Biology subjects

Genereux, J. C.

Publications and source records attributed to Genereux, J. C..

5 recordsLinked to original sources

bioRxiv2022

Hspa13 Regulates Endoplasmic Reticulum and Cytosolic Proteostasis Through Modulation of Protein Translocation

Most eukaryotic secretory proteins are co-translationally 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 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. We find that Hspa13 interacts primarily with the Sec61 translocon and its associated factors. Hspa13 overexpression inhibits translocation of the secreted protein transthyretin (TTR), leading to accumulation and aggregation of immature TTR 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, 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 manuscript has been deposited in the PRIDE archive and can be accessed at PXD033498.

cell biology↗

bioRxiv2022

ATF6 Activation Reduces Amyloidogenic Transthyretin Secretion Through Increased Interactions with Endoplasmic Reticulum Proteostasis Factors

The extracellular aggregation of destabilized transthyretin (TTR) variants is implicated in the onset and pathogenesis of familial TTR-related amyloid diseases. One strategy to reduce toxic, extracellular aggregation of TTR is to decrease the population of aggregation-prone protein secreted from mammalian cells. Stress-independent activation of the unfolded protein response (UPR)-associated transcription factor ATF6 preferentially decreases secretion and subsequent aggregation of destabilized, aggregation-prone TTR variants. However, the mechanism of this reduced secretion was previously undefined. Here, we implement a mass spectrometry-based interactomics approach to identify endoplasmic reticulum (ER) proteostasis factors involved in ATF6-dependent reductions of destabilized TTR secretion. We show that ATF6 activation reduces amyloidogenic TTR secretion and subsequent aggregation through a mechanism involving ER retention that is mediated by increased interactions with ATF6-regulated ER proteostasis factors including BiP and PDIA4. Intriguingly, the PDIA4-dependent retention of TTR is independent of both the single TTR cysteine residue and the redox activity of PDIA4, indicating that PDIA4 retains destabilized TTR in the ER through a redox-independent chaperoning activity. Our results define a mechanistic basis to explain the ATF6 activation-dependent reduction of destabilized, amyloidogenic TTR secretion that could be therapeutically accessed to improve treatments of TTR-related amyloid diseases.

cell biology↗

bioRxiv2022

Evaluating Client Protein Recovery by the Hsp40s DNAJB8 and DNAJB1 with AP-MS

Hsp40s, also termed J-domain proteins, play a central role in cellular protein homeostasis by promiscuously surveying the proteome for misfolded proteins. We have exploited this property to develop Hsp40 affinity profiling as a method for identifying proteins that misfold in response to cellular stresses. In this assay, we use the Hsp40 FlagDNAJB8H31Q as our recognition element for misfolded proteins. This protein is exogenously introduced into cells, promoting interactions without regard for native protein clients. Herein, we evaluate potential approaches to improve the performance of this 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 DNJAB8 under basal conditions. By contrast, crosslinking and J-domain ablation both substantially increase relative protein interactor recovery with the structurally distinct Class B Hps40 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. Raw data is accessible through the PRIDE Archive at PXD030633.

biochemistry↗

bioRxiv2021

Monitoring Protein Import into the Endoplasmic Reticulum in Living Cells with Proximity Labeling

The proper trafficking of eukaryotic proteins is essential to cellular function. 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. Herein, we use genetically encoded peroxidases to characterize protein import into the endoplasmic reticulum (ER). We show that the ERHRP/cytAPEX pair provides good selectivity and sensitivity for identifying protein mistargeting, using the known ER pQC substrate transthyretin (TTR). Although ERHRP labeling induces formation of detergent-resistant TTR aggregates, this is minimized by using low ERHRP expression, without loss of labeling efficiency. cytAPEX labeling recovers TTR that is mistargeted as a consequence of Sec61 inhibition or ER stress-induced ER pQC. Furthermore, we demonstrate that stress-free activation of the ER stress-associated transcription factor ATF6 recapitulates the TTR import deficiency of ER pQC. Hence, proximity labeling is an effective strategy for characterizing factors that influence ER protein import in living cells.

molecular biology↗

bioRxiv2021

Hsp40 Affinity to Identify Proteins Destabilized by Cellular Toxicant Exposure

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 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.

biochemistry↗