Biology subjects

Pitek, M.

Publications and source records attributed to Pitek, M..

3 recordsLinked to original sources

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↗

bioRxiv2025

Mechanism and energetics of JDP induced Hsp70's conformational transition towards catalytically active state

Hsp70 chaperones are crucial for maintaining protein homeostasis by regulating the stability and conformational states of client polypeptides through cycles of their binding and release. These cycles require conformational transitions of Hsp70 driven by ATP binding and hydrolysis. The ATPase activity of Hsp70 is controlled by J-domain protein (JDP) cochaperones, which allosterically stimulate ATP hydrolysis via interactions between their J-domains and Hsp70. The J-domain binds at the interface between the nucleotide (NBD) and substrate (SBD) binding domains of ATP bound Hsp70. Although, it was established that the JD interaction involves residues of helices II and III, and the interhelical loop critical for ATPase stimulation, the mechanism by which the allosteric signal induced by J-domain binding is transmitted to the distal nucleotide-binding pocket of Hsp70 remains unclear, as do the conformational changes leading to the ATP hydrolysis. Here, we addressed these questions by means of all-atom free energy simulations and dynamic network analysis, starting from the crystal structures of ATP-bound Hsp70 DnaK alone and in complex with the J-domain of DnaJ. We demonstrated that the presence of the J-domain results in the rearrangement of the nucleotide-binding pocket into a hydrolysis competent state, characterized by close contact between universally conserved T199 of NBD and {gamma}-phosphate of ATP. With network analysis we revealed that the allosteric signal for this rearrangement is transmitted along the {beta}-strand containing T199. Finally, we provide rationale for the signal transmission, where steric repulsion between the J-domains helix III and SBD induces a push of the T199 containing {beta}-strand. Overall, our study provides mechanistic insights into allosteric signal transmission within Hsp70, bridging the gap between J-domain binding and ATPase stimulation. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/655504v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1ff0f30org.highwire.dtl.DTLVardef@3e4157org.highwire.dtl.DTLVardef@133f159org.highwire.dtl.DTLVardef@12a35c2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Evolution towards simplicity in bacterial small heat shock protein system

Evolution can tinker with multi-protein machines and replace them with simpler single-protein systems performing equivalent functions in equally efficient manner. It is unclear how, on a molecular level, such simplification can arise. With ancestral reconstruction and biochemical analysis we have traced the evolution of bacterial small heat shock proteins (sHsp), which help to refold proteins from aggregates using either two proteins with different functions (IbpA and IbpB) or a secondarily single sHsp that performs both functions in an equally efficient way. Secondarily single sHsp evolved from IbpA, an ancestor specialized in strong substrate binding. Evolution of an intermolecular binding site drove the alteration of substrate binding properties, as well as formation of higher-order oligomers. Upon two mutations in the -crystallin domain, secondarily single sHsp interacts with aggregated substrates less tightly. Paradoxically, less efficient binding positively influences the ability of sHsp to stimulate substrate refolding, since the dissociation of sHps from aggregates is required to initiate Hsp70-Hsp100-dependent substrate refolding. After the loss of a partner, IbpA took over its role in facilitating the sHsp dissociation from an aggregate by weakening the interaction with the substrate, which became beneficial for the refolding process. We show that the same two amino acids introduced in modern-day system define whether the IbpA acts as a single sHsp or obligatorily cooperates with an IbpB partner. Our discoveries illuminate how one sequence has evolved to encode functions previously performed by two distinct proteins.

biochemistry↗