Biology subjects

Wydorski, P. M.

Publications and source records attributed to Wydorski, P. M..

6 recordsLinked to original sources

bioRxiv2025

DnaJB1 chaperone inhibits tau aggregation by recognizing its N-terminus

A network of protein folding and degradation machineries maintains protein homeostasis by preventing the accumulation of misfolded proteins and by facilitating their clearance. These systems are also crucial for the inhibition of protein aggregation in neurodegenerative diseases where misfolded proteins often aggregate into {beta}-rich amyloid fibrils. How these machineries selectively recognize pathological aggregates over normal conformations remains unclear. Here, we present the molecular logic for how a Hsp70 co-chaperone from the J-domain protein family, DnaJB1, binds pathological aggregates of the microtubule-associated protein tau through the recognition of the flexible N-terminus that comprises the disordered fuzzy coat of fibrils. We show that this interaction contributes to the regulation of tau assembly in cellular models of tau aggregation and depends on the presence of the negatively charged residues. We determined that DnaJB1 inhibits tau aggregation in vitro through these interactions, and found that this weak, transient binding can be enhanced by the presence of polyanionic factors such as heparin. As prospective client-binding sites, we identified the charged hinge between the two {beta}-sandwich C-terminal domains I and II, as well as the conserved J-domain of this chaperone. This work presents novel biochemical and structural insights into how the molecular chaperone DnaJB1 recognizes full-length forms of tau protein in a pathological context.

biochemistry↗

bioRxiv2023

Ensemble-based design of tau to inhibit aggregation while preserving biological activity.

The microtubule-associated protein tau is implicated in neurodegenerative diseases characterized by amyloid formation. Mutations associated with frontotemporal dementia increase tau aggregation propensity and disrupt its endogenous microtubule-binding activity. However, the structural relationship between aggregation propensity and biological activity remains unclear. We employed a multi-disciplinary approach, including computational modeling, NMR, cross-linking mass spectrometry, and cell models to engineer tau sequences that modulate its structural ensemble. Our findings show that substitutions near the conserved PGGG {beta}-turn motif informed by tau isoform context reduce tau aggregation in vitro and cells and can even counteract aggregation induced by turn destabilizing disease-associated proline-to-serine mutations. Engineered tau sequences maintain microtubule binding and explain why 3R isoforms exhibit reduced pathogenesis compared to 4R. We propose a simple mechanism to reduce the formation of pathogenic species while preserving biological function, thus offering insights for therapeutic strategies aimed at reducing tau protein misfolding in neurodegenerative diseases. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/571598v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@178a79org.highwire.dtl.DTLVardef@1842dc7org.highwire.dtl.DTLVardef@d9a32borg.highwire.dtl.DTLVardef@676dec_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

DNAJB8 oligomerization is mediated by an aromatic-rich motif that is dispensable for substrate activity

J-domain protein (JDP) molecular chaperones have emerged as central players that maintain a healthy proteome. The diverse members of the JDP family function as monomers/dimers and a small subset assemble into micron-sized oligomers. The oligomeric JDP members have eluded structural characterization due to their low-complexity, intrinsically disordered middle domains. This in turn, obscures the biological significance of these larger oligomers in protein folding processes. Here, we identified a short, aromatic motif within DNAJB8, that drives self-assembly through {nu}-{nu} stacking and determined its X-ray structure. We show that mutations in the motif disrupt DNAJB8 oligomerization in vitro and in cells. DNAJB8 variants that are unable to assemble bind to misfolded tau seeds more specifically and retain capacity to reduce protein aggregation in vitro and in cells. We propose a new model for DNAJB8 function in which the sequences in the low-complexity domains play distinct roles in assembly and substrate activity. HIGHLIGHTSDNAJB8 oligomerization is mediated by a short phenylalanine-based motif in the S/T domain Mutation of a single phenylalanine yields a monomeric form of DNAJB8 Monomeric DNABJ8 binds to an aggregation-prone substrate Monomeric DNAJB8 retains substrate aggregation prevention activity

biophysics↗

bioRxiv2022

Structural polymorphism of amyloid fibrils in cardiac ATTR amyloidosis revealed by cryo-electron microscopy

The deposition of amyloidogenic transthyretin (ATTR) in ATTR amyloidosis leads to an unexplained variety of clinical phenotypes, including cardiomyopathy. In brain amyloid conditions, there is an apparent association between the clinical phenotype and the amyloid fibril structure. Here, we question this phenotype-structure association in cardiac amyloidoses by determining the cryo-electron microscopy structures of fibrils extracted from the hearts of seven ATTR amyloidosis patients. We found that, in contrast to brain fibrils, cardiac ATTR fibrils display a structural polymorphism that is not genotype-specific, can co-exist within the same individual, and is independent of the cardiac phenotype. This polymorphism challenges the current paradigm of "one disease equals one fibril fold" proposed in tauopathies and synucleinopathies, and questions whether a similar structural heterogeneity occurs in other amyloidoses. One-Sentence SummaryUnlike brain amyloid fibrils, cardiac ATTR fibrils are polymorphic independent of genotype and even within the same patient.

molecular biology↗

bioRxiv2021

Dual domain recognition determines SARS-CoV-2 PLpro selectivity for human ISG15 and K48-linked di-ubiquitin

The Papain-like protease (PLpro) is a domain of a multi-functional, non-structural protein 3 of coronaviruses. PLpro cleaves viral polyproteins and posttranslational conjugates with poly-ubiquitin and protective ISG15, composed of two ubiquitin-like (UBL) domains. Across coronaviruses, PLpro showed divergent selectivity for recognition and cleavage of posttranslational conjugates despite sequence conservation. We show that SARS-CoV-2 PLpro binds human ISG15 and K48-linked di-ubiquitin (K48-Ub2) with nanomolar affinity and detect alternate weaker-binding modes. Crystal structures of untethered PLpro complexes with ISG15 and K48-Ub2 combined with solution NMR and cross-linking mass spectrometry revealed how the two domains of ISG15 or K48-Ub2 are differently utilized in interactions with PLpro. Analysis of protein interface energetics predicted differential binding stabilities of the two UBL/Ub domains that were validated experimentally. We emphasize how substrate recognition can be tuned to cleave specifically ISG15 or K48-Ub2 modifications while retaining capacity to cleave mono-Ub conjugates. These results highlight alternative druggable surfaces that would inhibit PLpro function.

biophysics↗

bioRxiv2020

DnaJC7 binds natively folded structural elements in tau to inhibit amyloid formation

Molecular chaperones, including Hsp70/Hsp40 families, play central roles in binding substrates to prevent their aggregation. How Hsp40s select different conformations of substrates remains poorly understood. Here, we report a novel interaction between the Hsp40 DnaJC7 and tau that efficiently suppresses tau aggregation in vitro and in cells. DnaJC7 binds preferentially to natively folded wild-type tau, but disease-associated mutants in tau reduce chaperone binding affinity. We identify that DnaJC7 uses a single TPR domain to recognize a {beta}-turn element in tau that contains the 275VQIINK280 amyloid motif. Wild-type tau {beta}-turn fragments, but not mutant fragments, can block full-length tau binding to DnaJC7. These data suggest DnaJC7 preferentially binds and stabilizes natively folded conformations of tau to prevent tau conversion into amyloids. This identifies a novel mechanism of tau aggregation regulation that can be exploited as both a diagnostic and a therapeutic intervention.

biophysics↗