Biology subjects

Bezsonova, I.

Publications and source records attributed to Bezsonova, I..

10 recordsLinked to original sources

bioRxiv2026

Mechanisms of USP7/MAGEL2 Complex Assembly and Its Mutational Disruption in Neurodevelopmental Diseases

The WASH complex regulates endosomal trafficking and is linked to several neurodevelopmental diseases, including Prader-Willi syndrome, Schaaf-Yang syndrome, and Hao-Fountain syndrome. Its function is tightly controlled by ubiquitination, maintained by the multi-subunit MUST complex containing both a ubiquitin ligase (MAGEL2/TRIM27) and a deubiquitinase (USP7). However, the mechanism underlying the MUST complex assembly remains poorly understood. In this study, we investigate the assembly of USP7 and MAGEL2 components of the MUST complex using NMR spectroscopy, isothermal titration calorimetry, X-ray crystallography, and cellular assays. We show that the USP7/MAGEL2 interaction is bipartite and multivalent. Two distinct domains of USP7, TRAF and UBL1-2, recognize two unstructured but evolutionarily conserved regions of MAGEL2, one of which contains multiple TRAF-binding sites. Furthermore, we determine the high-resolution crystal structure of the TRAF/MAGEL2 complex and identify Hao-Fountain syndrome-linked mutations in USP7 that disrupt USP7/MAGEL2 complex formation in vitro and in cells. These findings provide mechanistic insight into the pathogenic basis of Hao-Fountain syndrome and related Schaaf-Yang and Prader-Willi syndromes.

biochemistry↗

bioRxiv2026

High-pH NMR to Identify Macromolecular Hydrogen-Bonds and Foldons

Hydrogen bond (H-bond) restraints are critical for NMR structure determination, yet their experimental identification can be challenging for marginally stable structures that afford insufficient protection from (H/D) exchange in D2O. As an alternative, we explored the use of NMR between pH 10 and 11 conditions that promote rapid exchange, for identifying backbone amide protons involved in H-bonds. We analyzed [~]750 amide sites distributed across ten proteins with known structures. Persistence of amide protons at high pH in standard 2D 1H-15N HSQC spectra for 15N-labeled proteins in H2O, or TOCSY for unlabeled proteins, identifies H-bonds with [~]91% accuracy that exceeds the [~]80% accuracy of traditional H/D exchange experiments in D2O. For two -helical coiled coils and three globular proteins, we performed alkaline unfolding experiments taking advantage of amide NMR signal attenuation from unstructured polypeptides. Increasing the sample pH led to a progressive loss of native amide proton NMR signals, revealing an unfolding hierarchy where "foldons" remaining at the highest pH values had the most persistent H-bonds under EX1 exchange conditions. The foldons observed at high pH are consistent with partially folded structures previously characterized near neutral pH by native state hydrogen exchange, equilibrium unfolding, and protein fragment studies. For {beta}-sheet proteins, foldons correspond to regions with high inter-residue contact density, whereas in coiled coils they demarcate regions with high -helical propensity. High-pH NMR experiments provide a sensitive, fast, inexpensive, and broadly applicable approach to map H-bonding in marginally stable or partially folded proteins. Additionally, they offer the opportunity to explore uncharted protein dynamics and unfolding pathways under basic pH conditions.

biophysics↗

bioRxiv2026

A RAD18 SAP domain PIP motif enables PCNA mono-ubiquitination and USP1-BRCA1 synthetic lethality

The proliferating cell nuclear antigen (PCNA) sliding clamp is mono-ubiquitinated by RAD6-RAD18 in response to DNA damage, initiating the DNA damage tolerance pathway of translesion synthesis. The molecular basis by which RAD18 engages PCNA has, however, remained incompletely defined. Mono-ubiquitinated PCNA is subsequently poly-ubiquitinated with K48-linked chains that target PCNA for degradation. Ubiquitin-specific protease 1 (USP1) reverses PCNA mono- and poly-ubiquitination; accordingly, inhibiting USP1 causes the accumulation of mono-ubiquitinated PCNA at replication forks and a reduction in total PCNA levels. USP1 inhibitors promote the accumulation of ssDNA gaps (ssGAPs) in newly replicated DNA and are synthetic lethality in BRCA1-deficient cells. Here, we combine computational and structural approaches to identify and characterize a PCNA-interacting peptide (PIP) motif in RAD18. This PIP motif is required for RAD18-dependent DNA damage-induced PCNA ubiquitination and PCNA turnover. Mutation of the RAD18-PCNA interface reduces ssGAP accumulation and USP1 inhibitor sensitivity in BRCA1-deficient cells. Furthermore, cells adapted to prolonged USP1 inhibition exhibit reduced RAD18 levels, suggesting that deregulation of RAD18 contributes to a biologically relevant drug resistance mechanism. This resistance could be overcome by inhibiting the Ataxia telangiectasia and Rad3-related (ATR) kinase. Together, these findings define a molecular interface required for RAD18-dependent PCNA mono-ubiquitination and identify it as a key determinant of USP1-BRCA1 synthetic lethality.

biochemistry↗

bioRxiv2026

PXL: a Nucleic Acid-Binding Module of Promyelocytic Leukemia Protein

The promyelocytic leukemia protein (PML) is a stress-response factor that assembles into PML nuclear bodies, dynamic subnuclear compartments involved in tumor suppression and antiviral defense. The most abundant isoform, PML-1, has been linked to transcriptional regulation, genome stability, and antiviral responses, yet the molecular basis of these functions remains unclear. Here, we report that PML-1 contains a unique nucleic acid- binding module, PXL, and determine its three-dimensional structure by X-ray crystallography. Further biochemical, mutational, and cellular analyses, including RNA-seq, demonstrate that this module selectively binds single-stranded G-rich RNA and DNA motifs and modulates the transcriptome. These findings reveal an unexpected molecular function of PML and provide a framework for understanding its roles in nuclear organization and gene regulation.

biochemistry↗

bioRxiv2025

Functional Spectrum of USP7 Pathogenic Variants in Hao-Fountain Syndrome: Insights into the Enzyme's Activity, Stability, and Allosteric Modulation

Hao-Fountain syndrome is a rare neurodevelopmental disorder caused by mutations in the de-ubiquitinating enzyme USP7 (Ubiquitin Specific Protease 7). Due to the novelty of the disease and its poorly understood molecular mechanisms, treatments for the syndrome are currently lacking. This study examines the effects of 11 patient-derived variants located within the catalytic domain of USP7, focusing on their impact on the enzymes activity, thermodynamic stability, and substrate recognition. Our findings reveal a spectrum of functional consequences, ranging from complete inactivation to hyperactivation of USP7. Notably, we identify a specific subset of pathogenic variants whose catalytic activity can be significantly boosted using a novel allosteric activator. These results provide the first insight into USP7 malfunction in Hao-Fountain syndrome-linked variants and pave the way for improved prognostic approaches and targeted treatments in the future.

biochemistry↗

bioRxiv2025

Small-molecule allosteric activator of ubiquitin-specific protease 7 (USP7)

Ubiquitin-specific protease 7 (USP7) is a deubiquitylase essential for cell homeostasis, DNA repair, and regulation of both tumor suppressors and oncogenes. Inactivating USP7 mutations have been associated with Hao-Fountain Syndrome (HAFOUS), a rare neurodevelopmental disorder. Although a range of USP7 inhibitors have been developed over the last decade, in the context of HAFOUS as well as oncogene regulation, USP7 activators may represent a more relevant approach. To address this challenge, we report the discovery and characterization of a small-molecule activator of USP7 called MS-8. We showed that MS-8 activates USP7 by engaging the allosteric C-terminal binding pocket of USP7, thus mimicking the allosteric autoactivation by the USP7 C-terminal tail. We observed that MS-8 engages and activates mutant USP7 in a cellular context, impacting downstream proteins. Taken together, our study provides validation of the USP7 activator that paves the way towards novel activation-driven USP7 pharmacology.

biochemistry↗

bioRxiv2024

DNA polymerase η is regulated by mutually exclusive mono-ubiquitination and mono-NEDDylation

DNA polymerase eta (Pol {eta}) is a Y-family translesion polymerase responsible for synthesizing new DNA across UV-damaged templates. It is recruited to replication forks following mono-ubiquitination of the PCNA DNA clamp. This interaction is mediated by PCNA-interacting protein (PIP) motifs within Pol {eta}, as well as by its C-terminal ubiquitin-binding zinc finger (UBZ) domain. Previous work has suggested that Pol {eta} itself is mono-ubiquitinated at four C-terminal lysine residues, which is dependent on prior ubiquitin-binding by its UBZ domain. Here, we show that Pol {eta} can be modified at the same lysine residues by the ubiquitin-like protein, NEDD8. Like ubiquitination, this modification is driven by non-covalent interactions between NEDD8 and the UBZ domain. While only a small proportion of Pol {eta} is mono-NEDDylated under normal conditions, these levels rapidly increase by inhibiting the COP9 signalosome, suggesting that mono-NEDDylation is maintained under strong negative regulation. Finally, we provide data to support that mono-ubiquitination is important for Pol {eta} foci formation and suggest that NEDDylation disrupts this process. These results reveal a new mechanism of Pol {eta} regulation by ubiquitin-like proteins.

biochemistry↗

bioRxiv2023

Activation Dynamics of Ubiquitin Specific Protease 7

Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme responsible for the regulation of key human oncoproteins and tumor suppressors including Mdm2 and p53, respectively. Unlike other members of the USP family of proteases, the isolated catalytic domain of USP7 adopts an enzymatically inactive conformation that has been well characterized using X-ray crystallography. The catalytic domain also samples an active conformation, which has only been captured upon USP7 substrate-binding. Here, we utilized CPMG NMR relaxation dispersion studies to observe the dynamic motions of USP7 in solution. Our results reveal that the catalytic domain of USP7 exchanges between two distinct conformations, the inactive conformation populated at 95% and the active conformation at 5%. The largest structural changes are localized within functionally important regions of the enzyme including the active site, the ubiquitin-binding fingers, and the allosteric helix of the enzyme, suggesting that USP7 can adopt its active conformation in the absence of a substrate. Furthermore, we show that the allosteric L299A activating mutation disturbs this equilibrium, slows down the exchange, and increases the residence time of USP7 in its active conformation, thus, explaining the elevated activity of the mutant. Overall, this work shows that the isolated USP7 catalytic domain pre-samples its "invisible" active conformation in solution, which may contribute to its activation mechanism.

biophysics↗

bioRxiv2019

The HSV-1 immediate early protein ICP22 is a J-like protein required for Hsc70 reorganization during lytic infection

Molecular chaperones and co-chaperones are the most abundant cellular effectors of protein homeostasis, assisting protein folding and preventing aggregation of misfolded proteins. We have previously shown that HSV-1 infection results in the drastic spatial reorganization of the cellular chaperone Hsc70 into nuclear domains called VICE (Virus Induced Chaperone Enriched) domains and that this recruitment is dependent on the viral immediate early protein ICP22. In this paper, we present several lines of evidence supporting the notion that ICP22 functions as a virally encoded co-chaperone (J-protein/Hsp40) functioning together with its Hsc70 partner to recognize and manage aggregated and misfolded proteins. We show that ICP22 results in (i) nuclear sequestration of non-native proteins, (ii) reduction of cytoplasmic aggresomes in cells expressing aggregation-prone proteins and (iii) thermoprotection against heat-inactivation of firefly luciferase. (iv) Sequence homology analysis indicated that ICP22 contains an N-terminal J-domain and a C-terminal substrate binding domain, similar to type II cellular J-proteins. ICP22 may, thus, be functionally similar to J-protein/Hsp40 co-chaperones that function together with their HSP70 partners to prevent aggregation of non-native proteins. This is not the first example of a virus hijacking a function of a cellular chaperone, as SV40 T Antigen was previously shown to contain a J-domain; however, this the first known example of the acquisition of a complete J-like protein by a virus and suggests that HSV has taken advantage of the adaptable nature of J-proteins to evolve a multi-functional co-chaperone that functions with Hsc70 to promote lytic infection.\n\nIMPORTANCEViruses have evolved a variety of strategies to succeed in a hostile environment. The HSV immediate early protein ICP22 plays several roles in the virus life cycle including down-regulation of cellular gene expression, up-regulation of late viral gene expression, inhibition of apoptosis, prevention of aggregation of non-native proteins and the recruitment of a cellular heat shock protein, Hsc70, to nuclear domains. We present evidence that ICP22 resembles a cellular J-protein/HSP40 family co-chaperone, interacting specifically with Hsc70. This is the first known example of the acquisition of a complete J-like protein by a virus and suggests that HSV has evolved to manipulate the host proteostatic machinery during the establishment of lytic infection.

molecular biology↗

bioRxiv2018

Sequence variation of rare outer membrane protein β-barrel domains in clinical strains provides insights into the evolution of Treponema pallidum subsp. pallidum, the syphilis spirochete.

In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum (TPA), the syphilis spirochete, and identifying its surface-exposed {beta}-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of {beta}-barrel-encoding regions of tprC, tprD, and bamA, in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used tp0548 genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) {beta}-barrels in clinical strains could be grouped according to allelic variants in TPA reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored {beta}-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. Based upon structural models, non-conservative amino acid substitutions in predicted transmembrane {beta}-strands of TprC and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive TPA OMP diversity and that genetic exchange contributes to the evolutionary biology of TPA. They also set the stage for topology-based analysis of antibody responses against OMPs and help frame strategies for syphilis vaccine development.\n\nIMPORTANCEDespite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum (TPA), little is known about how their surface-exposed, {beta}-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the {beta}-barrel-encoding regions of three OMP loci, tprC, tprD, and bamA, in TPA from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within {beta}-barrel domains occurred predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function also were noted. Our findings suggest that selection pressures exerted by human populations drive TPA OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of TPA and frame strategies for vaccine development based upon conserved OMP extracellular loops.

microbiology↗