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Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2023)

Nearly all proteins are synthesized in the cytosol. A third of this proteome must be trafficked elsewhere, such as to membranes, to subcellular compartments, or outside of the cell. Proper trafficking of nascent protein is necessary for protein folding, maturation, quality control and cellular and organismal health. Genetic, environmental, and other stresses can induce protein mistargeting, and in turn threaten cellular protein homeostasis. Current methods for measuring protein mistargeting are difficult to translate to living cells, and thus the role of cellular signaling networks in stress-dependent protein mistargeting processes, such as ER pre-emptive quality control (ER pQC), are difficult to parse. Proximity labelling has been a powerful approach for characterizing subcellular proteomes. Herein, we use genetically encoded heme peroxidases to characterize protein import into the endoplasmic reticulum (ER). Firstly, we show that the ERHRP/cytAPEX pair provides good selectivity and sensitivity for multiplexed protein labelling, and for identifying protein mistargeting, using a known ER pQC substrate transthyretin (TTR). cytAPEX labelling recovers mistargeted TTR due to translocon blockade or ER pQC. Furthermore, we discover that stress-free activation of the ER unfolded protein response (UPR) sensor ATF6 mistargets TTR. We also evaluate ER stressors tunicamycin, Brefeldin A and DTT in HEK293T cells. Only thapsigargin induces substantial TTR mistargeting, while the result of other stressors remains ambiguous due to limited sensitivity. To enable a more quantitative platform for ER import, we integrate parallel reaction monitoring mass spectrometry. With this approach, we revisit the relationship between ER stressors and ER pQC. Indeed, only ER calcium depletion by thapsigargin or cyclopiazonic acid mistargets the most TTR, suggesting UPR itself is not sufficient. We also evaluate different normalization factors for relative quantification - MS1 total ion current, cytAPEX, pyruvate carboxylase, actin, etc. We consider that labelling activity (e.g. cytAPEX) as the most appropriate standard, because labelling activity may be subjected to change due to drug treatment. The last session includes efforts to expand this assay for proteome-wide protein import measurement. Our initial attempts may be obscured due to prolonged drug treatments, their pleiotropic effects, and a cell type that does not secrete much protein. To overcome those limitations, we generate HepG2 stable cell lines with either heme peroxidase. Drug treatment is shortened to 2 hours, and translation maintained by integrated stress response inhibitor ISRIB.

    • 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 (2023)
      Herbicides in the widely used chloroacetanilide class harbor a potent electrophilic moiety, which can damage proteins through nucleophilic substitution. In general, damaged proteins are subject to misfolding. Accumulation of misfolded proteins compromises cellular integrity by disrupting cellular proteostasis networks, which can further destabilize the cellular proteome. While direct conjugation targets can be discovered through affinity-based protein profiling, there are few approaches to probe how cellular exposure to toxicants impacts the stability of the proteome. We apply a quantitative proteomics methodology to identify chloroacetanilide-destabilized proteins in HEK293T cells based on their binding to the H31Q mutant of the human Hsp40 chaperone DNAJB8. We find that a brief cellular exposure to the chloroacetanilides acetochlor, alachlor, and propachlor induces misfolding of dozens of cellular proteins. These herbicides feature distinct but overlapping profiles of protein destabilization, highly concentrated in proteins with reactive cysteine residues. Consistent with the recent literature from the pharmacology field, reactivity is driven by neither inherent nucleophilic nor electrophilic reactivity but is idiosyncratic. We discover that propachlor induces a general increase in protein aggregation and selectively targets GAPDH and PARK7, leading to a decrease in their cellular activities. Hsp40 affinity profiling identifies a majority of propachlor targets identified by competitive activity-based protein profiling (ABPP), but ABPP can only identify about 10% of protein targets identified by Hsp40 affinity profiling. GAPDH is primarily modified by the direct conjugation of propachlor at a catalytic cysteine residue, leading to global destabilization of the protein. The Hsp40 affinity strategy is an effective technique to profile cellular proteins that are destabilized by cellular toxin exposure. Raw proteomics data is available through the PRIDE Archive at PXD030635.
        Cover page: Cellular Exposure to Chloroacetanilide Herbicides Induces Distinct Protein Destabilization Profiles.
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