation. This work shows the importance of IDRs in mediating protein condensation upon stress.Finally, we examined which newly translated proteins aggregate after heat-shock. We found these proteins were abundant, shorter, highly ordered, and more hydrophobic. Notably these proteins contained more β-sheets. As well, there was an enrichment for proteins with chaperone-binding motifs. Interestingly, these proteins were also more often components of stable protein complexes. We found a specific group of thermal labile proteins that takes longer to reach thermostable native state.Collectively, this thesis uncovered protein features associated with aggregating proteins using a common experimental framework and sheds light on how protein aggregates are formed and regulated. Our work provides a platform for future studies to better comprehend how protein homeostasis is regulated and to potentially prevent unwanted protein aggregation for therapeutic applications.
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Characterization of degradative protein quality control mechanisms using model substrates derived from temperature sensitive alleles (2017)
The purpose of protein homeostasis (proteostasis) is to maintain proteome integrity, thereby promoting viability at both the cellular and organism levels. Exposure to a range of acute stresses often produces misfolded proteins, which present a challenge to maintaining proteostatic balance. The accumulation of misfolded proteins can lead to the formation of potentially toxic protein aggregates, which are characteristic of a number of neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Therefore, a number of protein quality control pathways exist to promote protein folding by molecular chaperones or target terminally misfolded proteins for degradation via the ubiquitin proteasome system or autophagy. Within the cytosol the mechanisms responsible for targeting substrates for proteasomal degradation remain to be fully elucidated. In this thesis, we established and employed thermosensitive model substrates to screen for factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations in Saccharomyces cerevisiae. Using a genome- wide flow cytometry based screen we identified the prefoldin chaperone subunit Gim3 as well as the E3 ubiquitin ligase Ubr1. An absence of Gim3 leads to the accumulation of model substrates in cytosolic inclusions and their delayed degradation. We propose that Gim3 promotes degradation by maintaining substrate solubility. In the course of screening for factors involved in degradative protein quality control, we identified secondary mutations in the general stress response gene WHI2 among a number of E3 ligase deletion strains. We demonstrate that an absence of WHI2 is responsible for the observed impairment in the proteolytic degradation of Guk1-7. We propose a link between mutations in WHI2 to a deficiency in the Msn2/4 transcriptional response, thereby altering the cell’s capacity to degrade misfolded cytosolic proteins. Collectively, the data in this thesis generated with the Guk1-7 model substrate underscores how changes in the elaborate protein quality control network can perturb proteostasis. Given that proteostasis is altered in a number of diseases ranging from cancer to ageing, identifying the factors that mediate protein quality control and understanding the interplay between members of the proteostatic network are important not only for understanding the basic biological processes but also for potential therapeutic applications.
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Good riddance to bad proteins: Identification of novel protein quality control pathways targeting cytosolic misfolded proteins for degradation (2014)
Protein misfolding is cytotoxic and the accumulation of misfolded proteins threatens cell fitness and viability. Failure to eliminate these polypeptides has been associated with numerous diseases including neurodegenerative disorders. The ubiquitin proteasome system is a major pathway that degrades in the cell these unwanted proteins targeted by protein quality control. Several distinct protein quality control degradation pathways that employ different ubiquitin ligases have been discovered in recent years. Here, we present two novel protein quality control degradation pathways that require the ubiquitin ligases Hul5 and Rsp5 to target cytosolic misfolded proteins for degradation. We used quantitative mass spectrometry to determine that in Saccharomyces cerevisiae, heat-shock triggered a large increase in the level of ubiquitylation of mainly cytosolic proteins. We discovered that the Hul5 ubiquitin ligase participated in this ubiquitylation response. Hul5 was required to maintain cell fitness after heat-shock and to degrade short-lived misfolded proteins. In addition, the localization of Hul5 in the cytoplasm was important for its quality control function. We also showed that Hul5 targeted low-solubility cytosolic proteins in both heat-shock and unstressed conditions. These data indicate that Hul5 is involved in the degradation of cytosolic misfolded proteins.Beside the Hul5 pathway, we found that Rsp5 ubiquitin ligase also participated in the increase of ubiquitylation levels upon heat-shock. Our results indicated that Rsp5 employed a bipartite recognition mechanism to ubiquitylate heat-induced cytosolic targets via the interaction with the Hsp40 co-chaperone Ydj1 and the PY-motifs primarily found in structured regions of these proteins. Notably, we also found that the Rsp5-dependent pathway was dependent on both Ubp2 and Ubp3 deubiquitinases, which acted to mainly reduce the levels of K63-linked ubiquitin chains conjugated to cytosolic misfolded proteins upon heat-shock. The absence of either deubiquitinase led to reduced cell fitness under stress conditions underscoring the importance of the Rsp5-dependent pathway. All together, we identified the two major yeast ubiquitin ligases that mediated the increase in ubiquitylation of cytosolic misfolded proteins upon heat stress. Our work shed new light on protein quality control and how the cell can mediate the degradation of misfolded proteins.
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Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Identifying the key elements involved in cytosolic protein quality control (2024)
The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
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High throughput screen for improved recombinant laccase production in Saccharomyces cerevisiae (2023)
The yeast Saccharomyces cerevisiae is an attractive host for the recombinant production of proteins. Despite widespread, the typically low protein yield is a severe disadvantage compared to other host systems. To address this limitation, the objective of this research is to identify gene deletions that influence recombinant protein production in S. cerevisiae. Specifically, we have used an industrially relevant fungal laccase enzyme, Lcc1, isolated from Trametes trogii as our model recombinant protein. We utilized synthetic genetic array (SGA) methodology to produce a library of 4,790 laccase expressing gene deletion strains and a novel soft agarose overlay assay to assess quantities of active secreted laccase. We identified 66 gene deletions that resulted in increased secreted laccase. Gene ontology (GO) analysis revealed an enrichment for processes such as vacuolar targeting, proteolysis, autophagy, and vesicle trafficking. Further validation of the levels of secreted laccase from the identified mutant strains was done in liquid cultures resulting in a set of 17 gene deletions with significantly increased secreted laccase activity from that of a reference strain. Notably, deletions of the endoplasmic reticulum (ER) membrane localized flippase, ARV1, a component of the SKI complex, SKI3, and PMT2, an ER membrane protein-O-mannosyltransferase all resulted in an over 4-fold increase in secreted laccase activity. Through complementation experiments and independent gene deletions, we were able to verify the role of six gene deletions in increasing secreted laccase activity. We have also identified a set of 207 gene deletions which decrease secreted laccase activity that could serve as potential targets for overexpression. When assessed in liquid cultures, deletion of the ER co-chaperone SCJ1, resulted in almost no secreted laccase activity. However, upon overexpression of the co-chaperone, secreted laccase activity was also decreased compared to that of a reference strain suggesting the basal expression level is optimal to produce the recombinant laccase. Our results firstly demonstrate the application of high throughput screening techniques for investigating effects on recombinant protein production. Additionally, we have identified a set of gene deletions with uninvestigated roles in recombinant laccase production thus elaborating upon previously identified bottlenecks limiting recombinant protein production in S. cerevisiae.
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Investigating the mammalian heat-shock induced ubiquitination response (2018)
Aggregation of misfolded proteins in the cell is often indicative of a failing protein quality control system, whose responsibility is to mediate either the refolding or degradation of these aberrant proteins. Heat-shock (HS), which causes proteins to misfold, leads to a marked increase of the ubiquitination of proteins that display poor solubility in the cell. The E3 ubiquitin ligase Rsp5 has previously been shown to be the major ubiquitin ligase that targets cytosolic misfolded proteins upon HS in S. cerevisiae. Its closest mammalian orthologue, Nedd4-1, was also found to be responsible for an increased ubiquitination upon HS in HeLa and MEF cells, however further work is needed to better characterize this response. Such characterization included looking at which proteins get ubiquitinated upon HS to then determine whether they share common characteristics such as cellular localization, and if they are newly synthesized versus pre-existing. Using diGly enrichment coupled with mass spectrometry, we identified over 300 proteins that were further ubiquitinated upon HS. An important portion of these proteins are localized in the nucleus, whereas proteins associated to the mitochondria are markedly under-represented. As well, proteins involved in processes such as pre-mRNA processing, cell cycle and SUMOylation were found to be enriched. Using a pulse-SILAC approach, we found that a large portion of proteins ubiquitinated after HS were pre-existing, whereas some were newly synthesized including ribosomal proteins. We also investigated the potential role of other Nedd4 family members in the HS ubiquitination response in Hek293 cells. Using shRNA, we found that the knockdown of the Itch E3, but not of Nedd4-1, caused a decrease of the HS-induced ubiquitination response in these cells. Accordingly, we also showed a reduced cell viability upon Itch knocked down under HS.
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Characterization of the Potential Role of Myosin IIa in Parkin Translocation during Mitopahgy (2015)
Mitochondria are important organelles of eukaryotic cells that provide energy through cellular respiration. Cells have evolved several quality control mechanisms to preserve functional mitochondria and avoid cell damage. Damaged mitochondria are recognized and removed by mitophagy to avoid the production of reactive oxygen species. A major signal for the recognition of damaged mitochondria is the electrical membrane potential depolarization, which leads to the recruitment of PTEN-induced putative kinase 1 (PINK1), followed by the recruitment of the E3 ubiquitin ligase parkin at the outer mitochondrial membrane. Parkin ubiquitinates numerous mitochondrial outer membrane proteins and initiates mitophagy. Mutations in the gene encoding parkin are frequently found in familiar forms of Parkinson’s disease. Although several factors involved in parkin mitochondrial recruitment have been characterized, additional proteins may be involved. The aim of this work was to determine whether other factors may be involved in colocalizing parkin to damaged mitochondria. Following up a SILAC-immunoprecipitation experiment, we hypothesized that an unconventional myosin (myosin IIa) may be involved in the recruitment of parkin to the mitochondria. Myosins are a large family of actin-based cytoskeletal motors that use energy derived from ATP hydrolysis to generate movement. Non-conventional myosins are well studied for their contribution to synaptic function in neuronal cells. MYH9 was identified as potential interactor of parkin by mass spectrometry. This interaction was validated through co-immunoprecipitation and immunofluorescence experiments. In addition, myosin alteration with small chemicals that depolymerize actin filament or inhibit myosin activity, impaired parkin localization to mitochondria upon stress. This study potentially implicates myosin IIa as a modulator of parkin recruitment to the mitochondria, and may thus open the door to new therapeutic strategies for Parkinson’s disease.
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Feature analysis and in silico prediction of lower solubility proteins in three eukaryotic model systems (2015)
Regulation of protein solubility, or the ability of proteins to remain soluble withinthe cell, is an important part of protein homeostasis. This is highlighted with thedisruption of protein homeostasis and dysregulation of solubility being associatedwith various neurodegenerative diseases. Using quantitative mass spectrometryand computational analyses, we identify low solubility proteins under unstressedconditions in three eukaryotic model systems: yeast cells, human neuroblastomacells, and mouse brain tissue. Using an internal reference, we account for proteinabundance, and allow for the analysis of proteins based on their partitioning betweenthe soluble and insoluble fractions, rather than purely on their abundancewithin the insoluble fraction. We identified several intrinsic traits such as length,disorder, abundance, molecular recognition features, and low complexity regionswhich are correlated with protein solubility. These features have been previouslyshown to be associated with protein-protein interactions. This suggests that, underunstressed conditions, lower solubility in proteins may be linked to functional aggregation,rather than aberrant aggregation. We then present two predictors whichmay be used to predict the in vivo solubility of proteins, built using the many traitsexamined in this work. The linear regression model is able to give estimates ofprotein solubility, although proteins near the threshold between low and normalsolubility may be misclassified. The Support Vector Machine is able to reliablydistinguish between low and high solubility proteins, but is unable to reliably distinguishlow and normal solubility proteins. We have identified several traits thatdistinguish low solubility proteins from other proteins, as well as developed twomodels that are able to estimate the solubility of proteins.
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Characterizing cytosolic protein quality control pathways in saccharomyces cerevisiae using temperature-sensitive model substrates (2011)
A large pool of proteins localizes in the cytosol of eukaryotic cells. Proteins in the cell can be misfolded due to cellular stress, mutations, and transcriptional and translational errors. Several E3 ubiquitin ligases have been shown to target misfolded cytosolic proteins for degradation by the proteasome. In this study, we characterized a panel of thermosensitive mutant proteins in Saccharomyces cerevisiae from six essential genes. The wild-type alleles of these thermosensitive proteins are stable at restrictive temperature. In contrast, we found that roughly half the tested alleles are significantly degraded at non-permissive temperature in a proteasome-dependent manner. These unstable alleles thus display hallmarks of protein quality control substrates. We found that degradation of mutant protein Pro3-1p, one of the unstable alleles, is dependent on the dual action of Ubr1 and San1 ubiquitin ligases. The single deletions of the ligases do not affect the stability of Pro3-1p. In contrast, double deletion of UBR1 and SAN1 leads to significant stabilization of the mutant protein. The increase in stability of Pro3-1 is associated with suppression of thermosensitive phenotype at restrictive temperature, suggesting that the depletion of the essential protein due to its degradation leads to the loss of viability at restrictive temperature.
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Quantitative mass spectrometry to discover interactors of parkin E3 ubiquitin ligase, a protein implicated in early-onset Parkinson's disease (2010)
Ubiquitylation is a major post-translational modification based on a network of about six hundred E3 ubiquitin ligases in human. It is involved in several processes such as proteolysis, vesicle trafficking and DNA damage response. Mutations in PARK2, which encode parkin E3 ubiquitin ligase, account for half of autosomal recessive juvenile Parkinsonism cases, an early onset form of Parkinson’s disease. Multiple PARK2 mutations underlie the RING domain, which contains ligase activity. This finding suggests an inability for substrate ubiquitylation may trigger neurodegeneration. We used a quantitative proteomics approach to seek identifying parkin substrates and interactors. We first developed and tested new methods to enrich for ubiquitylated proteins that could potentially be used to study the influence of parkin on the ubiquitin proteome. In our first approach, ubiquitin conjugates were purified from SH-SY5Y neuroblastoma expressing His8-biotin-ubiquitin by tandem affinity purification. A second approach to purify ubiquitylated proteins was based on affinity chromatography using S5a proteasome receptor that bound to poly-ubiquitylated proteins. We determined that both approaches were not adequate for identifying low abundance parkin substrates. We then sought to identify which proteins were associated with parkin. Parkin interactors were enriched from SH-SY5Y expressing FLAG-parkin versus endogenous parkin by anti-FLAG immunoprecipitation in the context of SILAC. Proteins from the neuroendocrine chromogranin-secretogranin family were highly enriched suggesting a potential granin vesicle trafficking role for parkin. CCCP, a mitochondrial uncoupling agent was also employed to investigate parkin ligase interactors during mitochondrial stress since parkin localizes to mitochondria to promote mitophagy upon a reduction in mitochondrial membrane potential. Several actin related proteins were enriched from FLAG-parkin cells treated with CCCP including non-muscle unconventional signaling myosin suggesting a potential role for these proteins during parkin-mediated mitophagy.
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