Unanchored K63-linked polyubiquitin chains: a novel second messenger involved in G protein-coupled receptors early signaling events
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ABSTRACT:
PROVIDER: PRJNA1285873 | ENA |
REPOSITORIES: ENA
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Project description:GPCR rapidly transduced extracellular cues via their ability to activate effector proteins leading to the production of a variety of well-characterized second messenger molecules. We describe here that the E3 ubiquitin ligase tumor necrosis factor receptor associated factor 6 (TRAF6) is required for the rapid and transient production of unanchored lysine (K)63-linked polyubiquitin chains in primary and transformed cells exposed to vasoactive agonists Angiotensin II (Ang II), Lysophosphatidic acid (LPA) and Thrombin (Thr). We further show that unanchored K63-linked polyubiquitin chains accumulate in Transforming Growth Factor beta-activated kinase 1 (TAK1) and IkappaB kinase beta (IKKbeta) immunocomplexes and are required for the T-loop phosphorylation of TAK1 leading to IKKbeta, c- Jun N-terminal kinases (JNK1/2) and p38 mitogen-activated protein kinases (p38) phosphorylation and the induction of transcriptional events. Our results support a novel paradigm in field of GPCR signal transduction, identifying unanchored K63-linked polyubiquitin chains as novel second messenger molecules.
2025-07-07 | GSE301643 | GEO
Project description:Ubiquitin chains encode signals defining the fates of their modified proteins. Ubiquitin chain-mediated proteasomal degradation is a widespread eukaryotic regulatory mechanism. Yet, heterogeneity of intracellular ubiquitination has precluded systematically comparing degradation capacities of different ubiquitin chains. Here, we monitor cellular degradation and deubiquitination at high temporal resolution after delivery of bespoke ubiquitinated proteins into human cells. Comparing degradation of a model substrate modified with various K48, K63 or K48/K63-branched ubiquitin chains revealed fundamental differences in their intracellular degradation capacities. K48 chains with three or more ubiquitins triggered degradation within minutes. K63-ubiquitinated substrate was rapidly deubiquitinated rather than degraded. Surprisingly, degradation and deubiquitination behaviour of K48/K63-branched chains was determined by the identity of the substrate-anchored chain, establishing that branched chains are not the sum of their parts. Overall, our work reveals a degradation code for ubiquitin chains varying by linkage, length and topology and a functional hierarchy within branched ubiquitin chains.
2025-06-23 | PXD060731 | Pride
Project description:Assembly of polymeric ubiquitin (Ub) chains is an essential posttranslational protein modification that regulates widespread intracellular processes in eukaryotic cells. Factors and mechanisms that regulate the formation of Ub chains are key to the understanding of many cellular processes. The E2 enzyme Ubc1 (Ube2K) exclusively targets K48 in Ub for chain formation and has been linked to cell-cycle progression and proteostasis. Uniquely among E2 enzymes, it harbors a Ub binding UBA domain, which has been implicated in Ub chain formation but its function remained elusive. Through in vitro binding experiments, we unexpectedly found that the UBA domain enables preferential binding of K63-linked Ub chains. Based on structural modeling, extensive in vitro ubiquitination experiments and NMR binding studies, we propose a mechanism through which Ubc1 selectively forms K48/K63 branched Ub chains – an usual chain architecture, about whose prevalence and function little is known to date. Ultimately, we link UBA dependent activity of Ubc1 to its role in proteostasis through genetic experiments.
2021-09-09 | PXD018651 | Pride
Project description:The assembly of a specific polymeric ubiquitin (Ub) chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The ubiquitin-conjugating (E2) enzymes Ubc1 in yeast and Ube2K in mammals exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a Ub binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with Ub chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments and NMR studies, we propose that the UBA domain aligns Ubc1 with K63-linked polyubiquitin and facilitates the selective assembly of K48/K63-branched Ub molecules. Genetic experiments link the activity of the UBA domain and hence the formation of this unusual Ub chain topology to the maintenance of cellular proteostasis.
2021-03-02 | PXD022621 | Pride
Project description:SpyTagged branched and unbranched K48- and K63-linked Tetra-Ubiquitin chains were immobilized on SpyCatcher agarose (48Ub4; 63Ub4; [Ub]2-48,63Ub-48Ub; [Ub]2-48,63Ub-63Ub). Pulldown were performed from protease-inhibitor treated U2OS cell lines to identify specific binders of K48-K63-branched Ub4.
2024-05-07 | PXD046025 | Pride
Project description:Pseudomonas species employ complex, dynamic signalling networks to fine-tune responses to changing environments, with regulation taking place at the transcriptional, post-transcriptional and post-translational levels. Control of mRNA translation and hence protein abundance is a crucial element of this regulatory network. Previously, we identified the ribosomal modification protein RimK, which influences the transition between active and sessile bacterial lifestyles. RimK is an ATP-dependent glutamyl ligase that adds glutamate residues to the C-terminus of ribosomal protein RpsF. This in-turn induces specific changes in ribosome function and translational output. RimK activity is itself under complex, multifactorial control: by the bacterial second messenger cyclic-di-GMP; a phosphodiesterase trigger enzyme (RimA); and a polyglutamate-specific protease (RimB). Deletion of the rim operon affects phenotypes including attachment, motility and cytotoxicity, severely compromising rhizosphere colonisation by the soil bacterium Pseudomonas fluorescens. Using a combination of protein biochemistry, quantitative proteomics and ribosomal profiling experiments, we examined the relationship between ribosomal modification and downstream changes in the P. fluorescens proteome. RimK activity leads to active proteome remodelling by two main routes; indirectly, through changes in the abundance of the global translational regulator Hfq and directly, with translation of surface attachment factors, amino acid transporters and key secreted molecules linked specifically to RpsF modification. Our findings suggest post-translational ribosomal modification as a rapid-response mechanism to tune global gene translation and thereby protein abundance in response to environmental signals.
2020-06-30 | PXD017233 | Pride
Project description:Cyclic di-GMP (c-di-GMP) is a ubiquitous second messenger that regulates many biological processes in bacteria. The genome in Mycobacterium tuberculosis encodes a single copy of the diguanylate cyclase gene (dgc) responsible for c-di-GMP synthesis. To determine the role of c-di-GMP signaling in M. tuberculosis, the mutant strain of Δdgc was generated in the virulent H37Rv strain. We used whole genome microarray expression profiling as a discovery platform to identify the genes controlled by c-di-GMP in M. tuberculosis, providing molecular proof for the phenotypes modulated by the signaling.
2013-10-09 | GSE37973 | GEO
Project description:STING activation by cyclic dinucleotides in mammals induces Type I Interferon- and NFkB-related gene expression, and the lipidation of LC3B at Golgi membranes. While the mechanisms of the Interferon-related responses are well understood, the mechanism of NFkB activation mediated by STING activation, and how the multi-functional responses downstream of STING signaling are related remain unknown. We report that STING activation induces K63 and linear ubiquitin chain formation, and ubiquitin co-localizes with STING and LC3B at Golgi-derived vesicles. Loss of the LUBAC E3 ubiquitin ligase subunit, HOIP, prevents formation of the linear, but not K63-linked, ubiquitin chains. The ubiquitin and LC3B binding proteins, Optineurin and TNIP1, are co-localized with STING activation-induced, perinuclear LC3B foci via their UBAN domains, however loss of HOIP does not influence this localization, indicating they may bind the K63-linked ubiquitin chains. HOIP-mediated linear ubiquitin chains are well established to mediate NFkB downstream of TNFa and IL1beta receptor stimulation. Loss of HOIP in monocytic THP-1 cells inhibits STING-induced NFkB activation and NFkB-related gene transcription, as well as Interferon-related transcription. Further, the recently reported proton channel activity of STING that blocks LC3B lipidation at Golgi membranes is also important for both K63 and Linear ubiquitin chain formation, and NFkB signaling. Thus, STING induces HOIP and linear ubiquitin chain formation to activate NFkB.
| MSV000092940 | MassIVE
Project description:Ubiquitin-specific proteases (USPs) are the largest class of human deubiquitinases (DUBs) and comprise its phylogenetically most distant members USP53 and USP54, which are annotated as catalytically inactive pseudo-enzymes. Conspicuously, mutations in the USP domain of USP53 cause progressive familial intrahepatic cholestasis. Here we report the discovery that USP53 and USP54 are active DUBs with high specificity for K63-linked polyubiquitin. We demonstrate how USP53 patient mutations abrogate catalytic activity, implicating loss of DUB activity in USP53-mediated pathology. Depletion of USP53 increases K63-linked ubiquitination of tricellular junction components. Assays with substrate-bound polyubiquitin reveal that USP54 cleaves within K63-linked chains, whereas USP53 can en bloc deubiquitinate substrate proteins in a K63-linkage-dependent manner. Biochemical and structural analyses uncover underlying K63-specific S2-ubiquitin-binding sites within their catalytic domains. Collectively, our work revises the annotation of USP53 and USP54, provides reagents and a mechanistic framework to investigate K63-polyubiquitin decoding, and establishes K63-linkage-directed deubiquitination as novel DUB activity.
2024-11-11 | PXD054748 | Pride
Project description:Ubiquitin-interactor co-pulldown coupled with mass spectrometry used to elucidate K48- and K63-linked interactomes including those of Ub chains of varying length (Ub-Ub3) and heterotypic branched Ub chains. Ubiquitin-interactors were enriched from human HeLa and yeast s. cerevisiae cell lysate with either chloroacetamide (CAA) or N-ethylmaleimide (NEM) as DUB inhibitors.
2024-05-15 | PXD051890 | Pride