The demographics of the ubiquitin system Clague, Michael J; Heride, Claire; Urbé, Sylvie
Trends in cell biology,
07/2015, Letnik:
25, Številka:
7
Journal Article
Recenzirano
Highlights • Global proteome analysis provides an inventory of the ubiquitin system. • DUBs and E2s both outnumber RING E3s. • Key ubiquitin linkage-specific enzymes have been identified.
The post-translational modification of proteins regulates many biological processes. Their dysfunction relates to diseases. Ubiquitination is one of the post-translational modifications that target ...lysine residue and regulate many cellular processes. Three enzymes are required for achieving the ubiquitination reaction: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). E3s play a pivotal role in selecting substrates. Many structural studies have been conducted to reveal the molecular mechanism of the ubiquitination reaction. Recently, the structure of PCAF_N, a newly categorized E3 ligase, was reported. We present a review of the recent progress toward the structural understanding of E3 ligases.
The E3 ligase parkin ubiquitinates outer mitochondrial membrane proteins during oxidative stress and is linked to early‐onset Parkinson's disease. Parkin is autoinhibited but is activated by the ...kinase PINK1 that phosphorylates ubiquitin leading to parkin recruitment, and stimulates phosphorylation of parkin's N‐terminal ubiquitin‐like (pUbl) domain. How these events alter the structure of parkin to allow recruitment of an E2~Ub conjugate and enhanced ubiquitination is an unresolved question. We present a model of an E2~Ub conjugate bound to the phospho‐ubiquitin‐loaded C‐terminus of parkin, derived from NMR chemical shift perturbation experiments. We show the UbcH7~Ub conjugate binds in the open state whereby conjugated ubiquitin binds to the RING1/IBR interface. Further, NMR and mass spectrometry experiments indicate the RING0/RING2 interface is re‐modelled, remote from the E2 binding site, and this alters the reactivity of the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer. Our experiments provide evidence that parkin phosphorylation and E2~Ub recruitment act synergistically to enhance a weak interaction of the pUbl domain with the RING0 domain and rearrange the location of the RING2(Rcat) domain to drive parkin activity.
Synopsis
The multidomain E3 ubiquitin ligase parkin, which ubiquitinates outer mitochondrial membrane proteins in response to oxidative stress, otherwise resides in an auto‐inhibited state. Structural models incorporating new NMR and mass spectrometric data reveal how phosphorylation of its ubiquitin‐like (Ubl) domain and interaction with a phosphorylated ubiquitin allow the E3 ligase to recruit an E2~Ub conjugate needed for the ubiquitination process.
The phosphorylated parkin Ubl domain samples a large conformational space that includes binding to the RING0 domain of parkin.
A UbcH7‐Ub conjugate binds to parkin in an open configuration and causes re‐modeling of the C‐terminal catalytic region of parkin that facilitates pUbl interaction.
UbcH7‐Ub binding to phosphorylated parkin in complex with phospho‐ubiquitin causes large conformational changes in parkin.
Both UbcH7‐Ub and pUbl recruitment are needed to fully optimize parkin ubiquitination activity.
Modeling based on NMR and mass spectrometry data reveals how PINK1‐mediated phosphorylation allows E2 enzyme binding and subsequent remodeling of the autoinhibited multi‐domain E3 parkin into an active E3 ligase.
Eukaryotic cell biology depends on cullin-RING E3 ligase (CRL)-catalysed protein ubiquitylation
, which is tightly controlled by the modification of cullin with the ubiquitin-like protein NEDD8
. ...However, how CRLs catalyse ubiquitylation, and the basis of NEDD8 activation, remain unknown. Here we report the cryo-electron microscopy structure of a chemically trapped complex that represents the ubiquitylation intermediate, in which the neddylated CRL1
promotes the transfer of ubiquitin from the E2 ubiquitin-conjugating enzyme UBE2D to its recruited substrate, phosphorylated IκBα. NEDD8 acts as a nexus that binds disparate cullin elements and the RING-activated ubiquitin-linked UBE2D. Local structural remodelling of NEDD8 and large-scale movements of CRL domains converge to juxtapose the substrate and the ubiquitylation active site. These findings explain how a distinctive ubiquitin-like protein alters the functions of its targets, and show how numerous NEDD8-dependent interprotein interactions and conformational changes synergistically configure a catalytic CRL architecture that is both robust, to enable rapid ubiquitylation of the substrate, and fragile, to enable the subsequent functions of cullin-RING proteins.
The BRCA1-BARD1 tumour suppressor is an E3 ubiquitin ligase necessary for the repair of DNA double-strand breaks by homologous recombination
. The BRCA1-BARD1 complex localizes to damaged chromatin ...after DNA replication and catalyses the ubiquitylation of histone H2A and other cellular targets
. The molecular bases for the recruitment to double-strand breaks and target recognition of BRCA1-BARD1 remain unknown. Here we use cryo-electron microscopy to show that the ankyrin repeat and tandem BRCT domains in BARD1 adopt a compact fold and bind to nucleosomal histones, DNA and monoubiquitin attached to H2A amino-terminal K13 or K15, two signals known to be specific for double-strand breaks
. We further show that RING domains
in BRCA1-BARD1 orient an E2 ubiquitin-conjugating enzyme atop the nucleosome in a dynamic conformation, primed for ubiquitin transfer to the flexible carboxy-terminal tails of H2A and variant H2AX. Our work reveals a regulatory crosstalk in which recognition of monoubiquitin by BRCA1-BARD1 at the N terminus of H2A blocks the formation of polyubiquitin chains and cooperatively promotes ubiquitylation at the C terminus of H2A. These findings elucidate the mechanisms of BRCA1-BARD1 chromatin recruitment and ubiquitylation specificity, highlight key functions of BARD1 in both processes and explain how BRCA1-BARD1 promotes homologous recombination by opposing the DNA repair protein 53BP1 in post-replicative chromatin
. These data provide a structural framework to evaluate BARD1 variants and help to identify mutations that drive the development of cancer.
Post‐translational modification by ubiquitination determines intracellular location and fate of numerous proteins, thus impacting a diverse array of physiologic functions. Past dogma has been that ...ubiquitin was only coupled to substrates by isopeptide bonds to internal lysine residues or less frequently peptide bonds to the N‐terminus. Enigmatically, however, several proteins lacking lysines had been reported to retain ubiquitin‐dependent fates. Resolution of this paradox was afforded by recent observations that ubiquitination of substrates can also occur on cysteine or serine and threonine residues by thio‐ or oxy‐ester bond formation, respectively (collectively called esterification). Although chemically possible, these bonds were considered too labile to be of physiological relevance. In this review we discuss recent evidence for the ubiquitination of protein substrates by esterification and speculate on its mechanism and its physiological importance.
Protein ubiquitination is one of the most powerful posttranslational modifications of proteins, as it regulates a plethora of cellular processes in distinct manners. Simple monoubiquitination events ...coexist with more complex forms of polyubiquitination, the latter featuring many different chain architectures. Ubiquitin can be subjected to further posttranslational modifications (e.g., phosphorylation and acetylation) and can also be part of mixed polymers with ubiquitin-like modifiers such as SUMO (small ubiquitin-related modifier) or NEDD8 (neural precursor cell expressed, developmentally downregulated 8). Together, cellular ubiquitination events form a sophisticated and versatile ubiquitin code. Deubiquitinases (DUBs) reverse ubiquitin signals with equally high sophistication. In this review, we conceptualize the many layers of specificity that DUBs encompass to control the ubiquitin code and discuss examples in which DUB specificity has been understood at the molecular level. We further discuss the many mechanisms of DUB regulation with a focus on those that modulate catalytic activity. Our review provides a framework to tackle lingering questions in DUB biology.
HECT-family E3 ligases ubiquitinate protein substrates to control virtually every eukaryotic process and are misregulated in numerous diseases. Nonetheless, understanding of HECT E3s is limited ...by a paucity of selective and potent modulators. To overcome this challenge, we systematically developed ubiquitin variants (UbVs) that inhibit or activate HECT E3s. Structural analysis of 6 HECT-UbV complexes revealed UbV inhibitors hijacking the E2-binding site and activators occupying a ubiquitin-binding exosite. Furthermore, UbVs unearthed distinct regulation mechanisms among NEDD4 subfamily HECTs and proved useful for modulating therapeutically relevant targets of HECT E3s in cells and intestinal organoids, and in a genetic screen that identified a role for NEDD4L in regulating cell migration. Our work demonstrates versatility of UbVs for modulating activity across an E3 family, defines mechanisms and provides a toolkit for probing functions of HECT E3s, and establishes a general strategy for systematic development of modulators targeting families of signaling proteins.
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•High-affinity and selective UbV modulators for 20 HECT E3 ligases•UbV inhibitors hijack the E2 binding site•N-lobe exosite bound UbVs activate HECT E3 ligases•UbVs function in cells and intestinal organoids to reveal new roles of HECT E3 ligases
Zhang, Wu et al. generated ubiquitin variants (UbVs) that inhibit or activate the catalytic activities of HECT E3 ligases. These variants can be used to delve into E3 mechanisms and to probe new biological functions of HECT E3s.
The multi-domain deubiquitinase USP15 regulates diverse eukaryotic processes and has been implicated in numerous diseases. We developed ubiquitin variants (UbVs) that targeted either the catalytic ...domain or each of three adaptor domains in USP15, including the N-terminal DUSP domain. We also designed a linear dimer (diUbV), which targeted the DUSP and catalytic domains, and exhibited enhanced specificity and more potent inhibition of catalytic activity than either UbV alone. In cells, the UbVs inhibited the deubiquitination of two USP15 substrates, SMURF2 and TRIM25, and the diUbV inhibited the effects of USP15 on the transforming growth factor β pathway. Structural analyses revealed that three distinct UbVs bound to the catalytic domain and locked the active site in a closed, inactive conformation, and one UbV formed an unusual strand-swapped dimer and bound two DUSP domains simultaneously. These inhibitors will enable the study of USP15 function in oncology, neurology, immunology, and inflammation.
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•Tight and selective UbVs target USP15 catalytic and adaptor domains•UbV inhibitors lock the USP15 active site in an inactive conformation•A strand-swapped UbV dimer binds two DUSP domains simultaneously•Linear UbV dimers are potent and specific USP15 inhibitors in cells
Teyra et al. developed ubiquitin variants (UbVs) that targeted distinct domains of the deubiquitinase USP15 and revealed alternative inhibition and dimerization strategies. Linear UbV dimers exhibited improved potency and specificity of USP15 inhibition. UbVs can be used to study the catalytic mechanism and biological functions of USP15.
Ubiquitin E3 ligases control every aspect of eukaryotic biology by promoting protein ubiquitination and degradation. At the end of a three-enzyme cascade, ubiquitin ligases mediate the transfer of ...ubiquitin from an E2 ubiquitin-conjugating enzyme to specific substrate proteins. Early investigations of E3s of the RING (really interesting new gene) and HECT (homologous to the E6AP carboxyl terminus) types shed light on their enzymatic activities, general architectures, and substrate degron-binding modes. Recent studies have provided deeper mechanistic insights into their catalysis, activation, and regulation. In this review, we summarize the current progress in structure-function studies of ubiquitin ligases as well as exciting new discoveries of novel classes of E3s and diverse substrate recognition mechanisms. Our increased understanding of ubiquitin ligase function and regulation has provided the rationale for developing E3-targeting therapeutics for the treatment of human diseases.
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