Purzycki, D. | Search bioRxiv

bioRxiv2026

Multi-Step Hsp70 Recruitment by Class B J-Domain Proteins Drives Efficient Protein Disaggregation

Protein disaggregation by the Hsp70 system is essential for maintaining cellular proteostasis, especially under stress and proteotoxic diseases. J-Domain Proteins (JDPs) assist and regulate Hsp70 by stimulating substrate binding and ATP hydrolysis through their signature J-domain. In Class B JDPs, which play a key role in disaggregation of amyloids and amorphous aggregates, the J-domain is autoinhibited and requires activation via an auxiliary interaction with the EEVD motif of Hsp70. We reconstituted Hsp70 system from yeast and human to examine how the distinctive properties of Class B JDPs--autoinhibition and EEVD binding--promote the accumulation of Hsp70 molecules at the aggregate surface, facilitating aggregate disassembly. We revise the Hsp70 functional cycle to account for EEVD binding, showing that it enables the formation of a JDP-Hsp70-aggregate complex. This implies a model of multi-step Hsp70 clustering on aggregates, in which Class B JDPs in complex with aggregate-bound Hsp70 recruit additional Hsp70 molecules through an EEVD-independent mechanism. Experiments with the Hsp70 variant lacking EEVD validate this model but also reveal negative consequences of the stable Hsp70-JDP interaction that make the J-domain autoinhibition indispensable. Collectively, our results highlight EEVD binding and JDP autoinhibition as a coupled regulatory strategy leading to enhanced Hsp70-mediated disaggregation.

biochemistry↗

bioRxiv2025

Origin of Class B J-domain proteins involved in amyloid transactions

J-domain protein (JDP) chaperones function widely in proteostasis. Notably, eukaryotic class B JDPs of the cytosol/nucleus prevent assembly or drive disassembly of amyloid aggregates known to cause neurodegenerative diseases, yet their evolutionary origin is not known. Members of the most ubiquitous class B subgroup, canonical B (BC) JDPs, lack the signature zinc finger region (ZnF) of the more prevalent class A JDPs, while having other key features in common. Our phylogenetic analysis revealed that BC JDPs evolved more than once from class A duplicates, losing their ZnF. The cytonuclear BCs emerged at the base of eukaryotes. Cytonuclear class B (i.e. B(ST)) JDPs that have a substrate binding domain of unknown origin, distinct from that of As and BCs, emerged from a BC duplication at the base of metazoans and subsequently multiplied by duplications. Origin of B(ST)s, which are capable of suppressing formation of amyloid aggregates, predated the emergence of disease-causing amyloidogenic proteins. Using ancestral sequence resurrection, we tested when cytonuclear Bs evolved their amyloid related functions. We found that their common ancestor with As, AncAB that has a ZnF does not facilitate disassembly of amyloid fibrils, while AncB, which lacks a ZnF, is active. Overall, our findings are consistent with the idea that, though the ZnF of class A JDPs is important for some roles, its loss allowed evolution of novel functions, as illustrated by the ability of BC and B(ST) JDPs to control amyloid aggregate levels. Significance statementAcross procaryotes and eukaryotes J-domain proteins (JDPs) are key players in Hsp70 chaperone systems that maintain cellular protein homeostasis. The abundant class A and B JDPs have structural similarities, yet their origin has remained unresolved. Here we show that B JDPs independently evolved from As more than once. In each case A lost its zinc finger (ZnF) domain, suggesting that such loss has allowed evolution of new functions. Supporting this idea, biochemical resurrection of an ancestral eukaryotic B revealed that its ability to disassemble amyloid aggregates, differentiating it from As, evolved after ZnF loss. Later, the subset of Bs implicated in suppression of disease-causing amyloid aggregate formation originated from a duplicate of this B in the common ancestor of animals.

evolutionary biology↗