Biology subjects

Kulathu, Y.

Publications and source records attributed to Kulathu, Y..

12 recordsLinked to original sources

bioRxiv2026

FAF1 and FAF2 enhance human p97-UFD1-NPL4 complex unfoldase activity enabling rational design of p97 activators

VCP/p97 is an AAA+ ATPase that, together with its cofactors UFD1-NPL4 (p97-UN), binds and unfolds ubiquitylated substrates to maintain cellular homeostasis. The human p97-UN complex associates with additional cofactors, but how these cofactors modulate p97-UN activity is not fully understood. Here, we screen for cofactors that enhance p97-UN activity and identify FAF2 as the strongest activator. Using biochemical and structural approaches, we show how FAF2 engages p97-UN and polyubiquitin to promote unfolding. We define a conserved activation motif in FAF2 that contacts both UFD1 and the ubiquitin proximal to the initiator, stabilizing and initiating unfolding in a UFD1-dependent manner. We leverage the features of FAF2 AM to engineer de novo proteins that potently enhance unfolding, providing a rational strategy to boost p97 activity. Our findings reveal how cofactors can provide additional adaptive control, fine-tuning human p97 activity to unfold challenging substrates and those modified with short ubiquitin chains.

biochemistry↗

bioRxiv2025

Systematic Discovery of Motif-based Interactions of the Auxiliary Domains of USP Family Deubiquitinases

The ubiquitin-specific proteases (USPs) family is the largest family of human deubiquitinating enzymes (DUBs). While most USPs are agnostic to polyubiquitin linkage-type, their substrate specificity is thought to be mediated by the recognition of the ubiquitylated protein itself. In addition to their catalytic domain, USPs have one or more auxiliary domains (ADs) with key functions in regulating DUB activity and subcellular localization. We hypothesize that some ADs bind short linear motifs (SLiMs) typically found in intrinsically disordered regions of proteins to achieve targeting to substrates and multiprotein complexes. To test this hypothesis, we systematically assessed the potential of 29 USP-ADs and two full-length USPs for SLiM binding using a combination of proteomic-peptide phage display, peptide arrays and affinity measurements. We discovered SLiM-based interactions for 14 ADs from 9 USP-DUBs, including CYLD, USP11, USP19, USP20, USP22 and USP33, and define the consensus motif and properties of the SLiM-AD binding. Interestingly, we established that the zf-UBP and DUSP2 domains of USP20 and USP33 are SLiM binding ADs with similar binding profiles, explaining the functional redundancy between the two DUBs. Our work reveals unique motifs recognized by the auxiliary domains CAP-Gly, UBL, zf-UBP and DUSP, with potential functional implications for substrate recognition and complex assemblies.

biochemistry↗

bioRxiv2025

The EF-hand domain of MINDY3 is a Ubiquitin and RAD23 UBL-binding domain

The MINDY family of deubiquitinases (DUBs) are exemplified by their preference for cleaving K48-linked polyubiquitin. MINDY3 is architecturally distinct from other MINDY DUBs as its catalytic domain spans the entire length of the protein except for an atypical EF-hand insertion. We uncover this EF-hand (MINDY3EF-hand) to be a ubiquitin-binding domain with three distinct binding sites, enabling MINDY3 to bind and effectively cleave long polyubiquitin chains. Furthermore, the MINDY3EF-hand domain binds not only to polyubiquitin but also to the UBL domain of the proteasome shuttling and DNA repair factors RAD23A and RAD23B. The MINDY3EF-hand facilitates this interaction with RAD23s in cells and mediates MINDY3 recruitment to DNA damage sites, establishing this unique DUB as a potential regulator of cellular DNA damage responses. MINDY3 binds specifically to the UBL domain of RAD23s, and none of the other UBLs tested. The crystal structure of the MINDY3EF-hand:RAD23AUBL domain complex reveals the molecular basis for specificity. We find that MINDY3 can form a ternary complex with RAD23A/B and polyubiquitin, and our findings suggest a model wherein MINDY3 can deubiquitylate RAD23A/B-bound clients.

biochemistry↗

bioRxiv2025

ATXN3 regulates lysosome regeneration after damage by targeting K48-K63-branched ubiquitin chains

The cellular response to lysosomal damage involves fine-tuned mechanisms of membrane repair, lysosome regeneration and lysophagy, but how these different processes are coordinated is not fully understood. Here we show in human cells that the deubiquitinating enzyme ATXN3 helps restore integrity of the lysosomal system after damage by targeting K48-K63-linked branched ubiquitin chains on regenerating lysosomes. We find that ATXN3 translocates to lysosomes after different types of damage and there colocalizes with its partner, VCP/p97. ATXN3 recruitment occurs late after the initial repair of a subset of lysosomes and after phagophore formation on terminally damaged lysosomes. Of note, inactivation of ATXN3 by induced degradation, depletion or knock-out impairs clearance of damaged lysosomes and full restoration of lysosomal capacity. Mechanistically, ATXN3, along with VCP/p97, turns over K48-K63-branched ubiquitin conjugates on LAMP1-positive, phosphatidylinositol-(4,5)-bisphosphate-decorated compartments that are not yet fully re-acidified indicating involvement in lysosome regeneration. Our findings identify a key role of ATXN3 in restoring lysosomal function after lysosomal membrane damage and uncover K48-K63-branched ubiquitin chain-regulated regeneration as a critical element of the lysosomal damage stress response.

cell biology↗

bioRxiv2024

Cross-species interactome analysis uncovers a conserved selective autophagy mechanism for protein quality control in plants

Selective autophagy is a fundamental protein quality control pathway that safeguards proteostasis by degrading damaged or surplus cellular components, particularly under stress. This process is orchestrated by selective autophagy receptors (SARs) that direct specific cargo for degradation. While significant strides have been made in understanding the molecular framework of selective autophagy, the diversity of SAR repertoires across species remain largely unexplored. Through a comparative interactome analysis across five model organisms, we identified a suite of conserved and lineage-specific SAR candidates. Among these, we validated CESAR as a conserved SAR critical for proteostasis under heat stress. CESAR specifically facilitates the degradation of hydrophobic, ubiquitinated protein aggregates and is indispensable for heat stress tolerance. Our study offers a rich resource for SAR discovery and positions CESAR as a pivotal regulator of proteostasis, with broad implications for improving stress resilience in plants.

plant biology↗

bioRxiv2023

Comprehensive approach to study branched ubiquitinchains reveals roles for K48-K63 branches inVCP/p97-related processes

Branched ubiquitin (Ub) chains make up a significant proportion of Ub polymers in human cells and are formed when two or more sites on a single Ub molecule are modified with Ub creating bifurcated architectures. Despite their abundance, we have a poor understanding of the cellular functions of branched Ub signals that stems from a lack of facile tools and methods to study them. Here we develop a comprehensive pipeline to define branched Ub function, using K48-K63-branched chains as a case study. We discover branch-specific binders and, by developing a method that monitors cleavage of linkages within complex polyUb, we discover the VCP/p97-associated ATXN3, and MINDY family deubiquitinases to act as debranching enzymes. By engineering and utilizing a branched K48-K63-Ub chain-specific nanobody, we reveal roles for these chains in VCP/p97-related processes. In summary, we provide a blueprint to investigate branched Ub function that can be readily applied to study other branched chain types. HighlightsO_LIAssembly of defined branched ubiquitin chains enables identification of specific binding proteins C_LIO_LIDevelopment of quantitative DUB assay monitoring cleavage of individual Ub linkages within complex ubiquitin chains identifies debranching enzymes C_LIO_LIEngineering specific, high-affinity nanobody against branched K48-K63 ubiquitin reveals roles in VCP/p97 related processes and DNA damage responses C_LIO_LIGeneral blueprint of new methods and tools for in-depth characterization of branched ubiquitin chains and their underlying biology C_LI

biochemistry↗

bioRxiv2022

Human UFSP1 is an active protease that regulates UFM1 maturation and UFMylation

An essential first step in the posttranslational modification of proteins with UFM1, UFMylation, is the proteolytic cleavage of pro-UFM1 to expose a C-terminal glycine. Of the two UFM1-specific proteases (UFSPs) identified in humans, only UFSP2 is reported to be active since the annotated sequence of UFSP1 lacks critical catalytic residues. Nonetheless, efficient UFM1 maturation occurs in cells lacking UFSP2 suggesting the presence of another active protease. We hereby identify a long isoform of UFSP1 to be this protease. Cells lacking both UFSPs show complete loss of UFMylation resulting from an absence of mature UFM1. While UFSP2, but not UFSP1, removes UFM1 from the ribosomal subunit RPL26, UFSP1 acts earlier in the pathway to mature UFM1 and cleave a potential auto-inhibitory modification on UFC1, thereby controlling activation of UFMylation. In summary, our studies reveal important distinctions in substrate specificity and localization-dependent functions for the two proteases in regulating UFMylation.

biochemistry↗

bioRxiv2022

Non-canonical scaffold-type ligase complex mediates protein UFMylation

Protein UFMylation is emerging as a posttranslational modification essential for endoplasmic reticulum and cellular homeostasis. Despite its biological importance, we have a poor understanding of how UFM1 is conjugated onto substrates. Here, we use a rebuilding approach to define the minimal requirements of protein UFMylation. We find that the reported E3 ligase UFL1 is inactive on its own and identify UFBP1 to bind UFL1 to form an active E3 ligase complex. While UFC1 is an intrinsically Cys-reactive E2, we do not identify any catalytic cysteines on UFL1/UFBP1, suggesting a scaffold-type E3 ligase mechanism. Interestingly, the E3 ligase complex consists of winged-helix (WH) domain repeats that activate UFC1 for aminolysis. We identify the adaptor protein CDK5RAP3 to bind to and regulate E3 ligase activity potentially by preventing off-target UFMylation. In summary, our work identifies the minimal requirements for UFMylation and reveals regulatory principles of this atypical E3 ligase complex.

biochemistry↗

bioRxiv2021

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp3 Papain-like Protease

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom chemical library from which we identified Dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.

biochemistry↗

bioRxiv2021

Mechanism of activation and regulation of Deubiquitinase activity in MINDY1 and MINDY2

Of the eight distinct polyubiquitin chains that can be assembled, K48-linked ubiquitin is the most well-understood linkage and modification of proteins with K48 chains targets the modified protein for degradation. By removing ubiquitin from substrates or trimming ubiquitin chains, deubiquitinases (DUBs) can modulate the outcome of ubiquitylation. MINDY1 and MINDY2 are members of the MINDY family of DUBs that have exquisite specificity for cleaving K48-linked polyubiquitin. Being recently discovered DUBs, we have a poor understanding of their catalytic mechanism. By analysing crystal structures of MINDY1 alone and in complex with monoubiquitin or K48-linked ubiquitin chains, we here reveal how substrate interaction relieves autoinhibition and activates the DUB. Further, our analyses reveal a non-canonical catalytic triad composed of Cys-His-Thr and explain how these DUBs sense both ubiquitin chain length and linkage type to trim K48-linked ubiquitin chains. Our findings highlight the multiple layers of regulation modulating DUB activity in MINDY1 and MINDY2. SynopsisO_LIStructure of MINDY1 in complex with K48-linked diUb reveals how K48-linked polyUb is recognized and cleaved C_LIO_LIThe Cys loop mediates autoinhibition of the DUB and substrate binding at the S1 and S1 sites relieves autoinhibition and activates the enzyme for catalysis C_LIO_LIMINDY1 uses a non-canonical catalytic triad composed of Cys-His-Thr C_LIO_LIMINDY1 has five ubiquitin binding sites within its catalytic domain and switches from exo to endo cleavage in a ubiquitin chain length-dependent manner C_LI

biochemistry↗

bioRxiv2020

Characterization of protease activity of Nsp3 from SARS-CoV-2 and its in vitro inhibition by nanobodies

Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease PLpro domain that cleaves not only the viral protein but also polyubiquitin and the ubiquitin-like modifier ISG15 from host cells. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.

biochemistry↗

bioRxiv2019

UFMylation of MRE11 is essential for telomere length and hematopoietic stem cell survival

Genetic studies using knockout mouse models provide strong evidence for the essential role of the ubiquitin-like protein UFM1 for hematopoiesis, especially erythroid development, yet its biological roles in this process are largely unknown. Here we have identified a UFL1-dependent UFMylation of the MRE11 nuclease on the K281 and K282 residues. We show that Hela cells lacking the specific UFM1 E3 ligase display severe telomere shortening. We further demonstrate either by deleting UFM1 or by mutating MRE11 UFMylation sites that preventing MRE11 UFMylation impacts its interaction with the telomere protein TRF2. However, the MRE11 function in double-strand-break repair remains intact. We validate these results in vivo by showing that Zebrafish knockouts for the genes ufl1 and ufm1 have shorter telomeres in hematopoietic cells. Here we present UFMylation has a new mechanisms of regulation for telomere length maintenance with a role in hematopoiesis. Key pointsModification of MRE11 by UFM1 regulates telomere maintenance and cell death in HSCs Scientific categoryUFMylation, telomere maintenance, hematopoietic stem cell survival.

molecular biology↗