Elevated constitutive expression of Hsp40 chaperone Sis1 reduces TDP-43 aggregation-induced oxidative stress in Ire1 pathway dependent-manner in yeast TDP-43 proteinopathy model of amyotrophic lateral sclerosis.

Oxidative stress is a therapeutic target in TDP-43 proteinopathies like amyotrophic lateral sclerosis (ALS) and FTLD-TDP. TDP-43 over-expression causes oxidative stress in yeast model of ALS. Previously, we developed a red/white color conversion reporter assay using ade1 or ade2 mutant yeast to examine oxidative stress induced by expression of amyloidogenic proteins. Also, a previous study showed that overexpression of yeast Hsp40 chaperone Sis1 could mitigate the toxicity and proteosomal blockage induced by TDP-43 over-expression. Here, using the red/white reporter yeast assay and also by CellROX-staining, we found that an elevated expression of Sis1 mitigates the TDP-43-induced oxidative stress. Furthermore, as redox signalling and the ER stress response pathways cross-talk, we checked if the Sis1-mediated mitigation of the TDP-43-induced oxidative stress can also be observed in yeast deleted for ER stress response gene, IRE1. We find that in the yeast deleted for the IRE1 gene, the elevated expression of Sis1 fails to neutralize the TDP-43-induced oxidative stress. Taken together, Hsp40 chaperone modulation can be further examined towards therapeutic research on the TDP-43 proteinopathies.

Quantitative assessment of chaperone binding to amyloid aggregates identifies specificity of Hsp40 interaction with yeast prion fibrils.

Yeast self-perpetuating protein aggregates (yeast prions) provide a framework to investigate the interaction of misfolded proteins with the protein quality control machinery. The major component of this system that facilitates propagation of all known yeast amyloid prions is the Hsp104 chaperone that catalyzes fibril fragmentation. Overproduction of Hsp104 cures some yeast prions via a fragmentation-independent mechanism. Importantly, major cytosolic chaperones of the Hsp40 group, Sis1 and Ydj1, oppositely affect yeast prion propagation, and are capable of stimulating different activities of Hsp104. In this work, we developed a quantitative method to investigate the Hsp40 binding to amyloid aggregates. We demonstrate that Sis1 binds fibrils formed by the Sup35NM protein with higher affinity compared to Ydj1. Moreover, the interaction of Sis1 with the fibrils formed by the other yeast prion protein, Rnq1, is orders of magnitude weaker. We show that the deletion of the dimerization domain of Sis1 (crucial for the curing of [PSI+] by excess Hsp104) decreases its affinity to both Sup35NM and Rnq1 fibrils. Taken together, these results suggest that tight binding of Hsp40 to the amyloid fibrils is likely to enhance aggregate malpartition instead of fibril fragmentation.

Differential effects of Ydj1 and Sis1 on Hsp70-mediated clearance of stress granules in Saccharomyces cerevisiae.

Stress granules and P-bodies are conserved assemblies of nontranslating mRNAs in eukaryotic cells that can be related to RNA-protein aggregates found in some neurodegenerative diseases. Herein, we examine how the Hsp70/Hsp40 protein chaperones affected the assembly and disassembly of stress granules and P-bodies in yeast. We observed that Hsp70 and the Ydj1 and Sis1 Hsp40 proteins accumulated in stress granules and defects in these proteins led to decreases in the disassembly and/or clearance of stress granules. We observed that individual Hsp40 proteins have different effects on stress granules with defects in Ydj1 leading to accumulation of stress granules in the vacuole and limited recovery of translation following stress, which suggests that Ydj1 promotes disassembly of stress granules to promote translation. In contrast, defects in Sis1 did not affect recovery of translation, accumulated cytoplasmic stress granules, and showed reductions in the targeting of stress granules to the vacuole. This demonstrates a new principle whereby alternative disassembly machineries lead to different fates of components within stress granules, thereby providing additional avenues for regulation of their assembly, composition, and function. Moreover, a role for Hsp70 and Hsp40 proteins in stress granule disassembly couples the assembly of these stress responsive structures to the proteostatic state of the cell.

Normal levels of the antiprion proteins Btn2 and Cur1 cure most newly formed [URE3] prion variants.

[URE3] is an amyloid prion of the Saccharomyces cerevisiae Ure2p, a regulator of nitrogen catabolism. Overproduction of Btn2p, involved in late endosome to Golgi protein transport, or its paralog Cur1p, cures [URE3]. Btn2p, in curing, is colocalized with Ure2p in a single locus, suggesting sequestration of Ure2p amyloid filaments. We find that most [URE3] variants generated in a btn2 cur1 double mutant are cured by restoring normal levels of Btn2p and Cur1p, with both proteins needed for efficient curing. The [URE3] variants cured by normal levels of Btn2p and Cur1p all have low seed number, again suggesting a seed sequestration mechanism. Hsp42 overproduction also cures [URE3], and Hsp42p aids Btn2 overproduction curing. Cur1p is needed for Hsp42 overproduction curing of [URE3], but neither Btn2p nor Cur1p is needed for overproduction curing by the other. Although hsp42Δ strains stably propagate [URE3-1], hsp26Δ destabilizes this prion. Thus, Btn2p and Cur1p are antiprion system components at their normal levels, acting with Hsp42. Btn2p is related in sequence to human Hook proteins, involved in aggresome formation and other transport activities.

Backbone and sidechain NMR assignments of residues 1-81 from yeast Sis1 in complex with an Hsp70 C-terminal EEVD peptide.

Molecular chaperones aid proteins to fold and assemble without modifying their final structure, requiring, in several folding processes, the interplay between members of the Hsp70 and Hsp40 families. Here, we report the NMR chemical shift assignments for 1 H, 15 N, and 13 C nuclei of the backbone and side chains of the J-domain of the class B Hsp40 from Saccharomyces cerevisiae, Sis1, complexed with the C-terminal EEVD motif of Hsp70. The data revealed information on the structure and backbone dynamics that add significantly to the understanding of the J-domain-Hsp70-EEVD mechanism of interaction.

Transcriptional regulation of Sis1 promotes fitness but not feedback in the heat shock response.

The heat shock response (HSR) controls expression of molecular chaperones to maintain protein homeostasis. Previously, we proposed a feedback loop model of the HSR in which heat-denatured proteins sequester the chaperone Hsp70 to activate the HSR, and subsequent induction of Hsp70 deactivates the HSR (Krakowiak et al., 2018; Zheng et al., 2016). However, recent work has implicated newly synthesized proteins (NSPs) - rather than unfolded mature proteins - and the Hsp70 co-chaperone Sis1 in HSR regulation, yet their contributions to HSR dynamics have not been determined. Here, we generate a new mathematical model that incorporates NSPs and Sis1 into the HSR activation mechanism, and we perform genetic decoupling and pulse-labeling experiments to demonstrate that Sis1 induction is dispensable for HSR deactivation. Rather than providing negative feedback to the HSR, transcriptional regulation of Sis1 by Hsf1 promotes fitness by coordinating stress granules and carbon metabolism. These results support an overall model in which NSPs signal the HSR by sequestering Sis1 and Hsp70, while induction of Hsp70 - but not Sis1 - attenuates the response.

Calmodulin regulates protease versus co-chaperone activity of a metacaspase.

Metacaspases are ancestral homologs of caspases that can either promote cell death or confer cytoprotection. Furthermore, yeast (Saccharomyces cerevisiae) metacaspase Mca1 possesses dual biochemical activity: proteolytic activity causing cell death and cytoprotective, co-chaperone-like activity retarding replicative aging. The molecular mechanism favoring one activity of Mca1 over another remains elusive. Here, we show that this mechanism involves calmodulin binding to the N-terminal pro-domain of Mca1, which prevents its proteolytic activation and promotes co-chaperone-like activity, thus switching from pro-cell death to anti-aging function. The longevity-promoting effect of Mca1 requires the Hsp40 co-chaperone Sis1, which is necessary for Mca1 recruitment to protein aggregates and their clearance. In contrast, proteolytically active Mca1 cleaves Sis1 both in vitro and in vivo, further clarifying molecular mechanism behind a dual role of Mca1 as a cell-death protease versus gerontogene.

Differential Interactions of Molecular Chaperones and Yeast Prions.

Baker's yeast Saccharomyces cerevisiae is an important model organism that is applied to study various aspects of eukaryotic cell biology. Prions in yeast are self-perpetuating heritable protein aggregates that can be leveraged to study the interaction between the protein quality control (PQC) machinery and misfolded proteins. More than ten prions have been identified in yeast, of which the most studied ones include [PSI+], [URE3], and [PIN+]. While all of the major molecular chaperones have been implicated in propagation of yeast prions, many of these chaperones differentially impact propagation of different prions and/or prion variants. In this review, we summarize the current understanding of the life cycle of yeast prions and systematically review the effects of different chaperone proteins on their propagation. Our analysis clearly shows that Hsp40 proteins play a central role in prion propagation by determining the fate of prion seeds and other amyloids. Moreover, direct prion-chaperone interaction seems to be critically important for proper recruitment of all PQC components to the aggregate. Recent results also suggest that the cell asymmetry apparatus, cytoskeleton, and cell signaling all contribute to the complex network of prion interaction with the yeast cell.

Hsp40/JDP Requirements for the Propagation of Synthetic Yeast Prions.

Yeast prions are protein-based transmissible elements, most of which are amyloids. The chaperone protein network in yeast is inexorably linked to the spreading of prions during cell division by fragmentation of amyloid prion aggregates. Specifically, the core "prion fragmentation machinery" includes the proteins Hsp104, Hsp70 and the Hsp40/J-domain protein (JDP) Sis1. Numerous novel amyloid-forming proteins have been created and examined in the yeast system and occasionally these amyloids are also capable of continuous Hsp104-dependent propagation in cell populations, forming synthetic prions. However, additional chaperone requirements, if any, have not been determined. Here, we report the first instances of a JDP-Hsp70 system requirement for the propagation of synthetic prions. We utilized constructs from a system of engineered prions with prion-forming domains (PrDs) consisting of a polyQ stretch interrupted by a single heterologous amino acid interspersed every fifth residue. These "polyQX" PrDs are fused to the MC domains of Sup35, creating chimeric proteins of which a subset forms synthetic prions in yeast. For four of these prions, we show that SIS1 repression causes prion loss in a manner consistent with Sis1's known role in prion fragmentation. PolyQX prions were sensitive to Sis1 expression levels to differing degrees, congruent with the variability observed among native prions. Our results expand the scope known Sis1 functionality, demonstrating that Sis1 acts on amyloids broadly, rather than through specific protein-protein interactions with individual yeast prion-forming proteins.

Yeast J-protein Sis1 prevents prion toxicity by moderating depletion of prion protein.

[PSI+] is a prion of Saccharomyces cerevisiae Sup35, an essential ribosome release factor. In [PSI+] cells, most Sup35 is sequestered into insoluble amyloid aggregates. Despite this depletion, [PSI+] prions typically affect viability only modestly, so [PSI+] must balance sequestering Sup35 into prions with keeping enough Sup35 functional for normal growth. Sis1 is an essential J-protein regulator of Hsp70 required for the propagation of amyloid-based yeast prions. C-terminally truncated Sis1 (Sis1JGF) supports cell growth in place of wild-type Sis1. Sis1JGF also supports [PSI+] propagation, yet [PSI+] is highly toxic to cells expressing only Sis1JGF. We searched extensively for factors that mitigate the toxicity and identified only Sis1, suggesting Sis1 is uniquely needed to protect from [PSI+] toxicity. We find the C-terminal substrate-binding domain of Sis1 has a critical and transferable activity needed for the protection. In [PSI+] cells that express Sis1JGF in place of Sis1, Sup35 was less soluble and formed visibly larger prion aggregates. Exogenous expression of a truncated Sup35 that cannot incorporate into prions relieved [PSI+] toxicity. Together our data suggest that Sis1 has separable roles in propagating Sup35 prions and in moderating Sup35 aggregation that are crucial to the balance needed for the propagation of what otherwise would be lethal [PSI+] prions.

Impact of Amyloid Polymorphism on Prion-Chaperone Interactions in Yeast.

Yeast prions are protein-based genetic elements found in the baker's yeast Saccharomyces cerevisiae, most of which are amyloid aggregates that propagate by fragmentation and spreading of small, self-templating pieces called propagons. Fragmentation is carried out by molecular chaperones, specifically Hsp104, Hsp70, and Hsp40. Like other amyloid-forming proteins, amyloid-based yeast prions exhibit structural polymorphisms, termed "strains" in mammalian systems and "variants" in yeast, which demonstrate diverse phenotypes and chaperone requirements for propagation. Here, the known differential interactions between chaperone proteins and yeast prion variants are reviewed, specifically those of the yeast prions [PSI+], [RNQ+]/[PIN+], and [URE3]. For these prions, differences in variant-chaperone interactions (where known) with Hsp104, Hsp70s, Hsp40s, Sse1, and Hsp90 are summarized, as well as some interactions with chaperones of other species expressed in yeast. As amyloid structural differences greatly impact chaperone interactions, understanding and accounting for these variations may be crucial to the study of chaperones and both prion and non-prion amyloids.

Overexpression of a conserved HSP40 chaperone reduces toxicity of several neurodegenerative disease proteins.

TDP-43 and FUS are DNA/RNA binding proteins associated with neuronal inclusions in amyotrophic lateral sclerosis (ALS) patients. Other neurodegenerative diseases are also characterized by neuronal protein aggregates, e.g. Huntington's disease, associated with polyglutamine (polyQ) expansions in the protein huntingtin. Here we discuss our recent paper establishing similarities between aggregates of TDP-43 that have short glutamine and asparagine (Q/N)-rich modules and are soluble in detergents, with those of polyQ and PIN4C that have large Q/N-rich domains and are detergent-insoluble. We also present new, similar data for FUS. Together, we show that like overexpression of polyQ or PIN4C, overexpression of FUS or TDP-43 causes inhibition of the ubiquitin proteasome system (UPS) and toxicity, both of which are mitigated by overexpression of the Hsp40 chaperone Sis1. Also, in all cases toxicity is enhanced by the [PIN+] prion. In addition, we show that the Sis1 mammalian homolog DNAJBI reduces toxicity arising from overexpressed FUS and TDP-43 respectively in human embryonic kidney cells and primary rodent neurons. The common properties of these proteins suggest that heterologous aggregates may enhance the toxicity of a variety of disease-related aggregating proteins, and further that chaperones and the UPS may be key therapeutic targets for diseases characterized by protein inclusions.

The story of stolen chaperones: how overexpression of Q/N proteins cures yeast prions.

Prions are self-seeding alternate protein conformations. Most yeast prions contain glutamine/asparagine (Q/N)-rich domains that promote the formation of amyloid-like prion aggregates. Chaperones, including Hsp104 and Sis1, are required to continually break these aggregates into smaller "seeds." Decreasing aggregate size and increasing the number of growing aggregate ends facilitates both aggregate transmission and growth. Our previous work showed that overexpression of 11 proteins with Q/N-rich domains facilitates the de novo aggregation of Sup35 into the [PSI(+)] prion, presumably by a cross-seeding mechanism. We now discuss our recent paper, in which we showed that overexpression of most of these same 11 Q/N-rich proteins, including Pin4C and Cyc8, destabilized pre-existing Q/N rich prions. Overexpression of both Pin4C and Cyc8 caused [PSI(+)] aggregates to enlarge. This is incompatible with a previously proposed "capping" model where the overexpressed Q/N-rich protein poisons, or "caps," the growing aggregate ends. Rather the data match what is expected of a reduction in prion severing by chaperones. Indeed, while Pin4C overexpression does not alter chaperone levels, Pin4C aggregates sequester chaperones away from the prion aggregates. Cyc8 overexpression cures [PSI(+)] by inducing an increase in Hsp104 levels, as excess Hsp104 binds to [PSI(+)] aggregates in a way that blocks their shearing.