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Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2020)

The integrity of the proteome must be maintained to ensure normal protein function and prevent cellular toxicity. Proteostasis factors scan the proteome to promote protein folding, trafficking, disassembly, and degradation. In all forms of life and cellular environments, the Hsp70/Hsp40 chaperone machinery rescues misfolded proteins, with over 40 human Hsp40s responsible for identifying and recruiting misfolded protein substrates to Hsp70. This Hsp40 diversity could be responsible for matching client recognition to Hsp70 functional diversity. To evaluate the client diversity of class B Hsp40s, we applied tandem mass tag-affinity purification-mass spectrometry (TMT-AP-MS) to characterize the interactor profiles of human Hsp40s. To increase interactor recovery yield, we used mutations that inactivate handoff of clients to Hsp70s. We found > 400 high-confidence interactors of DNAJB8 in HEK293T cells by using crosslinking with high stringency washing conditions to decrease false positives, TMT labeling to allow head-to-head comparison across different samples, and identification of specific interactors based on integrated TMT intensity correlation between prey and bait levels.

With bait correlation coupled with TMT-AP-MS we could assess the substrate profiles of different Hsp40 members. DNAJB1 and DNAJB8 are two Hsp40s that differ in size, structure, and functionality, that are ideal candidates for probing differential substrate binding across human class B Hsp40s. We first evaluated the proteome-wide effect of crosslinking and J-domain activity on interactor recovery for each Hsp40. After finding crosslinking to be unnecessary and J-domain activity to be neutral for recovery of interactors, we profiled substrates of DNAJB1WT and DNAJB8WT. Only 19 interactors, largely Hsp70 chaperones, were identified for DNAJB1WT. Approximately half of the 249 interactors for DNAJB8WT were shared with DNAJB8H31Q clients. However, heat shock, which induces protein misfolding, increases global client binding to DNAJB8H31Q but not to DNAJB8WT, suggesting that the J-domain mutation is necessary to maintain increased client binding under stress conditions. We demonstrate an effective technique that could probe client interactions for Hsp40 co-chaperones, which could lead to insight into how proteostasis machineries subdivide their large substrate pool between different fates and minimize the gap in understanding of the role of Hsp40s in Hsp70 functional diversity.

    Cover page: Cellular Protein Client Recovery of Human HSP40s
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2021)
    Environmental toxins and toxicants can damage proteins and threaten cellular proteostasis. Most current methodologies to identify misfolded proteins in cells survey the entire proteome for sites of changed reactivity. We describe and apply a quantitative proteomics methodology to identify destabilized proteins based on their binding to the human Hsp40 chaperone DNAJB8. These protein targets are validated by an orthogonal limited proteolysis assay using parallel reaction monitoring. We find that a brief exposure of HEK293T cells to meta-arsenite increases the affinity of two dozen proteins to DNAJB8, including known arsenite-sensitive proteins. In particular, arsenite treatment destabilizes both the pyruvate dehydrogenase complex E1 subunit and several RNA-binding proteins. This platform can be used to explore how environmental toxins impact cellular proteostasis and to identify the susceptible proteome.
      Cover page: Hsp40 Affinity to Identify Proteins Destabilized by Cellular Toxicant Exposure.
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2022)
      Most eukaryotic secretory proteins are cotranslationally translocated through Sec61 into the endoplasmic reticulum (ER). Because these proteins have evolved to fold in the ER, their mistargeting is associated with toxicity. Genetic experiments have implicated the ER heat shock protein 70 (Hsp70) Hspa13/STCH as involved in processing of nascent secretory proteins. Herein, we evaluate the role of Hspa13 in protein import and the maintenance of cellular proteostasis in human cells, primarily using the human embryonic kidney 293T cell line. We find that Hspa13 interacts primarily with the Sec61 translocon and its associated factors. Hspa13 overexpression inhibits translocation of the secreted protein transthyretin, leading to accumulation and aggregation of immature transthyretin in the cytosol. ATPase-inactive mutants of Hspa13 further inhibit translocation and maturation of secretory proteins. While Hspa13 overexpression inhibits cell growth and ER quality control, we demonstrate that HSPA13 knockout destabilizes proteostasis and increases sensitivity to ER disruption. Thus, we propose that Hspa13 regulates import through the translocon to maintain both ER and cytosolic protein homeostasis. The raw mass spectrometry data associated with this article have been deposited in the PRIDE archive and can be accessed at PXD033498.
        Cover page: Heat shock protein Hspa13 regulates endoplasmic reticulum and cytosolic proteostasis through modulation of protein translocation.
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2024)
        The identification and quantification of misfolded proteins from complex mixtures is important for biological characterization and disease diagnosis, but remains a major bioanalytical challenge. We have developed Hsp40 Affinity Profiling as a bioanalytical approach to profile protein stability in response to cellular stress. In this assay, we ectopically introduce the Hsp40 FlagDNAJB8H31Q into cells and use quantitative proteomics to determine how protein affinity for DNAJB8 changes in the presence of cellular stress, without regard for native clients. Herein, we evaluate potential approaches to improve the performance of this bioanalytical assay. We find that although intracellular crosslinking increases recovery of protein interactors, this is not enough to overcome the relative drop in DNAJB8 recovery. While the J-domain promotes Hsp70 association, it does not affect the yield of protein association with DNAJB8 under basal conditions. By contrast, crosslinking and J-domain ablation both substantially increase relative protein interactor recovery with the structurally distinct Class B Hsp40 DNAJB1 but are completely compensated by poorer yield of DNAJB1 itself. Cellular thermal stress promotes increased affinity between DNAJB8H31Q and interacting proteins, as expected for interactions driven by recognition of misfolded proteins. DNAJB8WT does not demonstrate such a property, suggesting that under stress misfolded proteins are handed off to Hsp70. Hence, we find that DNAJB8H31Q is still our most effective recognition element for the recovery of destabilized client proteins following cellular stress.
          Cover page: Factors affecting protein recovery during Hsp40 affinity profiling.
          • Article
          • Peer Reviewed
          UC Riverside Previously Published Works (2020)
          The quantitative multiplexing capacity of isobaric tandem mass tags (TMT) has increased the throughput of affinity purification mass spectrometry (AP-MS) to characterize protein interaction networks of immunoprecipitated bait proteins. However, variable bait levels between replicates can convolute interactor identification. We compared the Student's t-test and Pearson's R correlation as methods to generate t-statistics and assessed the significance of interactors following TMT-AP-MS. Using a simple linear model of protein recovery in immunoprecipitates to simulate reporter ion ratio distributions, we found that correlation-derived t-statistics protect against bait variance while robustly controlling type I errors (false positives). We experimentally determined the performance of these two approaches for determining t-statistics under two experimental conditions: irreversible prey association to the Hsp40 mutant DNAJB8H31Q followed by stringent washing, and reversible association to 14-3-3ζ with gentle washing. Correlation-derived t-statistics performed at least as well as Student's t-statistics for each sample and with substantial improvement in performance for experiments with high bait-level variance. Deliberately varying bait levels over a large range fails to improve selectivity but does increase the robustness between runs. The use of correlation-derived t-statistics should improve identification of interactors using TMT-AP-MS. Data are available via ProteomeXchange with identifier PXD016613.
          • 1 supplemental PDF
          Cover page: Bait Correlation Improves Interactor Identification by Tandem Mass Tag-Affinity Purification-Mass Spectrometry
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