Thibault Mayor

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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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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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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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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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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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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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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