J-domain proteins (JDPs) are known to drive the functional specificity of Hsp70 chaperone machines. Here we report on the latest findings presented at the third international JDP workshop held in 2025 in Gdansk, Poland. Investigators from many different disciplines, including structural biology, genetics, chemical biology, translational research, computational sciences, and biophysics, took part in the meeting. This article includes short summaries of the seminars presented by many of the speakers, which provided exciting new insights into the chaperone-dependent and chaperone-independent functions of JDPs, some of which go beyond Hsp70-dependent functions. We also provide a revised classification of members of the (human) JDP family that emerged from open discussion at the meeting. This workshop continues to serve as the premier venue for discussions of JDP evolution, structure, function, and roles in health, aging, and disease.

Cells safeguard the functionality of the proteome using complex pathways of protein quality control. The centerpiece of this proteostasis network is a large set of molecular chaperones and proteases that impact the entire lifespan of proteins by controlling protein folding and degradation. Dysfunction of the proteostasis network is associated with many diseases and age-associated functional decline of neurons, including Alzheimer's and Parkinson's diseases, as well as several motor neuron diseases. The 2025 EMBO workshop "Protein quality control: from molecular mechanisms to aging and disease" gathered the large and interdisciplinary community of researchers that study protein quality control, from its fundamental molecular mechanisms via higher-order organization in organisms to its impact on and use in the medical field. Here we summarize the workshop and report research findings.

Cotranslational protein folding depends on general chaperones that engage highly diverse nascent chains at the ribosomes. Here we discover a dedicated ribosome-associated chaperone, Chp1, that rewires the cotranslational folding machinery to assist in the challenging biogenesis of abundantly expressed eukaryotic translation elongation factor 1A (eEF1A). Our results indicate that during eEF1A synthesis, Chp1 is recruited to the ribosome with the help of the nascent polypeptide-associated complex (NAC), where it safeguards eEF1A biogenesis. Aberrant eEF1A production in the absence of Chp1 triggers instant proteolysis, widespread protein aggregation, activation of Hsf1 stress transcription and compromises cellular fitness. The expression of pathogenic eEF1A2 variants linked to epileptic-dyskinetic encephalopathy is protected by Chp1. Thus, eEF1A is a difficult-to-fold protein that necessitates a biogenesis pathway starting with dedicated folding factor Chp1 at the ribosome to protect the eukaryotic cell from proteostasis collapse.

J-domain proteins (JDPs) are the largest family of chaperones in most organisms, but much of how they function within the network of other chaperones and protein quality control machineries is still an enigma. Here, we report on the latest findings related to JDP functions presented at a dedicated JDP workshop in Gdansk, Poland. The report does not include all (details) of what was shared and discussed at the meeting, because some of these original data have not yet been accepted for publication elsewhere or represented still preliminary observations at the time.

Lipid droplets (LDs) are fat storage organelles critical for energy and lipid metabolism. Upon nutrient exhaustion, cells consume LDs via gradual lipolysis or via lipophagy, the en bloc uptake of LDs into the vacuole. Here, we show that LDs dock to the vacuolar membrane via a contact site that is required for lipophagy in yeast. The LD-localized LDO proteins carry an intrinsically disordered region that directly binds vacuolar Vac8 to form vCLIP, the vacuolar-LD contact site. Nutrient limitation drives vCLIP formation, and its inactivation blocks lipophagy, resulting in impaired caloric restriction-induced longevity. We establish a functional link between lipophagy and microautophagy of the nucleus, both requiring Vac8 to form respective contact sites upon metabolic stress. In sum, we identify the tethering machinery of vCLIP and find that Vac8 provides a platform for multiple and competing contact sites associated with autophagy.

The resilience of cellular proteostasis declines with age, which drives protein aggregation and compromises viability. The nucleus has emerged as a key quality control compartment that handles misfolded proteins produced by the cytosolic protein biosynthesis system. Here, we find that age-associated metabolic cues target the yeast protein disaggregase Hsp104 to the nucleus to maintain a functional nuclear proteome during quiescence. The switch to respiratory metabolism and the accompanying decrease in translation rates direct cytosolic Hsp104 to the nucleus to interact with latent translation initiation factor eIF2 and to suppress protein aggregation. Hindering Hsp104 from entering the nucleus in quiescent cells results in delayed re-entry into the cell cycle due to compromised resumption of protein synthesis. In sum, we report that cytosolic-nuclear partitioning of the Hsp104 disaggregase is a critical mechanism to protect the latent protein synthesis machinery during quiescence in yeast, ensuring the rapid restart of translation once nutrients are replenished.

Heat shock factor 1 (HSF1) responds to stress to mount the heat shock response (HSR), a conserved transcriptional program that allows cells to maintain proteostasis by upregulating heat shock proteins (HSPs). The homeostatic stress regulation of HSF1 plays a key role in human physiology and health but its mechanism has remained difficult to pinpoint. Recent work in the budding yeast model has implicated stress-inducible chaperones of the HSP70 family as direct negative regulators of HSF1 activity. Here, we have investigated the latency control and activation of human HSF1 by HSP70 and misfolded proteins. Purified oligomeric HSF1-HSP70 (HSPA1A) complexes exhibited basal DNA binding activity that was inhibited by increasing the levels of HSP70 and, importantly, misfolded proteins reverted the inhibitory effect. Using site-specific UV photo-crosslinking, we monitored HSP70-HSF1 complexes in HEK293T cells. While HSF1 was bound by the substrate binding domain of HSP70 in unstressed cells, activation of HSF1 by heat shock as well as by inducing the misfolding of newly synthesized proteins resulted in release of HSF1 from the chaperone. Taken our results together, we conclude that latent HSF1 populate dynamic complexes with HSP70, which are sensitive to increased levels of misfolded proteins that compete for binding to the HSP70 substrate binding domain. Thus, human HSF1 is activated by various stress conditions that all titrate available HSP70.

Heat Shock Factor 1 (Hsf1) in yeast drives the basal transcription of key proteostasis factors and its activity is induced as part of the core heat shock response. Exploring Hsf1 specific functions has been challenging due to the essential nature of the HSF1 gene and the extensive overlap of target promoters with environmental stress response (ESR) transcription factors Msn2 and Msn4 (Msn2/4). In this study, we constructed a viable hsf1 increment strain by replacing the HSF1 open reading frame with genes that constitutively express Hsp40, Hsp70, and Hsp90 from Hsf1-independent promoters. Phenotypic analysis showed that the hsf1 increment strain grows slowly, is sensitive to heat as well as protein misfolding and accumulates protein aggregates. Transcriptome analysis revealed that the transcriptional response to protein misfolding induced by azetidine-2-carboxylic acid is fully dependent on Hsf1. In contrast, the hsf1 increment strain responded to heat shock through the ESR. Following HS, Hsf1 and Msn2/4 showed functional compensatory induction with stronger activation of the remaining stress pathway when the other branch was inactivated. Thus, we provide a long-overdue genetic test of the function of Hsf1 in yeast using the novel hsf1 increment construct. Our data highlight that the accumulation of misfolded proteins is uniquely sensed by Hsf1-Hsp70 chaperone titration inducing a highly selective transcriptional stress response.

Protein quality control (PQC) degrons are short protein segments that target misfolded proteins for proteasomal degradation, and thus protect cells against the accumulation of potentially toxic non-native proteins. Studies have shown that PQC degrons are hydrophobic and rarely contain negatively charged residues, features which are shared with chaperone-binding regions. Here we explore the notion that chaperone-binding regions may function as PQC degrons. When directly tested, we found that a canonical Hsp70-binding motif (the APPY peptide) functioned as a dose-dependent PQC degron both in yeast and in human cells. In yeast, Hsp70, Hsp110, Fes1, and the E3 Ubr1 target the APPY degron. Screening revealed that the sequence space within the chaperone-binding region of APPY that is compatible with degron function is vast. We find that the number of exposed Hsp70-binding sites in the yeast proteome correlates with a reduced protein abundance and half-life. Our results suggest that when protein folding fails, chaperone-binding sites may operate as PQC degrons, and that the sequence properties leading to PQC-linked degradation therefore overlap with those of chaperone binding. 

While proteins populating their native conformations constitute the functional entities of cells, protein aggregates are traditionally associated with cellular dysfunction, stress and disease. During recent years, it has become clear that large aggregate-like protein condensates formed via liquid-liquid phase separation age into more solid aggregate-like particles that harbor misfolded proteins and are decorated by protein quality control factors. The constituent proteins of the condensates/aggregates are disentangled by protein disaggregation systems mainly based on Hsp70 and AAA ATPase Hsp100 chaperones prior to their handover to refolding and degradation systems. Here, we discuss the functional roles that condensate formation/aggregation and disaggregation play in protein quality control to maintain proteostasis and why it matters for understanding health and disease.