Explore the vast range of titles available.

Key Points The reversible conjugation of ubiquitin chains to protein substrates regulates almost every cellular process. Ubiquitin chains can be assembled via one of the seven ubiquitin Lys residues (which are Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 or Lys63) or via the amino terminus (Met1). Atypical ubiquitin chains are those not linked via canonical Lys48 linkages or Lys63 linkages. Available structures of ubiquitin chains suggest that each linkage type adopts unique conformations. Ubiquitin-binding domains (UBDs) and deubiquitinases (DUBs) exploit the distinct features of polyubiquitin to achieve specificity. All linkage types coexist in cells, and the abundance of particular atypical linkages changes in response to specific stimuli and can be altered in disease states. The enzymatic assembly of ubiquitin chains requires the action of E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes and E3 ubiquitin-ligating enzymes. E2 enzymes and certain classes of E3 ligases determine the chain linkage type. DUBs negatively regulate ubiquitylation by hydrolyzing ubiquitin chains. Although many DUBs are promiscuous, certain DUBs such as members of the ovarian tumour (OTU) family have evolved distinct mechanisms to achieve linkage selectivity. New tools such as the application of chemical biology techniques to achieve site-specific ubiquitylation, the generation of ubiquitin polymers of defined linkage types, linkage-specific antibodies and ubiquitin sensors will advance the field of ubiquitin research. Little is known about the biological roles of atypical ubiquitylation events. The reported physiological roles highlight the fact that the differently linked polyubiquitin chains are independent post-translational modifications. Ubiquitin can form eight structurally distinct chain types. Recent advances have elucidated the mechanisms of linkage-specific chain assembly, recognition and hydrolysis. The cellular roles of the six 'atypical' ubiquitin chains (linked via Lys6, Lys11, Lys27, Lys29, Lys33 or Met1 of ubiquitin) are beginning to emerge, highlighting how they can each act as independent post-translational modifications. Ubiquitylation is one of the most abundant and versatile post-translational modifications (PTMs) in cells. Its versatility arises from the ability of ubiquitin to form eight structurally and functionally distinct polymers, in which ubiquitin moieties are linked via one of seven Lys residues or the amino terminus. Whereas the roles of Lys48- and Lys63-linked polyubiquitin in protein degradation and cellular signalling are well characterized, the functions of the remaining six 'atypical' ubiquitin chain types (linked via Lys6, Lys11, Lys27, Lys29, Lys33 and Met1) are less well defined. Recent developments provide insights into the mechanisms of ubiquitin chain assembly, recognition and hydrolysis and allow detailed analysis of the functions of atypical ubiquitin chains. The importance of Lys11 linkages and Met1 linkages in cell cycle regulation and nuclear factor-κB activation, respectively, highlight that the different ubiquitin chain types should be considered as functionally independent PTMs.

Protein ubiquitination is a dynamic multifaceted post-translational modification involved in nearly all aspects of eukaryotic biology. Once attached to a substrate, the 76-amino acid protein ubiquitin is subjected to further modi- fications, creating a multitude of distinct signals with distinct cellular outcomes, referred to as the ＇ubiquitin code＇. Ubiquitin can be ubiquitinated on seven lysine （Lys） residues or on the N-terminus, leading to polyubiquitin chains that can encompass complex topologies. Alternatively or in addition, ubiquitin Lys residues can be modified by ubiq- uitin-like molecules （such as SUMO or NEDD8）. Finally, ubiquitin can also be acetylated on Lys, or phosphorylated on Ser, Thr or Tyr residues, and each modification has the potential to dramatically alter the signaling outcome. While the number of distinctly modified ubiquitin species in cells is mind-boggling, much progress has been made to characterize the roles of distinct ubiquitin modifications, and many enzymes and receptors have been identified that create, recognize or remove these ubiquitin modifications. We here provide an overview of the various ubiqnitin modifications present in cells, and highlight recent progress on ubiquitin chain biology. We then discuss the recent findings in the field of ubiquitin acetylation and phosphorylation, with a focus on Ser65-phosphorylation and its role in mitophagy and Parkin activation.

Mutations in the protein Parkin are associated with Parkinson's disease (PD), the second most common neurodegenerative disease in men. Parkin is an E3 ubiquitin (Ub) ligase of the structurally uncharacterized RING‐in‐between‐RING(IBR)‐RING (RBR) family, which, in an HECT‐like fashion, forms a catalytic thioester intermediate with Ub. We here report the crystal structure of human Parkin spanning the Unique Parkin domain (UPD, also annotated as RING0) and RBR domains, revealing a tightly packed structure with unanticipated domain interfaces. The UPD adopts a novel elongated Zn‐binding fold, while RING2 resembles an IBR domain. Two key interactions keep Parkin in an autoinhibited conformation. A linker that connects the IBR with the RING2 over a 50‐Å distance blocks the conserved E2∼Ub binding site of RING1. RING2 forms a hydrophobic interface with the UPD, burying the catalytic Cys431, which is part of a conserved catalytic triad. Opening of intra‐domain interfaces activates Parkin, and enables Ub‐based suicide probes to modify Cys431. The structure further reveals a putative phospho‐peptide docking site in the UPD, and explains many PD‐causing mutations. The complete structural view of a RING‐IBR‐RING (RBR) ubiquitin ligase domain reveals an unexpected catalytic triad and explains the effects of various Parkin mutations underlying Parkinson's disease.

This study provides insights into conformational changes that lead to phospho-ubiquitin-induced PARKIN activation and how PARKIN is recruited to phospho-ubiquitin chains on mitochondria; the crystal structure of PARKIN in complex with phospho-ubiquitin also indicates that the pocket within PARKIN where phospho-ubiquitin binds carries amino acid residues that are mutated in patients with autosomal-recessive juvenile Parkinsonism. PARKIN activation mechanism revealed The enzymatic duo PARKIN and PINK1 are notable not only because they regulate the process of mitophagy, whereby the cell degrades its damaged mitochondria, but also because they are mutated in autosomal-recessive juvenile Parkinson disease (AR-JP). At a molecular level, PINK1 activates PARKIN by phosphorylating both the ubiquitin (Ub)-like (Ubl) domain of PARKIN and Ub molecules. David Komander and colleagues provide insights into conformational changes that lead to phosphoUb-induced PARKIN activation and how PARKIN recruits phosphoUb chains on mitochondria. The crystal structure of PARKIN in complex with phosphoUb also indicates that the pocket within PARKIN where phosphoUb binds carries amino acid residues that are mutated in patients with AR-JP. The E3 ubiquitin ligase PARKIN (encoded by PARK2 ) and the protein kinase PINK1 (encoded by PARK6 ) are mutated in autosomal-recessive juvenile Parkinsonism (AR-JP) and work together in the disposal of damaged mitochondria by mitophagy 1 , 2 , 3 . PINK1 is stabilized on the outside of depolarized mitochondria and phosphorylates polyubiquitin 4 , 5 , 6 , 7 , 8 as well as the PARKIN ubiquitin-like (Ubl) domain 9 , 10 . These phosphorylation events lead to PARKIN recruitment to mitochondria, and activation by an unknown allosteric mechanism 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 . Here we present the crystal structure of Pediculus humanus PARKIN in complex with Ser65-phosphorylated ubiquitin (phosphoUb), revealing the molecular basis for PARKIN recruitment and activation. The phosphoUb binding site on PARKIN comprises a conserved phosphate pocket and harbours residues mutated in patients with AR-JP. PhosphoUb binding leads to straightening of a helix in the RING1 domain, and the resulting conformational changes release the Ubl domain from the PARKIN core; this activates PARKIN. Moreover, phosphoUb-mediated Ubl release enhances Ubl phosphorylation by PINK1, leading to conformational changes within the Ubl domain and stabilization of an open, active conformation of PARKIN. We redefine the role of the Ubl domain not only as an inhibitory 13 but also as an activating element that is restrained in inactive PARKIN and released by phosphoUb. Our work opens up new avenues to identify small-molecule PARKIN activators.

The deubiquitylating enzymes (DUBs, also known as deubiquitylases or deubiquitinases) maintain the dynamic state of the cellular ubiquitome by releasing conjugated ubiquitin from proteins. In light of the many cellular functions of ubiquitin, DUBs occupy key roles in almost all aspects of cell behaviour. Many DUBs show selectivity for particular ubiquitin linkage types or positions within ubiquitin chains. Others show chain-type promiscuity but can select a distinct palette of protein substrates via specific protein–protein interactions established through binding modules outside of the catalytic domain. The ubiquitin chain cleavage mode or chain linkage specificity has been related directly to biological functions. Examples include regulation of protein degradation and ubiquitin recycling by the proteasome, DNA repair pathways and innate immune signalling. DUB cleavage specificity is also being harnessed for analysis of ubiquitin chain architecture that is assembled on specific proteins. The recent development of highly specific DUB inhibitors heralds their emergence as a new class of therapeutic targets for numerous diseases.By opposing protein ubiquitylation, deubiquitylating enzymes (DUBs) regulate various cellular processes, including protein degradation, the DNA damage response, cell signalling and autophagy. Many DUBs show high specificity for ubiquitin chain architecture and/or the protein substrate that they recognize, and have emerged as exciting therapeutic targets within the field of proteostasis.

Key Points Ubiquitylation is a reversible post-translational modification involved in a myriad of cellular functions. A superfamily of approximately 100 ubiquitin-specific proteases, called deubiquitylating enzymes, deubiquitinases or DUBs, remove ubiquitin from target proteins, disassemble polymeric ubiquitin chains and process ubiquitin precursor polypeptides to maintain ubiquitin homeostasis in cells. Most DUBs are Cys proteases; a small group are metalloproteases. DUBs are classified into five families (ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs), Ovarian tumour proteases (OTUs), Josephins and JAB1/MPN/Mov34 metalloenzymes (JAMMs, also known as MPN+) that are structurally unrelated, but all interact with a common hydrophobic patch on ubiquitin. Multiple layers of regulation modulate the activity and specificity of these enzymes. Specificity also entails recognition of and selective activity towards particular ubiquitin chain types, at least eight of which are now known to coexist in yeast and mammalian cells. DUBs might function to regulate both the stability and the activity of target proteins, which include oncogenes and tumour suppressors. Their wide-ranging involvement in key regulatory processes makes DUBs attractive targets for drug therapy. A large superfamily of deubiquitinases (DUBs) has a key role in both determining protein stability and terminating ubiquitin-dependent signal transduction. Structural and biochemical studies have started to reveal the underlying principles by which DUB substrate specificity is achieved. Ubiquitylation is a reversible protein modification that is implicated in many cellular functions. Recently, much progress has been made in the characterization of a superfamily of isopeptidases that remove ubiquitin: the deubiquitinases (DUBs; also known as deubiquitylating or deubiquitinating enzymes). Far from being uniform in structure and function, these enzymes display a myriad of distinct mechanistic features. The small number (<100) of DUBs might at first suggest a low degree of selectivity; however, DUBs are subject to multiple layers of regulation that modulate both their activity and their specificity. Due to their wide-ranging involvement in key regulatory processes, these enzymes might provide new therapeutic targets.

Structural and biochemical analyses of human USP30 explain the basis of Lys6-linkage preference and regulation by PINK1 and Parkin, shedding light onto how USP30 can act as a brake on mitophagy. Damaged mitochondria undergo mitophagy, a specialized form of autophagy that is initiated by the protein kinase PINK1 and the ubiquitin E3 ligase Parkin. Ubiquitin-specific protease USP30 antagonizes Parkin-mediated ubiquitination events on mitochondria and is a key negative regulator of mitophagy. Parkin and USP30 both show a preference for assembly or disassembly, respectively, of Lys6-linked polyubiquitin, a chain type that has not been well studied. Here we report crystal structures of human USP30 bound to monoubiquitin and Lys6-linked diubiquitin, which explain how USP30 achieves Lys6-linkage preference through unique ubiquitin binding interfaces. We assess the interplay between USP30, PINK1 and Parkin and show that distally phosphorylated ubiquitin chains impair USP30 activity. Lys6-linkage-specific affimers identify numerous mitochondrial substrates for this modification, and we show that USP30 regulates Lys6-polyubiquitinated TOM20. Our work provides insights into the architecture, activity and regulation of USP30, which will aid drug design against this and related enzymes.

UbiCRest is a simple PAGE-based method that leverages the varying specificities of deubiquitinating enzymes (DUBs) to generate information about the composition and architecture of ubiquitin chains. Protein ubiquitination is a versatile protein modification that regulates virtually all cellular processes. This versatility originates from polyubiquitin chains, which can be linked in eight distinct ways. The combinatorial complexity of eight linkage types in homotypic (one chain type per polymer) and heterotypic (multiple linkage types per polymer) chains poses significant problems for biochemical analysis. Here we describe UbiCRest, in which substrates (ubiquitinated proteins or polyubiquitin chains) are treated with a panel of linkage-specific deubiquitinating enzymes (DUBs) in parallel reactions, followed by gel-based analysis. UbiCRest can be used to show that a protein is ubiquitinated, to identify which linkage type(s) are present on polyubiquitinated proteins and to assess the architecture of heterotypic polyubiquitin chains. DUBs used in UbiCRest can be obtained commercially; however, we include details for generating a toolkit of purified DUBs and for profiling their linkage preferences in vitro . UbiCRest is a qualitative method that yields insights into ubiquitin chain linkage types and architecture within hours, and it can be performed on western blotting quantities of endogenously ubiquitinated proteins.

Polyubiquitin (pUb) chains formed between the C terminus of ubiquitin and lysine 63 (K63) or methionine 1 (M1) of another ubiquitin have been implicated in the activation of the canonical IκB kinase (IKK) complex. Here, we demonstrate that nearly all of the M1-pUb chains formed in response to interleukin-1, or the Toll-Like Receptors 1/2 agonist Pam ₃CSK ₄, are covalently attached to K63-pUb chains either directly as K63-pUb/M1-pUb hybrids or indirectly by attachment to the same protein. Interleukin-1 receptor (IL-1R)-associated kinase (IRAK) 1 is modified first by K63-pUb chains to which M1-pUb linkages are added subsequently, and myeloid differentiation primary response gene 88 (MyD88) and IRAK4 are also modified by both K63-pUb and M1-pUb chains. We show that the heme-oxidized IRP2 ubiquitin ligase 1 interacting protein (HOIP) component of the linear ubiquitin assembly complex catalyzes the formation of M1-pUb chains in response to interleukin-1, that the formation of K63-pUb chains is a prerequisite for the formation of M1-pUb chains, and that HOIP interacts with K63-pUb but not M1-pUb linkages. These findings identify K63-Ub oligomers as a major substrate of HOIP in cells where the MyD88-dependent signaling network is activated. The TGF-beta–activated kinase 1 (TAK1)-binding protein (TAB) 2 and TAB3 components of the TAK1 complex and the NFκB Essential Modifier (NEMO) component of the canonical IKK complex bind to K63-pUb chains and M1-pUb chains, respectively. The formation of K63/M1-pUb hybrids may therefore provide an elegant mechanism for colocalizing both complexes to the same pUb chain, facilitating the TAK1-catalyzed activation of IKKα and IKKβ. Our study may help to resolve the debate about the relative importance of K63-pUb and M1-pUb chains in activating the canonical IKK complex.

Ubiquitin can form different polymeric chains, either linear or using each of its seven lysine residues. The best studied are Lys48 and Lys63 chains, but Lys11 chains have been shown to be abundant in yeast. Now a procedure to obtain large amounts of Lys11 chains is described, allowing the structural characterization of this linkage and the identification of a Lys11-specific deubiquitinase, Cezanne. Ubiquitin is a versatile cellular signaling molecule that can form polymers of eight different linkages, and individual linkage types have been associated with distinct cellular functions. Though little is currently known about Lys11-linked ubiquitin chains, recent data indicate that they may be as abundant as Lys48 linkages and may be involved in vital cellular processes. Here we report the generation of Lys11-linked polyubiquitin in vitro , for which the Lys11-specific E2 enzyme UBE2S was fused to a ubiquitin binding domain. Crystallographic and NMR analyses of Lys11-linked diubiquitin reveal that Lys11-linked chains adopt compact conformations in which Ile44 is solvent exposed. Furthermore, we identify the OTU family deubiquitinase Cezanne as the first deubiquitinase with Lys11-linkage preference. Our data highlight the intrinsic specificity of the ubiquitin system that extends to Lys11-linked chains and emphasize that differentially linked polyubiquitin chains must be regarded as independent post-translational modifications.