In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells.

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells

Human Hsp70 Disaggregase reverses Parkinson’s-linked α-Synuclein Amyloid Fibrils

Intracellular amyloid fibrils linked to neurodegenerative disease typically accumulate in an age-related manner, suggesting inherent cellular capacity for counteracting amyloid formation in early life. Metazoan molecular chaperones assist native folding and block polymerization of amyloidogenic proteins, preempting amyloid fibril formation. Chaperone capacity for amyloid disassembly, however, is unclear. Here, we show that a specific combination of human Hsp70 disaggregase-associated chaperone components efficiently disassembles α-synuclein amyloid fibrils characteristic of Parkinson’s disease in vitro. Specifically, the Hsc70 chaperone, the class B J-protein DNAJB1, and an Hsp110 family nucleotide exchange factor (NEF) provide ATP-dependent activity that disassembles amyloids within minutes via combined fibril fragmentation and depolymerization. This ultimately generates non-toxic α-synuclein monomers. Concerted, rapid interaction cycles of all three chaperone components with fibrils generate the power stroke required for disassembly. This identifies a powerful human Hsp70 disaggregase activity that efficiently disassembles amyloid fibrils and points to crucial yet undefined biology underlying amyloid-based diseases

Second Virtual International Symposium on Cellular and Organismal Stress Responses, September 8–9, 2022 [Meeting Review]

The Second International Symposium on Cellular and Organismal Stress Responses took place virtually on September 8–9, 2022. This meeting was supported by the Cell Stress Society International (CSSI) and organized by Patricija Van Oosten-Hawle and Andrew Truman (University of North Carolina at Charlotte, USA) and Mehdi Mollapour (SUNY Upstate Medical University, USA). The goal of this symposium was to continue the theme from the initial meeting in 2020 by providing a platform for established researchers, new investigators, postdoctoral fellows, and students to present and exchange ideas on various topics on cellular stress and chaperones. We will summarize the highlights of the meeting here and recognize those that received recognition from the CSSI

Hidden information on protein function in censuses of proteome foldedness.

Methods that assay protein foldedness with proteomics have generated censuses of apparent protein folding stabilities in biological milieu. However, different censuses poorly correlate with each other. Here, we show that the reason for this is that methods targeting foldedness through monitoring amino acid sidechain reactivity also detect changes in conformation and ligand binding, which can be a substantial fraction of the data. We show that the reactivity of only one quarter of cysteine or methionine sidechains in proteins in a urea denaturation curve of mammalian cell lysate can be confidently explained by a two-state unfolding isotherm. Contrary to that expected from unfolding, up to one third of the cysteines decreased reactivity. These cysteines were enriched in proteins with functions relating to unfolded protein stress. One protein, chaperone HSPA8, displayed changes arising from ligand and cofactor binding. Unmasking this hidden information using the approaches outlined here should improve efforts to understand both folding and the remodeling of protein function directly in complex biological settings

Ydj1 Protects Nascent Protein Kinases from Degradation and Controls the Rate of Their Maturation▿

Ydj1 is a Saccharomyces cerevisiae Hsp40 molecular chaperone that functions with Hsp70 to promote polypeptide folding. We identified Ydj1 as being important for maintaining steady-state levels of protein kinases after screening several chaperones and cochaperones in gene deletion mutant strains. Pulse-chase analyses revealed that a portion of Tpk2 kinase was degraded shortly after synthesis in a ydj1Δ mutant, while the remainder was capable of maturing but with reduced kinetics compared to the wild type. Cdc28 maturation was also delayed in the ydj1Δ mutant strain. Ydj1 protects nascent kinases in different contexts, such as when Hsp90 is inhibited with geldanamycin or when CDC37 is mutated. The protective function of Ydj1 is due partly to its intrinsic chaperone function, but this is minor compared to the protective effect resulting from its interaction with Hsp70. SIS1, a type II Hsp40, was unable to suppress defects in kinase accumulation in the ydj1Δ mutant, suggesting some specificity in Ydj1 chaperone action. However, analysis of chimeric proteins that contained the chaperone modules of Ydj1 or Sis1 indicated that Ydj1 promotes kinase accumulation independently of its client-binding specificity. Our results suggest that Ydj1 can both protect nascent chains against degradation and control the rate of kinase maturation

Monitoring Protein Misfolding by Site-Specific Labeling of Proteins <i>In Vivo</i>

<div><p>Incorporating fluorescent amino acids by suppression of the TAG amber codon is a useful tool for site-specific labeling of proteins and visualizing their localization in living cells. Here we use a plasmid encoded orthogonal tRNA/aminoacyl-tRNA synthetase pair to site-specifically label firefly luciferase with the environmentally sensitive fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2- aminopropanoic acid (ANAP) and explore the detectability of conformational changes in labeled luciferase in the yeast cytoplasm. We find that ANAP labeling efficiency is greatly increased in [<i>PSI</i>⁺] cells and show that analysis of the ANAP fluorescence emission by confocal imaging allows for tracking the thermal unfolding and aggregation of luciferase <i>in vivo</i>. Furthermore we demonstrate that flow cytometry can be used to study conformational changes in luciferase and chaperone-mediated refolding in quantitative terms and at the level of single cells. This experimental setup for the first time allows for the direct analysis of the folding state of a protein in living cells and may serve as valuable new tool for examining mechanisms of protein folding, misfolding and aggregation.</p></div

Hsp110 Chaperones Control Client Fate Determination in the Hsp70–Hsp90 Chaperone System

The Hsp110 family of protein chaperones was known to promote maturation of Hsp90 client proteins. The yeast Hsp110 ortholog Sse1 is now shown to influence the decision to fold or degrade substrates of the Hsp70–Hsp90 chaperone system when maturation is compromised