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Single-molecule FRET assays used to probe the conformational dynamics of ubiquitin chains reveal that conformational selection is an important mechanism of ubiquitin chain recognition.

Small molecules are identified that inhibit the ubiquitin-specific protease USP7 with high affinity and specificity as explained by co-crystal structures, and are shown to reduce tumour growth in mice.

Stabilization of a transient protein kinase–substrate complex using a nanobody provides molecular details about how the Parkinson’s disease-linked protein kinase PINK1 phosphorylates ubiquitin, and suggests new pharmacological strategies.

A UBD containing ankyrin repeats determines the linkage specificity of TRABID.

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.

The structures of the deubiquitinating enzyme Cezanne alone or in complex with its substrate or product are solved, showing how Cezanne specifically targets Lys11-linked polyubiquitin.

A novel antiviral targeting the SARS-CoV-2 PLpro protease shows strong efficacy in a mouse model, preventing lung pathology and reducing brain dysfunction. The study provides proof-of-principle that PLpro inhibition may be a viable strategy for preventing and treating long COVID.

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.

Analysis of over 6000 single-site variants of SARS-CoV-2 Papain-Like protease uncovers critical mutations involved in enzymatic function and highlight the potential for drug-escape.

Ovarian tumour deubiquitinases are cysteine proteases that cleave polyubiquitin chains. Here the authors show that these enzymes are susceptible to reversible oxidation, and present crystal structures which reveal how the reversibly oxidized catalytic cysteine residue is stabilized by the active site.

The TAK1 kinase binds K63-linked ubiquitin specifically via its TAB2 subunit. The structure of the TAB2 NZF domain in complex with K63-linked ubiquitins now indicates that this domain interacts with neighboring ubiquitins through distinct sites, explaining the basis of specific recognition.

Most insights into deubiquitinase (DUB) substrate specificity originate from studies with isolated di-ubiquitins (diUb), but in cells diUbs with different linkage types coexist. Here, the authors develop a mass spectrometric DUB activity assay that can probe all diUbs simultaneously under substrate competition conditions.

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 (Ub) monomers can form different types of chains, depending on which of its seven lysine residues are involved in the linkages. Now linkage-specific deubiquitinases are used to explore the architecture of heterotypic Ub chains, and Lys6 chains are shown to feature an asymmetric interface and a different conformation for Ub.

Salmonella Typhimurium E3 ligase LUBAC generates linear polyubiquitin patches in the ubiquitin coat that serves as a signalling platform for the recruitment of Optineurin and Nemo for xenophagy and local activation of NF-κB, respectively.

Unphosphorylated PINK1 of Pediculus humanus corporis forms a dimerized state before undergoing trans-autophosphorylation, and phosphorylated PINK1 undergoes a conformational change in the N-lobe to produce its phosphorylated, ubiquitin-binding state.

Structures of the deubiquitinating enzyme Rpn11 of the proteasomal 19S regulatory particle reveal its role in hydrolyzing the proximal ubiquitin from a protein that is about to be degraded.

OTULIN is a linear ubiquitin hydrolase that regulates ubiquitin homeostasis. Here the authors identify the adaptor of the endosomal retromer complex sorting nexin 27 (SNX27) as a binding partner of OTULIN and determine the structure of the OTULIN-SNX27 complex, which reveals a secondary interface through which OTULIN non-catalytically antagonizes SNX27 retromer assembly and cargo loading.

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.

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.

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.

The multiple lysines that can link ubiquitin chains have created challenges in studying these important biopolymers. The ribosomal incorporation of a protected lysine analog now allows the specific construction of native K6- and K29-linked diubiquitin chains, enabling structural analysis and deubiquitinase profiling.

CYLD is a deubiquitinase known to act as a tumour suppressor in different models of carcinogenesis. Here, the authors show that CYLD suppresses carcinogen-induced tumorigenesis by deubiquitinating p53 and promoting its stabilization and activation in response to DNA damage.

The mechanisms underlying the activity of non-receptor tyrosine kinase, TNK1, in cancers are unclear. Here the authors show that MARK mediates 14-3-3 and TNK1 interaction which restrains TNK1 activity, while the release of TNK1 from 14-3-3 leads to TNK1 activation through its interaction with ubiquitin and thus results in TNK1-mediated tumor growth in vivo

The OTU domain deubiquitinase TRABID specifically hydrolyzes atypical Lys29- and Lys33-linked diubiquitin chains. Structural analysis of TRABID reveals an unpredicted ankyrin-repeat domain that binds ubiquitin and is crucial for TRABID efficiency and linkage specificity in vitro and in vivo.

A ubiquitin analog used as an activity-based probe of ubiquitin conjugation enzymes, E1, E2 and E3 covalently traps these enzymes without transfer to substrates. The probes can be used in structural and functional studies and to visualize enzyme activity in cells.

The modification of proteins with the small protein ubiquitin constitutes a Daedalian system of posttranslational modifications in every eukaryotic cell, which is often referred to as the ubiquitin code¹. Here we consider the scale and complexity of the ubiquitin system in light of recent developments.

The AGC kinase subfamily of protein kinases contains 60 members, including PKA, PKG and PKC. Research has shed light onto the architecture and regulatory mechanisms of these kinases, which mediate important cellular functions and are dysregulated in many human diseases.

Structural mass spectrometry of full-length human parkin and a structure of the activated parkin core reveal large-scale domain rearrangements involved in activation of parkin by PINK1.

Chlamydia trachomatis DUB1 uses a single catalytic centre to carry out dual lysine deubiquitinase and acetyltransferase activity. Deubiquitination is required for Golgi fragmentation during bacterial infection.

Enzymatic cleavage within ubiquitin molecules followed by quantitative mass-spectrometry simplifies complex ubiquitin chains and enables mapping of polyubiquitin architectures.

Deubiquitinating enzymes (DUBs) function in opposition to E3 ubiquitin ligases by removing ubiquitin from substrates to control protein and organelle homeostasis and responses to cellular stimuli. In this Review, Dewson et al. describe the many associations of DUBs with the hallmarks of cancer, with a view to identifying those DUBs most likely to impact cancer-associated phenotypes if targeted with selective inhibition.

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.

Pili are crucial virulence factors in many Gram-negative pathogens; these surface structures enable bacteria to interact with, and attach to structures in, their environment and can also facilitate horizontal gene transfer. In this Review, Waksmanet al. examine recent advances in our structural understanding of various pilus systems in Gram-negative bacteria and discuss their functional implications.