Import into the endoplasmic reticulum (ER) is a necessary process for the third of the eukaryotic proteome that participate in the secretory pathway. Proteins that take up residence in the ER or in a cellular membrane must successfully enter the Sec61 translocon in order to gain access to the ER lumenal complexes responsible for protein maturation.

Our studies identify HSPA13, a truncated ER-residing Hsp70 protein, as an inhibitor of ER import and protein processing. We demonstrate this effect by introducing a new peroxidase-based biotinylation assay for selectively labeling proteins on either side of the ER membrane. We utilize the secretory protein transthyretin (TTR) as a model substrate and show that TTR is inefficiently translocated. Furthermore, we discover that TTR that mistargets to the cytosol experiences slow degradation. We utilize ultracentrifugation to demonstrate a loss of protein solubility that is dependent on HSPA13.

Additionally, we explore HSPA13 function by identifying interactors using affinity purification followed by liquid chromatography coupled with tandem mass spectrometry. Among the discovered interactors were members of protein complexes that are involved with signal sequence cleavage and glycosylation of nascent protein entering the ER. We discover that mutations targeting HSPA13 ATPase activity increase HSPA13 regulatory functions, namely inhibition of ER protein translocation, maturation and secretion. We also associate the regulatory activity of HSPA13 to cytotoxicity, which is enhanced with HSPA13 ATPase mutants.

HSPA13 is conserved in metazoans and presumably provides an unidentified role in cell homeostasis. We employed the CRISPR/Cas9 gene editing system to develop HSPA13 knockout cells which were found to have impaired proteostasis which was observed when they were challenged with ER calcium disruption. Cells lacking HSPA13 also mishandled TTR, which provided additional evidence of compromised protein regulation.

In summary, this dissertation describes the effects of HSPA13 that diminish ER protein translocation and maturation, perhaps through direct interaction with the machinery involved with both processes. Additionally, overexpression or depletion of HSPA13 destabilizes proteostasis and confers deleterious effects.

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.

Proteins in their native or optimal conformational states can perform essential cellularfunctions to assist in the stability of the proteome and the overall health of the cell. However, if a protein misfolds, it instead can gain a new toxic function. Misfolded proteins can aggregate into more stable toxic precursors that can cause cellular degradation and ultimately induce health disorders such as Parkinson’s Disease, Alzheimer’s Disease, and Huntington’s Disease. Cellular stresses are significant factors in protein misfolding. These stimuli can change protein structure through several mechanisms such as covalent modification of cysteines or oxidation of methionine residues. The immediate consequence of posttranslational modifications of a protein from stress is difficulty in refolding. The long term consequence is that these altered proteins can instead gain new toxic functions as they proceed to aggregate. Therefore, changes to protein structures induced by stresses can create cellular havoc as the cell scrambles to recover from the affected misfolded proteins. Several analytical assays can measure the effects of stress on protein stability. viii Flourescence assays show direct changes to a protein based on gain or loss of signal. Mass Spectrometry surveys entire proteomes by measuring a protein’s ability to react after exposure to a cellular stress. Herein, we describe a quantitative proteomics approach that measures proteins binding to Hsp40 chaperone DNAJB8, a protein quality control factor designed to recognize misfolded proteins. We aimed to use the chaperone to identify destabilized proteins and deduce toxic cellular mechanisms arising from environmental stresses. We found several ribosomal proteins such as TAR DNA-Binding protein (TDP-43) and Pyruvate Dehydrogenase E1 subunit (PDHA1) misfolded after arsenite exposure. Later, we found potential biomarkers in GAPDH and PARK7 involved in driving the cellular toxicity of propachlor. Led by limited proteolysis, we conducted several validation experiments to show that DNAJ8 recognized significantly destabilized proteins after exposure to environmental toxins. In total, we hope to show the effectiveness of this approach in exploring how environmental toxins can impact cellular proteostasis and identifying the resulting susceptible proteome.

Proteins play major roles and participate in multiple pathways in the cell. Thus, cells have to rely on proper folding and localization of the proteome to avoid dysfunction. One of the key steps in maturation for secretory proteins is translocation into the endoplasmic reticulum (ER) rather than mistargeting to and accumulating in the cytosol. However, under ER stress, ER translocation becomes less efficient leading to higher accumulation of proteins in the cytosol. This potentially poses a threat to the cytosol. In our study, we identified an ER chaperone HSPA13 as a regulatory factor of protein translocation into the ER. Overexpression of HSPA13 in HEK293T cells impairs ER and cytosolic proteostasis by inhibiting ER translocation of substrates, disrupting their secretory pathway and promoting their intracellular aggregation. Additionally, we found that mistargeted protein in the cytosol is triaged on the basis of its stability. Highly destabilized mistargeted proteins are rapidly processed and degraded by the proteasome, while more stable proteins are degraded less efficiently. Furthermore, in the case of transthyretin, the processing is mediated by selective N-terminal proteolysis. Thus, we have found that mistargeted ER-directed proteins are subject to quality control in the cytosol.

Influenza infection requires viral escape from early endosomes into the cytosol, which is enabled by an acid-induced irreversible conformational transformation in the viral protein hemagglutinin. Despite the direct relationship between this conformational change and infectivity, label-free methods for characterizing this and other protein conformational changes in biological mixtures are limited. While the chemical reactivity of the protein backbone and side-chain residues is a proxy for protein conformation, coupling this reactivity to quantitative mass spectrometry is a challenge in complex environments. Herein, we developed electrophilic amidination coupled with reaction monitoring to detect the fusion-associated conformational transformation in recombinant hemagglutinin (rHA). We identified rHA peptides that are differentially amidinated between the pre- and post-fusion states, and validated that this difference relies upon the fusion-associated conformational switch. We further demonstrate that we can distinguish the fusion profile in a matrix of digested cellular lysate.

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.