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Tonne–Kalscheuer syndrome (TOKAS) is an X-linked intellectual disability syndrome associated with variable clinical features including craniofacial abnormalities, hypogenitalism and diaphragmatic hernia. TOKAS is caused exclusively by variants in the gene encoding the E3 ubiquitin ligase gene RLIM, also known as RNF12. Here we report identification of a novel RLIM missense variant, c.1262A>G p.(Tyr421Cys) adjacent to the regulatory basic region, which causes a severe form of TOKAS resulting in perinatal lethality by diaphragmatic hernia. Inheritance and X-chromosome inactivation patterns implicate RLIM p.(Tyr421Cys) as the likely pathogenic variant in the affected individual and within the kindred. We show that the RLIM p.(Tyr421Cys) variant disrupts both expression and function of the protein in an embryonic stem cell model. RLIM p.(Tyr421Cys) is correctly localised to the nucleus, but is readily degraded by the proteasome. The RLIM p.(Tyr421Cys) variant also displays significantly impaired E3 ubiquitin ligase activity, which interferes with RLIM function in Xist long-non-coding RNA induction that initiates imprinted X-chromosome inactivation. Our data uncover a highly disruptive missense variant in RLIM that causes a severe form of TOKAS, thereby expanding our understanding of the molecular and phenotypic spectrum of disease severity.

Polycomb Group (PcG) proteins organize chromatin at multiple scales to regulate gene expression. A conserved Sterile Alpha Motif (SAM) in the Polycomb Repressive Complex 1 (PRC1) subunit Polyhomeotic (Ph) has been shown to play an important role in chromatin compaction and large-scale chromatin organization. Ph SAM forms helical head to tail polymers, and SAM-SAM interactions between chromatin-bound Ph/PRC1 are believed to compact chromatin and mediate long-range interactions. To understand the underlying mechanism, here we analyze the effects of Ph SAM on chromatin in vitro. We find that incubation of chromatin or DNA with a truncated Ph protein containing the SAM results in formation of concentrated, phase-separated condensates. Ph SAM-dependent condensates can recruit PRC1 from extracts and enhance PRC1 ubiquitin ligase activity towards histone H2A. We show that overexpression of Ph with an intact SAM increases ubiquitylated H2A in cells. Thus, SAM-induced phase separation, in the context of Ph, can mediate large-scale compaction of chromatin into biochemical compartments that facilitate histone modification., The conserved SAM motif of Polycomb Repressive Complex 1 subunit Ph has been shown to play an important role in chromatin organization. Here, the authors study the effect of Ph SAM on chromatin in vitro, showing that it induces the formation of concentrated, phase-separated condensates, which enhance the ubiquitin ligase activity of PRC1.

Pseudogenes are mutated copies of protein-coding genes that cannot be translated into proteins, but a small subset of pseudogenes has been detected at the protein level. Although ubiquitin pseudogenes represent one of the most abundant pseudogene families in many organisms, little is known about their expression and signaling potential. By re-analyzing public RNA-sequencing and proteomics datasets, we here provide evidence for the expression of several ubiquitin pseudogenes including UBB pseudogene 4 (UBBP4), which encodes UbKEKS (Q2K, K33E, Q49K, N60S). The functional consequences of UbKEKS conjugation appear to differ from canonical ubiquitylation. Quantitative proteomics shows that UbKEKS modifies specific proteins including lamins. Knockout of UBBP4 results in slower cell division, and accumulation of lamin A within the nucleolus. Our work suggests that a subset of proteins reported as ubiquitin targets may instead be modified by ubiquitin variants that are the products of wrongly annotated pseudogenes and induce different functional effects., 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.

Cardiogenesis requires the orchestrated spatiotemporal tuning of BMP signalling upon the balance between induction and counter-acting suppression of the differentiation of the cardiac tissue. SMADs are key intracellular transducers and the selective degradation of SMADs by the ubiquitin–proteasome system is pivotal in the spatiotemporal tuning of BMP signalling. However, among three SMADs for BMP signalling, SMAD1/5/9, only the specific E3 ligase of SMAD9 remains poorly investigated. Here, we report for the first time that SMAD9, but not the other SMADs, is ubiquitylated by the E3 ligase ASB2 and targeted for proteasomal degradation. ASB2, as well as Smad9, is conserved among vertebrates. ASB2 expression was specific to the cardiac region from the very early stage of cardiac differentiation in embryogenesis of mouse. Knockdown of Asb2 in zebrafish resulted in a thinned ventricular wall and dilated ventricle, which were rescued by simultaneous knockdown of Smad9. Abundant Smad9 protein leads to dysregulated cardiac differentiation through a mechanism involving Tbx2, and the BMP signal conducted by Smad9 was downregulated under quantitative suppression of Smad9 by Asb2. Our findings demonstrate that ASB2 is the E3 ligase of SMAD9 and plays a pivotal role in cardiogenesis through regulating BMP signalling.

The proteasome, the primary protease for ubiquitin-dependent proteolysis in eukaryotes, is usually found as a mixture of 30S, 26S, and 20S complexes. These complexes have common catalytic sites, which makes it challenging to determine their distinctive roles in intracellular proteolysis. Here, we chemically synthesize a panel of homogenous ubiquitinated proteins, and use them to compare 20S and 26S proteasomes with respect to substrate selection and peptide-product generation. We show that 20S proteasomes can degrade the ubiquitin tag along with the conjugated substrate. Ubiquitin remnants on branched peptide products identified by LC-MS/MS, and flexibility in the 20S gate observed by cryo-EM, reflect the ability of the 20S proteasome to proteolyze an isopeptide-linked ubiquitin-conjugate. Peptidomics identifies proteasome-trapped ubiquitin-derived peptides and peptides of potential 20S substrates in Hi20S cells, hypoxic cells, and human failing-heart. Moreover, elevated levels of 20S proteasomes appear to contribute to cell survival under stress associated with damaged proteins., The 20S particle is part of the 26S proteasome, but also exists as a free complex. Here, the authors outline signature activities of the 20S and combine chemical, structural, functional and proteomic assays to show that the 20S can degrade ubiquitin tags along with conjugated substrates.

Strong evidence supports that protein ubiquitination is a critical regulator of fear memory formation. However, as this work has focused on protein degradation, it is currently unknown whether polyubiquitin modifications that are independent of the proteasome are involved in learning-dependent synaptic plasticity. Here, we present the first evidence that atypical linear (M1) polyubiquitination, the only ubiquitin chain that does not occur at a lysine site and is largely independent of the proteasome, is critically involved in contextual fear memory formation in the amygdala in a sex-specific manner. Using immunoblot and unbiased proteomic analyses, we found that male (49) and female (14) rats both had increased levels of linear polyubiquitinated substrates following fear conditioning, though none of these protein targets overlapped between sexes. In males, target protein functions involved cell junction and axonal guidance signaling, while in females the primary target was Adiponectin A, a critical regulator of neuroinflammation, synaptic plasticity, and memory, suggesting sex-dependent functional roles for linear polyubiquitination during fear memory formation. Consistent with these increases, in vivo siRNA-mediated knockdown of Rnf31, an essential component of the linear polyubiquitin E3 complex LUBAC, in the amygdala impaired contextual fear memory in both sexes without affecting memory retrieval. Collectively, these results provide the first evidence that proteasome-independent linear polyubiquitination is a critical regulator of fear memory formation, expanding the potential roles of ubiquitin-signaling in learning-dependent synaptic plasticity. Importantly, our data identify a novel sex difference in the functional role of, but not a requirement for, linear polyubiquitination in fear memory formation.

Ras/MAPK (mitogen active protein kinase) signaling plays contradictory roles in adipocyte differentiation and is tightly regulated during adipogenesis. However, mechanisms regulating adipocyte differentiation involving Ras protein stability regulation are unknown. Here, we show that WD40 repeat protein 76 (WDR76), a novel Ras regulating E3 linker protein, controls 3T3-L1 adipocyte differentiation through HRas stability regulation. The roles of WDR76 in obesity and metabolic regulation were characterized using a high-fat diet (HFD)-induced obesity model using Wdr76−/− mice and liver-specific Wdr76 transgenic mice (Wdr76Li−TG). Wdr76−/− mice are resistant to HFD-induced obesity, insulin resistance and hyperlipidemia with an increment of HRas levels. In contrast, Wdr76Li-TG mice showed increased HFD-induced obesity, insulin resistance with reduced HRas levels. Our findings suggest that WDR76 controls HFD-induced obesity and hepatic steatosis via HRas destabilization. These data provide insights into the links between WDR76, HRas, and obesity.

RNF183 is a ubiquitin ligase containing RING-finger and transmembrane domains, and its expression levels are increased in patients with inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis, and in 2,4,6-trinitrobenzene sulfonic acid-induced colitis mice. Here, we further demonstrate that RNF183 was induced to a greater degree in the dextran sulfate sodium (DSS)-treated IBD model at a very early stage than were inflammatory cytokines. In addition, fluorescence-activated cell sorting and polymerase chain reaction analysis revealed that RNF183 was specifically expressed in epithelial cells of DSS-treated mice, which suggested that increased levels of RNF183 do not result from the accumulation of immune cells. Furthermore, we identified death receptor 5 (DR5), a member of tumour necrosis factor (TNF)-receptor superfamily, as a substrate of RNF183. RNF183 mediated K63-linked ubiquitination and lysosomal degradation of DR5. DR5 promotes TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis signal through interaction with caspase-8. Inhibition of RNF183 expression was found to suppress TRAIL-induced activation of caspase-8 and caspase-3. Thus, RNF183 promoted not only DR5 transport to lysosomes but also TRAIL-induced caspase activation and apoptosis. Together, our results provide new insights into potential roles of RNF183 in DR5-mediated caspase activation in IBD pathogenesis.

Autophagy receptor p62/SQSTM1 promotes the assembly and removal of ubiquitylated proteins by forming p62 bodies and mediating their encapsulation in autophagosomes. Here we show that under nutrient-deficient conditions, cellular p62 specifically undergoes acetylation, which is required for the formation and subsequent autophagic clearance of p62 bodies. We identify K420 and K435 in the UBA domain as the main acetylation sites, and TIP60 and HDAC6 as the acetyltransferase and deacetylase. Mechanically, acetylation at both K420 and K435 sites enhances p62 binding to ubiquitin by disrupting UBA dimerization, while K435 acetylation also directly increases the UBA-ubiquitin affinity. Furthermore, we show that acetylation of p62 facilitates polyubiquitin chain-induced p62 phase separation. Our results suggest an essential role of p62 acetylation in the selective degradation of ubiquitylated proteins in cells under nutrient stress, by specifically regulating the assembly of p62 bodies., The autophagy receptor p62 mediates the assembly and removal of ubiquitylated protein aggregates by forming p62 bodies. Here, the authors identify an acetylation-dependent mechanism that regulates formation and autophagic clearance of p62 bodies under nutrient-deficient conditions.

The multi-subunit C-terminal to LisH (CTLH) complex is the mammalian homologue of the yeast Gid E3 ubiquitin ligase complex. In this study, we investigated the human CTLH complex and characterized its E3 ligase activity. We confirm that the complex immunoprecipitated from human cells comprises RanBPM, ARMC8 α/β, muskelin, WDR26, GID4 and the RING domain proteins RMND5A and MAEA. We find that loss of expression of individual subunits compromises the stability of other complex members and that MAEA and RMND5A protein levels are interdependent. Using in vitro ubiquitination assays, we demonstrate that the CTLH complex has E3 ligase activity which is dependent on RMND5A and MAEA. We report that the complex can pair with UBE2D1, UBE2D2 and UBE2D3 E2 enzymes and that recombinant RMND5A mediates K48 and K63 poly-ubiquitin chains. Finally, we show a proteasome-dependent increase in the protein levels of CTLH complex member muskelin in RMND5A KO cells. Furthermore, muskelin ubiquitination is dependent on RMND5A, suggesting that it may be a target of the complex. Overall, we further the characterization of the CTLH complex as an E3 ubiquitin ligase complex in human cells and reveal a potential autoregulation mechanism.

BRAF plays an indispensable role in activating the MEK/ERK pathway to drive tumorigenesis. Receptor tyrosine kinase and RAS-mediated BRAF activation have been extensively characterized, however, it remains undefined how BRAF function is fine-tuned by stimuli other than growth factors. Here, we report that in response to proinflammatory cytokines, BRAF is subjected to lysine 27-linked poly-ubiquitination in melanoma cells by the ITCH ubiquitin E3 ligase. Lysine 27-linked ubiquitination of BRAF recruits PP2A to antagonize the S365 phosphorylation and disrupts the inhibitory interaction with 14–3–3, leading to sustained BRAF activation and subsequent elevation of the MEK/ERK signaling. Physiologically, proinflammatory cytokines activate ITCH to maintain BRAF activity and to promote proliferation and invasion of melanoma cells, whereas the ubiquitination-deficient BRAF mutant displays compromised kinase activity and reduced tumorigenicity. Collectively, our study reveals a pivotal role for ITCH-mediated BRAF ubiquitination in coordinating the signals between cytokines and the MAPK pathway activation in melanoma cells., 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.

S-phase kinase associated protein 2 (Skp2), a member of the F-box family that constitute the largest known class of ubiquitin E3 specificity components, is responsible for recognizing and recruiting cyclin-dependent kinase inhibitor p27 for its ubiquitination in the presence of the small accessory protein cyclin-dependent kinase regulatory subunit 1(Cks1). Skp2 is overexpressed in esophageal carcinoma tissues and closely related with tumor poor prognosis, and perturbation of the Skp2-Cks1 interaction by inhibitors or RNAi could inhibit the proliferation and metastasis of tumor cells. Therefore, inhibition of Skp2 function by small-molecule compounds targeting Skp2-Cks1 interaction is emerging as a promising and novel anti-cancer strategy. In this study, we establish an improved high-throughput screening platform to screen Skp2 inhibitors targeting Skp2-Cks1interaction, which may provide a new therapeutic approach for the clinic.

p62/SQSTM1 is known to act as a key mediator in the selective autophagy of protein aggregates, or aggrephagy, by steering ubiquitinated protein aggregates towards the autophagy pathway. Here, we use a yeast two-hybrid screen to identify the prefoldin-like chaperone UXT as an interacting protein of p62. We show that UXT can bind to protein aggregates as well as the LB domain of p62, and, possibly by forming an oligomer, increase p62 clustering for its efficient targeting to protein aggregates, thereby promoting the formation of the p62 body and clearance of its cargo via autophagy. We also find that ectopic expression of human UXT delays SOD1(A4V)-induced degeneration of motor neurons in a Xenopus model system, and that specific disruption of the interaction between UXT and p62 suppresses UXT-mediated protection. Together, these results indicate that UXT functions as an autophagy adaptor of p62-dependent aggrephagy. Furthermore, our study illustrates a cooperative relationship between molecular chaperones and the aggrephagy machinery that efficiently removes misfolded protein aggregates., p62/SQSTM1 acts as a key mediator in the selective autophagy of protein aggregates, or aggrephagy. Here the authors identify the prefoldin-like chaperone UXT as an autophagy adaptor of p62 dependent aggrephagy and show that ectopic UXT expression delays motor neuron degeneration in a Xenopus model.

The proteasome is responsible for selective degradation of proteins. It exists in mammalian cells under four main subtypes, which differ by the combination of their catalytic subunits: the standard proteasome (β1–β2–β5), the immunoproteasome (β1i–β2i–β5i) and the two intermediate proteasomes (β1–β2–β5i and β1i–β2–β5i). The efficiency of the four proteasome subtypes to degrade ubiquitinated or oxidized proteins remains unclear. Using cells expressing exclusively one proteasome subtype, we observed that ubiquitinated p21 and c-­myc were degraded at similar rates, indicating that the four 26S proteasomes degrade ubiquitinated proteins equally well. Under oxidative stress, we observed a partial dissociation of 26S into 20S proteasomes, which can degrade non-ubiquitinated oxidized proteins. Oxidized calmodulin and hemoglobin were best degraded in vitro by the three β5i-containing 20S proteasomes, while their native forms were not degraded. Circular dichroism analyses indicated that ubiquitin-independent recognition of oxidized proteins by 20S proteasomes was triggered by the disruption of their structure. Accordingly, β5i-containing 20S proteasomes degraded unoxidized naturally disordered protein tau, while 26S proteasomes did not. Our results suggest that the three β5i-containing 20S proteasomes, namely the immunoproteasome and the two intermediate proteasomes, might help cells to eliminate proteins containing disordered domains, including those induced by oxidative stress.

IpaH enzymes are bacterial E3 ligases targeting host proteins for ubiquitylation. Two autoinhibition modes of IpaH enzymes have been proposed based on the relative positioning of the Leucine-rich repeat domain (LRR) with respect to the NEL domain. In mode 1, substrate-binding competitively displaces the interactions between theLRR and NEL to relieve autoinhibition. However, the molecular basis for mode 2 is unclear. Here, we present the crystal structures of Shigella IpaH9.8 and the LRR of IpaH9.8 in complex with the substrate of human guanylate-binding protein 1 (hGBP1). A hydrophobic cluster in the C-terminus of IpaH9.8LRR forms a hydrophobic pocket involved in binding the NEL domain, and the binding is important for IpaH9.8 autoinhibition. Substrate-binding destabilizes the hydrophobic cluster by inducing conformational changes of IpaH9.8LRR. Arg166 and Phe187 in IpaH9.8LRR function as sensors for substrate-binding. Collectively, our findings provide insights into the molecular mechanisms for the actication of IpaH9.8 in autoinhibition mode 2., Ye, Xiong et al. present crystal structures of bacterial E3 ubiquitin ligase IpaH9.8 and IpaH9.8LRR–hGBP1. They find that substrate-binding destabilizes the hydrophobic cluster to relieve the autoinhibition of IpaH9.8. This study provides insights into the mechanisms underlying substrate-induced activation of IpaH9.8.