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Riechmann et al. uncover structural features governing ubiquitin transfer from ubiquitin-activating E1 enzymes UBA1 and UBA6 to specific E2 ubiquitin-conjugating enzymes, revealing a hierarchy of E2 activity with cognate E1s.

Heterobifunctional small degrader molecules that tether endogenous E3 ubiquitin ligases are promising substrates for targeted protein degradation, however, the precise formation of the E3 ligase–target complex remains challenging and limits their application. Here, the authors introduce indirect ubiquitination of the target proteins via non-covalent interactions that bypass E3 ligases, enabling efficient protein degradation.

Ye et al. define the structure and mechanisms of a bacterial pathway that performs ubiquitination-like protein conjugation, revealing new insights into the evolution and biological roles of ubiquitination pathways across kingdoms.

Ribosome stalling during translation threatens proteostasis and requires targeted clearance of nascent chains. Here, the authors show how the yeast RQC complex recruits Cdc48 via Ltn1 and Rqc1 to extract ubiquitylated peptides, revealing the structural basis of stalled nascent chain removal.

This study demonstrates that SMURF1-mediated K63-linked ubiquitination of RIPK3 suppresses necroptosis by blocking necrosome formation, while USP5 removes these ubiquitin chains to promote necroptosis. Their antagonistic regulation precisely controls necroptosis.

DNA damage tolerance is regulated by ubiquitination of PCNA. Here, the authors present kinetic and structural studies showing that USP1/UAF1 prefers trimming K63- and K48-ubiquitin chains down over cleavage of monoubiquitinated PCNA. Mutant analysis suggests evolutionary preservation of this mechanism.

The nucleoporin RanBP2 functions in cellular transport, mitosis and SUMO conjugation. Here, the authors determine cryo-EM structures of RanBP2 complexed with SUMO1-RanGAP1, Ubc9, Crm1, Ran and use cell and biochemical assays to probe its functions.

Understanding the catalytic and regulatory mechanisms of the ubiquitin system is key to tapping into its therapeutic potential in diseases where its dysfunction is implicated. Here, the authors uncover a set of catalytically dead ‘pseudo’ ubiquitin ligases with possible regulatory roles.

Here Tai et al. unveil elevated formation of the C-terminal binding protein (CtBP)–tripartite-motif-containing protein 28 (TRIM28) complex in breast cancer cells and mutual protection between CtBP and TRIM28. This complex is required for mammary gland development and its high levels lead to breast cancer metastasis by repressing autophagy.

Oxyanion stabilization is essential for E2 enzyme activity in ubiquitin and Ub-like protein conjugation. Here, the authors demonstrate that E2 enzymes (1) employ C-alpha hydrogen bonding for oxyanion stabilization and (2) feature an additional oxyanion hole that facilitates cis conjugation, together highlighting the adaptability of E2 enzymes.

The Cdc48 enzyme is an abundant and essential enzyme that functions in many cellular pathways as a protein unfoldase. Here, the authors determine an ensemble of Cdc48 structures that capture snapshots of its unfolding action using a ‘hand-over-hand’ mechanism.

The bacterial pathogen Legionella pneumophila secretes effectors that affect protein ubiquitination within host cells, involving production of phosphoribosyl ubiquitin (PR-Ub). Here, the authors identify additional effectors that convert PR-Ub back into functional ubiquitin, thus maintaining ubiquitin homeostasis in infected cells.

Kelch-domain KLHDCX E3 ligases bind substrate C-terminal glycines. This study reveals substrate selectivity by E3s with similar structures; C-degrons are perceived by a “C-terminus anchor motif”, whose display on different Kelch propeller blades along with distal interactions establish specificity.

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.

Semisynthetic methods to make ubiquitin conjugates have yielded broad conclusions for epigenetics. A robust intein-mediated chemical crosslinking strategy now expands our understanding by showing that a methyltransferase is surprisingly tolerant of changes to ubiquitin location and composition.

The molecular mechanisms ensuring early face, brain, and skin formation are unclear. Here, the authors uncover a posttranslational pathway that controls cytoskeletal signaling circuits to coordinate ectodermal patterning and neurulation.

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.

The authors provide a litmus test for the recognition mechanism of transiently binding proteins based on nuclear magnetic resonance and find a conformational selection binding mechanism through concentration-dependent kinetics of ubiquitin and SH3.

Absence of regulatory T cells results in a severe inflammatory disease which leads to death in infancy in both human patients and in mouse models. Authors show here that in mice, conditional deletion of Rbx1, the RING component of Cullin-RING ligases in regulatory T cells causes a similar phenotype, due to the disrupted degradation of important regulatory proteins.

Misfolded proteins are ubquitinated and subsequently condensed by cargo receptors for selective autophagy. Here, the authors use in vitro reconstitution to elegantly dissect how the receptors p62/SQSTM1, NBR1 and TAX1BP1 contribute to p62-ubiquitin condensate formation and degradation by autophagy.

Ufmylation is a well-established ubiquitin-like protein modification, but its mechanism is largely unclear. Here, the authors present a crystal structure of the ufmylation-specific E1-E2 complex, revealing differences to the ubiquitination machinery and mechanistic details of the ufmylation process.

Ufmylation is a ubiquitylation-like protein modification but only a few ufmylation substrates and functions have been discovered so far. Here, the authors demonstrate ufmylation of histone H4 upon DNA damage and show that this modification is involved in the amplification of ATM activation.

The Lys48-linked polyubiquitin-mediated proteasomal degradation in yeast depends on Cdc48 and its cofactors Ufd1 and Npl4. Here, the authors present crystal structures of Npl4 bound to Lys48-linked diubiquitin and the Npl4-binding motif of Ufd1, providing insights into the reaction mechanism of the Cdc48- Ufd1/Npl4 complex.

Sequestration of reactants in lipid vesicles is a strategy prevalent in biological systems to raise the rate and specificity of chemical reactions. Here, the authors show that micelle-assisted reactions facilitate native chemical ligation between a peptide-thioester and a Cys-peptide modified by a lipid-like moiety.

Ubiquitin pseudogenes are present in many organisms but whether they encode functional proteins has remained unclear. Here, the authors show that human UBB pseudogene 4 produces ubiquitin variants with amino acid compositions and cellular functions that are distinct from canonical ubiquitin.

Posttranslational modification of proteins by small ubiquitin-related modifier is a response to stress signalling in plants. Here, theArabdiposisprotein SIZ1 is shown to cause SUMOylation of nitrate reductases 1 and 2 and to increase their activity, suggesting that SIZ1 controls nitrate uptake via SUMOylation.

mRNA deadenylation, the first step in regulated degradation, is mediated by the action of the CCR4-NOT and PAN2-PAN3 complexes. Here the authors show that the RNA-binding E3 ubiquitin-ligase MEX-3C associates with the CCR4-NOT complex and ubiquitinates the catalytic subunit CNOT7 to regulate its deadenylation activity.

Ubiquitin is a stable and soluble protein, but it is commonly found in inclusion bodies in neurodegenerative disorders and cancer. Here, Morimoto et al. report that increasing ubiquitin chain length leads to the formation of amyloid-like fibrils, which are degraded by an autophagy mechanism.

Ubiquitin is not only a posttranslational modifier but itself is subject to modifications, such as acetylation. Characterization of distinct acetylated ubiquitin variants reveals that each acetylation site has a particular impact on ubiquitin structure and its protein-protein interaction properties.

Ubiquitin (Ub) and Ub-like modifiers (Ubls) can form chains of various topologies, but preparing defined chains for functional studies remains challenging. Here, the authors develop chemoenzymatic tools to tailormake Ub/Ubl chains and study the involvement of specific Ub/SUMO chains in DNA repair.

BRAF drives MEK/ERK activation to facilitate tumorigenesis. Here, the authors show that in response to pro-inflammatory cytokines, ITCH mediates a non-proteolytic ubiquitination and activation of BRAF, which in turn sustains MEK/ERK signaling to facilitate melanoma cell growth.

The cell cycle regulatory E3 ligase APC/C cooperates with UBE2C to prime substrates with ubiquitin and UBE2S to extend the ubiquitin chains. Careful analysis reveals that binding of the UBE2S to APC/C accelerates the rate-limiting step of APC/C–UBE2C.

Many pathogens manipulate ubiquitin-mediated signaling to evade host cell defense. Here, the authors characterize the structure and enzymatic activity of a deubiquitylase domain from the causative pathogen of scrub typhus, providing evidence for a distinct mechanism of ubiquitin chain selectivity.

Ubiquitin-specific protease USP4 regulates several cellular signalling pathways. Here, Clerici et al.show that the DUSP–Ubl domain of USP4 is required for full catalytic activity, by enhancing the release of ubiquitin from the catalytic site after substrate hydrolysis.

Ubiquitin (Ub) receptors are responsible for the recognition of ubiquitylated proteins. Here the authors describe the crystal structure of the ubiquitylated form of the Ub-receptor Rpn10, which suggest that ubiquitylation of Rpn10 promotes its dissociation from the proteasome.

The mechanism by which the noncanonical E1-like enzyme Atg7 activates ubiquitin-like Atg8 to trigger autophagy has not been well understood. The crystal structures of the N-terminal domain of Atg7 alone and C-terminal domain of Atg7 in complex with Atg8 show that this probably proceeds without the need for dramatic conformational rearrangements by Atg7, distinct from other E1 enzymes.

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.

A new method based on protein engineering to trap an intact complex between Siz1, SUMO-bound E2, and PCNA for structure determination.

Stabilization of ubiquitin's β1-β2 region by computational design and phage display, targeting both buried and surface residues, yields a ubiquitin variant that specifically inhibits the deubiquitinase USP7 in vitro and in cells.

Although Archaea encode proteasomes highly related to those of eukaryotes, archaeal ubiquitin-like proteins are less conserved and not known to function in protein conjugation, complicating our understanding of the origins of ubiquitination. Two small archaeal modifier proteins, SAMP1 and SAMP2, structurally similar to ubiquitin, are now reported to form protein conjugates in the archaeon Haloferax volcanii.

The deubiquitinating enzyme OTUB1 binds charged E2 intermediates and prevents ubiquitin transfer. OTUB1 can also bind uncharged E2, and this interaction is now shown to stimulate OTUB1's deubiquitination activity. Thus, OTUB1–E2 complexes might regulate levels of ubiquitin conjugation in response to available free ubiquitin and the ratio of charged to uncharged E2.

Degrons are determinants within proteins that direct programmed degradation by the ubiquitinproteasome system. Here, the authors propose a three-part degron architecture which contains an E3-ligase recognition motif, a ubiquitination site(s), and a disordered site to initiate degradation.

Two carboxy-terminal motifs are necessary for Srs2 to recognize SUMO-conjugated PCNA, which is involved in DNA replication and repair; one motif is specific to SUMO, the other to PCNA.

Phagocytes employ multiple bactericidal mechanisms to kill microorganisms, including the generation of toxic superoxide and other reactive oxygen species. Here the authors utilize a multi-omics approach to identify and characterize new regulatory nodes implicated in mucosal immunity that control phagocyte oxidative burst.

Ubiquitin, an important post-translational modification that regulates a variety of biological processes is found in free and conjugated (monoubiquitin and polyubiquitin) forms in the cell. A method for precisely measuring these cellular pools using protein standard absolute quantification mass spectrometry is described; the approach should yield insights into ubiquitin signaling.

Ubiquitin modification also occurs in archaea. Here, the authors characterize an archaeal ancestral ubiquitination system, present the crystal structure of the archaeal deubiquitinase Rpn11 from Caldiarchaeum subterraneum bound to ubiquitin and provide insights into evolutionary relationships.