By binding to a multitude of polypeptide substrates, Hsp70-based molecular chaperone systems perform a range of cellular functions. All J-protein co-chaperones play the essential role, via action of their J-domains, of stimulating the ATPase activity of Hsp70, thereby stabilizing its interaction with substrate. In addition, J-proteins drive the functional diversity of Hsp70 chaperone systems through action of regions outside their J-domains. Targeting to specific locations within a cellular compartment and binding of specific substrates for delivery to Hsp70 have been identified as modes of J-protein specialization. To better understand J-protein specialization, we concentrated on Saccharomyces cerevisiae SIS1, which encodes an essential J-protein of the cytosol/nucleus. We selected suppressors that allowed cells lacking SIS1 to form colonies. Substitutions changing single residues in Ydj1, a J-protein, which, like Sis1, partners with Hsp70 Ssa1, were isolated. These gain-of-function substitutions were located at the end of the J-domain, suggesting that suppression was connected to interaction with its partner Hsp70, rather than substrate binding or subcellular localization. Reasoning that, if YDJ1 suppressors affect Ssa1 function, substitutions in Hsp70 itself might also be able to overcome the cellular requirement for Sis1, we carried out a selection for SSA1 suppressor mutations. Suppressing substitutions were isolated that altered sites in Ssa1 affecting the cycle of substrate interaction. Together, our results point to a third, additional means by which J-proteins can drive Hsp70’s ability to function in a wide range of cellular processes—modulating the Hsp70-substrate interaction cycle.

SummaryThe amyloid‐based prions of Saccharomyces cerevisiae are heritable aggregates of misfolded proteins, passed to daughter cells following fragmentation by molecular chaperones including the J‐protein Sis1, Hsp70 and Hsp104. Overexpression of Hsp104 efficiently cures cell populations of the prion [PSI +] by an alternative Sis1‐dependent mechanism that is currently the subject of significant debate. Here, we broadly investigate the role of J‐proteins in this process by determining the impact of amyloid polymorphisms (prion variants) on the ability of well‐studied Sis1 constructs to compensate for Sis1 and ask whether any other S. cerevisiae cytosolic J‐proteins are also required for this process. Our comprehensive screen, examining all 13 members of the yeast cytosolic/nuclear J‐protein complement, uncovered significant variant‐dependent genetic evidence for a role of Apj1 (antiprion DnaJ) in this process. For strong, but not weak [PSI +] variants, depletion of Apj1 inhibits Hsp104‐mediated curing. Overexpression of either Apj1 or Sis1 enhances curing, while overexpression of Ydj1 completely blocks it. We also demonstrated that Sis1 was the only J‐protein necessary for the propagation of at least two weak [PSI +] variants and no J‐protein alteration, or even combination of alterations, affected the curing of weak [PSI +] variants, suggesting the possibility of biochemically distinct, variant‐specific Hsp104‐mediated curing mechanisms.

Rapid thermal annealing of electron irradiated nanoscale type Ib diamond particles facilitates formation of various nitrogen-related fluorescent color centers, providing either red, yellow, green, or blue fluorescence for downstream multiplex imaging applications.

Prions are self-propagating protein isoforms that are typically amyloid. In Saccharomyces cerevisiae, amyloid prion aggregates are fragmented by a trio involving three classes of chaperone proteins: Hsp40s, also known as J-proteins, Hsp70s, and Hsp104. Hsp104, the sole Hsp100-class disaggregase in yeast, along with the Hsp70 Ssa and the J-protein Sis1, is required for the propagation of all known amyloid yeast prions. However, when Hsp104 is ectopically overexpressed, only the prion [PSI + ] is efficiently eliminated from cell populations via a highly debated mechanism that also requires Sis1. Recently, we reported roles for two additional J-proteins, Apj1 and Ydj1, in this process. Deletion of Apj1, a J-protein involved in the degradation of sumoylated proteins, partially blocks Hsp104-mediated [PSI + ] elimination. Apj1 and Sis1 were found to have overlapping functions, as overexpression of one compensates for loss of function of the other. In addition, overexpression of Ydj1, the most abundant J-protein in the yeast cytosol, completely blocks Hsp104-mediated curing. Yeast prions exhibit structural polymorphisms known as "variants"; most intriguingly, these J-protein effects were only observed for strong variants, suggesting variant-specific mechanisms. Here, we review these results and present new data resolving the domains of Apj1 responsible, specifically implicating the involvement of Apj1's Q/S-rich low-complexity domain.

Chromosomal instability (CIN) is a cancer hallmark associated with cancer metastasis and immune evasion. Yet, it is unclear how CIN modulates the tumor-microenvironment (TME). Here we show that CIN results in a protumor TME with enrichment of immune-suppressive macrophages, a granulocytic infiltrate, and exhausted T cells. Using ContactTracing, a newly developed computational tool to infer conditionally dependent cell-cell interactions from single cell RNA sequence data, we identify tumor ligands induced by the ER stress response in cancer cells as central mediators of immune suppression. Mechanistically, CIN-dependent chronic activation of the cytosolic DNA sensing cGAS-STING pathway promotes ER-stress-dependent transcription. Suppression of CIN or depletion of cancer cell STING reduces ER-stress and restores CIN-induced changes on the TME. Correspondingly, chronic STING activation in human breast cancer patients is associated with reduced tumor infiltrating lymphocytes and increased metastasis. Remarkably, pharmacologic inhibition of chronically active STING or depletion of downstream ER stress signaling suppresses metastasis in syngeneic models of melanoma, breast, and colorectal cancers, thereby demonstrating a viable therapeutic strategy for chromosomally unstable cancers. Citation Format: Jun Li, Melissa Hubisz, Ethan Earlie, Mercedes A. Duran, Emanuele Lettera, Su M. Phyu, Amit D. Amin, Matthew Deyell, Erina Kamiya, Karolina Budre, Julie-Ann Cavallo, Christopher Garris, Hannah Wen, Benjamin Izar, Eileen Parkes, Ashley Laughney, Samuel Bakhoum. Chromosomal instability shapes the tumor microenvironment through a chronic ER-stress response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3822.

Hsp70s are a versatile class of molecular chaperones, essential for a variety of cellular functions ranging from protein folding and transport to iron‐sulfur cluster biogenesis. Hsp70s function with obligate Hsp40 co‐chaperones, also called J‐proteins, which stimulate Hsp70 ATPase activity via their characteristic J‐domains. In Saccharomyces cerevisiae, the J‐protein Sis1 is essential for cell viability and the propagation of prions. However, the means by which Sis1 drives specific functions of Hsp70 are largely unknown. We recently reported the discovery of gain‐of‐function substitutions in both the J‐protein Ydj1 and the Hsp70 Ssa1 which allow cell survival without Sis1. Here we present additional data that support the ability of the suppressor mutants to substitute for Sis1‐dependent roles, specifically the maintenance of yeast prions. Specifically, we tested the ability of the two most robust suppressor mutations in both Ydj1 and Ssa1 to maintain two prions, [PSI‐+] and [RNQ+], and included both phenotypically strong and weak variants of [PSI‐+] in our analysis. Interestingly, we found similar results for all mutants: both Ydj1 mutants could substitute for the propagation of strong [PSI‐+], but not weak [PSI‐+] nor [RNQ+]. Similarly, we found that the Ssa1 mutants could not support the maintenance of weak [PSI‐+] nor [RNQ+]. Unfortunately, due to the severe [PSI‐+] toxicity in the compromised strain bearing strong [PSI‐+], we could not confidently determine the ability of the Ssa1 mutants to support strong [PSI‐+] propagation. Further investigation of additional Ydj1 and Ssa1 mutants may provide greater resolution regarding the specific functions required of J‐proteins by distinct prions for propagation.Support or Funding InformationThis work was supported by National Institutes of Health (https://www.nih.gov/) grants GM31107 and GM27870 (EAC) and R15GM110606 (JKH). This work was also supported by the Lafayette College Chemistry Department, the EXCEL research scholarship program, and the John F and Dorothy M Dorflinger Summer Research Endowment Fund (JKH).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The amyloid‐based prions of Saccharomyces cerevisiae are heritable aggregates of misfolded protein, passed to daughter cells following fragmentation by a set of molecular chaperones which includes the J‐protein Sis1, Hsp70, and Hsp104. Overexpression of Hsp104 efficiently cures the prion [PSI+], a phenomenon which has promoted the exploration of Hsp104 as a potential therapeutic agent for neurodegenerative diseases. However, the mechanism of [PSI+] elimination by Hsp104 overexpression has been the subject of significant debate for the past two decades and has garnered significant interest in the recent literature as multiple conflicting models have been proposed. Yeast prion propagation is inexorably reliant on the function of molecular chaperones of the Hsp100, Hsp70, and Hsp40 classes. Specifically, four Hsp40s (also called J‐proteins) have been implicated in various aspects of yeast prion biology: Sis1, Ydj1, Apj1, and Swa2. We found that overexpression of Sis1 or Apj1 accelerates strong [PSI+] elimination by Hsp104 overexpression, yet Ydj1 overexpression has a profound and opposing effect, completely blocking Hsp104‐mediated curing, indicating that Apj1 and Sis1 likely have similar and partially overlapping roles in this process. Interestingly results for weak variants of [PSI+] indicated potentially no role for J‐proteins in curing as no J‐protein alteration whatsoever affected the ability of Hsp104 to cure these variants. Additional experiments to determine the specific J‐protein domains responsible for various effects, as well as J‐protein requirements in cell backgrounds harboring both [PSI+] and [RNQ+] are underway. Overall our data support the hypothesis that Hsp104‐mediated curing may occur by biochemically distinct, variant‐specific mechanisms, only some of which involve J‐proteins.Support or Funding InformationThis work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM110606. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.