Biology subjects

Lazar, T.

Publications and source records attributed to Lazar, T..

9 recordsLinked to original sources

bioRxiv2026

Multivalent ubiquitin recognition regulates multiple stages of Tau self-assembly

Liquid-liquid phase separation (LLPS) has emerged as an important intermediate in the pathological self-assembly of Tau, yet the molecular mechanisms that regulate this process remain incompletely understood. Although ubiquitin is a prominent component of pathological inclusions in tauopathies and other neurodegenerative diseases, its presence has largely been linked to targeting aberrant proteins for proteasomal or autophagic clearance. Here, we identify Tau as a ubiquitin-binding protein and demonstrate that multivalent, non-covalent ubiquitin recognition regulates multiple stages of Tau self-assembly. Biochemical, biophysical, structural, and computational analyses identified four ubiquitin-binding elements within the Tau microtubule-binding repeat region that engage the canonical hydrophobic patch of ubiquitin, providing a structural basis for multivalent recognition of mono- and poly-ubiquitin species. Functionally, mono- and poly-ubiquitin suppress Tau condensate formation while enhancing the liquid-like properties of condensates that persist. In contrast, mono-ubiquitin selectively enhances aggregation of the disease-linked Tau-P301L variant under conditions without detectable LLPS, demonstrating that ubiquitin exerts distinct effects depending on the molecular context and stage of Tau self-assembly. Together, these findings establish non-covalent ubiquitin recognition as a previously unrecognized mechanism regulating Tau phase behavior and aggregation. More broadly, they expand the role of ubiquitin beyond protein quality control, with ubiquitin architecture providing an additional layer of regulation over Tau condensate formation, dynamics, maturation, and aggregation.

biophysics↗

bioRxiv2025

Negative design enables cell-free expression and folding of designed transmembrane β-Barrels

De novo design of membrane proteins (MPs) is a rapidly growing field with transformative potential for synthetic biology. Yet, progress has lagged behind that of soluble proteins, largely due to limited understanding of the fundamental principles governing MP folding, stability, and solubility--and their integration into computational models. Here, we use a cell-free expression system to bypass the cytotoxicity of failed or insoluble designs and investigate how sequence features influence the folding of synthetic transmembrane {beta}-barrels (TMBs). We find that even small, idealized TMBs challenge classical protein design workflows: sequences optimized solely for thermodynamic stability misfold and aggregate, preventing membrane insertion. Instead of bulk hydrophobicity, aggregation and {beta}-sheet propensity emerge as key determinants of membrane association. By designing better folding variants of a synthetic TMB, we demonstrate that suppressing aggregation-prone intermediates through local destabilization of {beta}-strands ("negative design") significantly improves folding efficiency. Strikingly, even substitutions typically considered highly destabilizing, such as prolines or polar threonines exposed to the bilayer core, can improve folding when strategically positioned, without significantly compromising thermodynamic stability. Based on these findings, we propose a framework for joint optimization of native stability and folding pathways for future MP and nanopore design. Significance StatementMembrane proteins are essential to biology and biotechnology, yet designing them from scratch remains challenging. Using synthetic transmembrane {beta}-barrels and a cell-free expression system with lipid vesicles, we show that conventional design strategies focused solely on native state stabilization can lead to misfolding and aggregation. By incorporating "negative design" features--specific mutations that locally disrupt {beta}-strand structure--we improve folding efficiency without compromising stability. Remarkably, a protein language model outperformed traditional energy-based methods in predicting these beneficial mutations. Our findings highlight the critical role of folding kinetics in membrane protein design and introduce new principles for engineering synthetic membrane proteins and {beta}-barrel nanopores.

biophysics↗

bioRxiv2025

Definition and discovery of tandem SH3-binding motifs interacting with members of the p47phox-related protein family

SH3 domains are widespread protein modules that mostly bind to proline-rich short linear motifs (SLiMs). Most known SH3 domain-motif interactions and canonical or non-canonical recognition specificities are described for individual SH3 domains. Although cooperation and coordinated motif binding between tandem SH3 domains has already been described for members of the p47phox-related protein family, individual cases have never been collected and analyzed collectively, which precluded the definition of the binding preferences and targeted discovery of further instances. Here, we apply an integrative approach that includes data collection, curation, bioinformatics analyses and state-of-the-art structure prediction methods to fill these gaps. We define the optimal binding preference of tandemly arranged SH3 domains as [PAVIL]PPR[PR][^DE][^DE], and propose potential new instances of this SLiM among the family members and their binding partners. Structure predictions suggest the possibility of a novel, reverse binding mode for certain motif instances. A search of the human proteome with the sequence signatures of SH3 tandemization and follow-up structure analyses suggest that SH3 tandemization could be specific for this family. In all, our comprehensive analysis of this unique SH3 binding mode enabled description of the binding preference and identification of novel interesting cases proposed for experimental validation.

molecular biology↗

bioRxiv2025

Expression and Purification of Full-Length hnRNPA2B1 for Biophysical Characterization of Liquid-Liquid Phase Separation

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) is a multifunctional RNA-binding protein involved in RNA maturation and mRNA transport. It has recently been shown to undergo liquid-liquid phase separation (LLPS), contributing to the assembly of membraneless organelles. Moreover, dysregulation of LLPS is associated with the formation of pathogenic protein aggregates, in which hnRNPA2B1 is frequently found. Despite its biological and pathological relevance, studies on the full-length protein remain limited due to its intrinsically disordered, low-complexity domain, which renders hnRNPA2B1 highly aggregation-prone and difficult to purify. In this study, we report the successful expression and purification of full-length hnRNPA2B1 with high purity and minimal nucleic acid contamination. By optimizing buffer conditions, specifically ionic strength and pH, we maintain the protein in solution following cleavage of its solubility tag. Preliminary in vitro characterization under near-physiological conditions reveals that purified hnRNPA2B1 undergoes LLPS, forming dynamic liquid-like droplets that grow and mature into amorphous aggregates. Our approach provides a robust method for purifying hnRNPA2B1 suitable for LLPS and aggregation studies. This strategy may also be useful to purify other aggregation-prone, intrinsically disordered proteins.

biochemistry↗

bioRxiv2023

In-depth characterization of HINT1 pathogenic variants

BackgroundBiallelic loss-of-function variants in HINT1 cause neuromyotonia-associated axonal neuropathy (NMAN). Affected patients present from an early onset with a motor-greater-than-sensory polyneuropathy that is currently incurable. NMAN is a global cause of inherited peripheral neuropathy with higher prevalence in Europe and Asia. Nearly 30 distinct NMAN-associated variants have been reported to date, mostly in sporadic patients and small families. There is limited functional characterization of most of them, resulting in limited knowledge on their pathogenic mode of action and hindering the development of therapeutical strategies. MethodsWe systematically (re-)evaluated the pathogenicity of all reported HINT1 variants associated with NMAN using several in silico pathogenicity predictors used in the standard of care genetic testing. Fifteen missense and one truncating variants were further mapped onto the HINT1 crystal structure and grouped according to their spatial distribution. We combined several structural modeling tools to assess the impact of the variants on protein stability in both monomeric and dimeric forms. These variants underwent further functional validation by immunoblotting in both HeLa HINT1 knockout cell lines and in a S. cerevisiae strain deficient for the HINT1 orthologue. Finally, we tested the in vivo functionality of each variant by its ability to rescue yeast growth under stress conditions. ResultsOur combinatorial approach allowed the systematic characterization and (re-)evaluation of all known HINT1 variants associated with NMAN, enabling improved pathogenicity classification. Additionally, this standardized functional assessment allowed their categorization into four structure-based groups: 1) truncating variants; 2) variants of the catalytic pocket; 3) variants at the dimer interface; 4) variants in the distal {beta}-hairpin and nearby loop. Functional tests in yeast and mammalian disease models demonstrated their detrimental- yet differential -effect on HINT1 function, enabling structure-function correlations. ConclusionsOur standardized experimental pipeline allows for the characterization of newly discovered HINT1 variants in the context of NMAN, facilitating their pathogenicity interpretation. This work highlights the value of combining structural and functional approaches to understand better the underlying disease mechanisms. Our findings provide the rational basis for patient stratification and the development of personalized treatment strategies.

neuroscience↗

bioRxiv2023

Phase-separating fusion proteins drive cancer by dysregulating transcription through ectopic condensates

Numerous cellular processes rely on biomolecular condensates formed through liquid-liquid phase separation (LLPS), thus, perturbations of LLPS underlie various diseases. We found that proteins initiating LLPS are frequently implicated in somatic cancers, even surpassing their involvement in neurodegeneration. Cancer-associated LLPS scaffolds are connected to all cancer hallmarks and tend to be oncogenes with dominant genetic effects lacking therapeutic options. Since most of them act as oncogenic fusion proteins (OFPs), we undertook a systematic analysis of cancer driver OFPs by assessing their module-level molecular functions. We identified both known and novel combinations of molecular functions that are specific to OFPs and thus have a high potential for driving tumorigenesis. Protein regions driving condensate formation show an increased association with DNA- or chromatin-binding domains of transcription regulators within OFPs, indicating a common molecular mechanism underlying several soft tissue sarcomas and hematologic malignancies where phase-separation-prone OFPs form abnormal, ectopic condensates along the DNA, and thereby dysregulate gene expression programs.

cancer biology↗

bioRxiv2023

C9orf72-linked arginine-rich dipeptide repeats aggravate pathological phase separation of G3BP1

Ras GTPase-activating protein-binding protein 1 (G3BP1) is the key protein driving the formation of cytoplasmic stress granules (SGs) by liquid-liquid phase separation (LLPS). It is a switch-like protein held in a closed and inactive state by intramolecular electrostatic interactions competitively opened by RNA, activating the protein and initiating its LLPS. Here we show that C9orf72-derived arginine-rich dipeptide repeats PR30 and GR30 (R-DPRs) present in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), also bind to G3BP1, switching it to an LLPS-competent open state much more effectively than RNA. Whereas RNA binds G3BP1 with micromolar affinity, and cannot initiate LLPS without crowding agents, R-DPRs exhibit a thousand-fold stronger binding to G3BP1, eliciting rapid LLPS even without crowding. The pathogenic effect of R-DPRs is also underscored by the slow transition of R-DPR-G3BP1 liquid droplets to aggregated, ThS-positive states that can recruit the ALS-linked protein hnRNPA2. Deletion constructs and molecular simulations show that R-DPR binding and LLPS are mediated via binding through the negatively charged intrinsically disordered region 1 (IDR1) of the protein, allosterically regulated by the positively charged IDR3. Bioinformatic analyses point to the strong mechanistic parallels of these effects with the interaction of R-DPRs with nuclear nucleophosmin (NPM1) and also suggest that R-DPRs also interact with many other similar nucleolar and stress-granule proteins, extending the underlying mechanism of R-DPR toxicity in cells.

molecular biology↗

bioRxiv2021

Concentration and dosage sensitivity of proteins driving liquid-liquid phase separation

Liquid-liquid phase separation (LLPS) is a molecular process that leads to the formation of membraneless organelles (MLOs), i.e. functionally specialized liquid-like cellular condensates formed by proteins and nucleic acids. Integration of data on LLPS-associated proteins from dedicated databases revealed only modest overlap between them and resulted in a confident set of 89 human LLPS driver proteins. Since LLPS is highly concentration-sensitive, the underlying experiments are often criticized for applying higher-than-physiological protein concentrations. To clarify this issue, we performed a naive comparison of in vitro applied and quantitative proteomics-derived protein concentrations and discuss a number of considerations that rationalize the choice of apparently high in vitro concentrations in most LLPS studies. The validity of in vitro LLPS experiments is further supported by in vivo phase-separation experiments and by the observation that the corresponding genes show a strong propensity for dosage sensitivity. This observation implies that the availability of the respective proteins is tightly regulated in cells to avoid erroneous condensate formation. In all, we propose that although local protein concentrations are practically impossible to determine in cells, proteomics-derived cellular concentrations should rather be considered as lower limits of protein concentrations, than strict upper bounds, to be respected by in vitro experiments.

molecular biology↗

bioRxiv2020

Distance-based metrics for comparing conformational ensembles of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) are proteins whose native functional states represent ensembles of highly diverse conformations. Such ensembles are a challenge for quantitative structure comparisons as their conformational diversity precludes optimal superimposition of the atomic coordinates, necessary for deriving common similarity measures such as the root-mean-square deviation (RMSD) of these coordinates. Here we introduce superimposition-free metrics, which are based on computing matrices of C-C distance distributions within ensembles and comparing these matrices between ensembles. Differences between two matrices yield information on the similarity between specific regions of the polypeptide, whereas the global structural similarity is captured by the ens_dRMS, defined as the root-mean-square difference between the medians of the C-Cdistance distributions of two ensembles. Together, our metrics enable rigorous investigations of structure-function relationships in conformational ensembles of IDPs derived using experimental restraints or by molecular simulations, and for proteins containing both structured and disordered regions. Statement of SignificanceImportant biological insight is obtained from comparing the high-resolution structures of proteins. Such comparisons commonly involve superimposing two protein structures and computing the residual root-mean-square deviation of the atomic positions. This approach cannot be applied to intrinsically disordered proteins (IDPs) because IDPs do not adopt well-defined 3D structures, rather, their native functional state is defined by ensembles of heterogeneous conformations that cannot be meaningfully superimposed. We report new measures that quantify the local and global similarity between different conformational ensembles by evaluating differences between the distributions of residue-residue distances and their statistical significance. Applying these measures to IDP ensembles and to a protein containing both structured and intrinsically disordered domains provides deeper insights into how structural features relate to function.

bioinformatics↗