Biology subjects

Tomiczek, B.

Publications and source records attributed to Tomiczek, B..

7 recordsLinked to original sources

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↗

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↗

bioRxiv2022

SbCYP79A61 Produces Phenylacetaldoxime, a Precursor of Benzyl Cyanide and Phenylacetic Acid in Sorghum bicolor

Aldoximes are amino acid derivatives that serve as intermediates for numerous specialized metabolites including cyanogenic glycosides, glucosinolates, and auxins. Aldoxime formation is mainly catalyzed by cytochrome P450 monooxygenases of the 79 family (CYP79s) that can have broad or narrow substrate specificity. Aldoxime biosynthesis in the cereal sorghum (Sorghum bicolor) is not well characterized. This study identified nine CYP79-encoding genes in the genome of sorghum. A phylogenetic analysis of CYP79 showed that SbCYP79A61 formed a subclade with maize ZmCYP79A61, previously characterized to be involved in aldoxime biosynthesis. Functional characterization of this sorghum enzyme using transient expression in Nicotiana benthamiana and stable overexpression in Arabidopsis thaliana revealed that SbCYP79A61 catalyzes the production of phenylacetaldoxime (PAOx) from phenylalanine, but unlike the maize enzyme, displays no detectable activity against tryptophan. Additionally, targeted metabolite analysis after stable isotope feeding assays revealed that PAOx can serve as a precursor of phenylacetic acid (PAA) in sorghum and identified benzyl cyanide as an intermediate of PAOx-derived PAA biosynthesis in both sorghum and maize. Taken together, our results demonstrate that SbCYP79A61 produces PAOx in sorghum and may serve in the biosynthesis of other nitrogen-containing phenylalanine-derived metabolites involved in mediating biotic and abiotic stresses. HighlightIdentification of SbCYP79A61, an enzyme catalyzing the production of phenylacetaldoxime (PAOx) in sorghum, and identification of benzyl cyanide as an intermediate of PAOx-derived phenylacetic acid (PAA) biosynthesis in monocots.

plant biology↗

bioRxiv2020

Two-step mechanism of J-domain action in driving Hsp70 function

J-domain proteins (JDPs), obligatory Hsp70 cochaperones, play critical roles in protein homeostasis. They promote key allosteric transitions that stabilize Hsp70 interaction with substrate polypeptides upon hydrolysis of its bound ATP. Although a recent crystal structure revealed the physical mode of interaction between a J-domain and an Hsp70, the structural and dynamic consequences of J-domain action once bound and how Hsp70s discriminate among its multiple JDP partners remain enigmatic. We combined free energy simulations, biochemical assays and evolutionary analyses to address these issues. Our results indicate that the invariant aspartate of the J-domain perturbs a conserved intramolecular Hsp70 network of contacts that crosses domains. This perturbation leads to destabilization of the domain-domain interface - thereby promoting the allosteric transition that triggers ATP hydrolysis. While this mechanistic step is driven by conserved residues, evolutionarily variable residues are key to initial JDP/Hsp70 recognition - via electrostatic interactions between oppositely charged surfaces. We speculate that these variable residues allow an Hsp70 to discriminate amongst JDP partners, as many of them have coevolved. Together, our data points to a two-step mode of J-domain action, a recognition stage followed by a mechanistic stage. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/901538v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@ba0d3aorg.highwire.dtl.DTLVardef@ba9193org.highwire.dtl.DTLVardef@ab7974org.highwire.dtl.DTLVardef@1b2de33_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2018

OMA standalone: orthology inference among public and custom genomes and transcriptomes

Genomes and transcriptomes are now typically sequenced by individual labs, but analysing them often remains challenging. One essential step in many analyses lies in identifying orthologs--corresponding genes across multiple species--but this is far from trivial. The OMA (Orthologous MAtrix) database is a leading resource for identifying orthologs among publicly available, complete genomes. Here, we describe the OMA pipeline available as a standalone program for Linux and Mac. When run on a cluster, it has native support for the LSF, SGE, PBS Pro, and Slurm job schedulers and can scale up to thousands of parallel processes. Another key feature of OMA standalone is that users can combine their own data with existing public data by exporting genomes and pre-computed alignments from the OMA database, which currently contains over 2100 complete genomes. We compare OMA standalone to other methods in the context of phylogenetic tree inference, by inferring a phylogeny of the Lophotrochozoa, a challenging clade within the Protostomes. We also discuss other potential applications of OMA standalone, including identifying gene families having undergone duplications/losses in specific clades, and identifying potential drug targets in non-model organisms. OMA Standalone is available at http://omabrowser.org/standalone under the permissible open source Mozilla Public License Version 2.0.

bioinformatics↗