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Deubiquitinases (DUBs) antagonize protein ubiquitination by removing ubiquitin from substrates. Identifying the physiological substrates of each DUB is critical for understanding DUB function and the principles that govern the specificity of this class of enzymes. Since multiple DUBs can act on the same substrate, it can be challenging to identify substrates using inactivating a single enzyme. Here, we outline a method that enables the identification of proteins whose stability depends on DUB activity and an approach to profile DUB specificity in Xenopus egg extract. By coupling broad DUB inhibition with quantitative proteomics, we circumvent DUB redundancy to identify DUB substrates. By adding back recombinant DUBs individually to the extract, we pinpoint DUBs sufficient to counteract proteasomal degradation of these newly identified substrates. We apply this method to Xenopus egg extract but suggest that it can also be adapted to other cell lysates.

Currently, no ideal treatment exists to combat skeletal muscle disuse-induced atrophy and loss of strength. Because the activity of AMP-activated protein kinase (AMPK) in rat soleus muscle is suppressed at the early stages of disuse, we hypothesized that pre-treatment of rats with metformin (an AMPK activator) would exert beneficial effects on skeletal muscle during disuse. Muscle disuse was performed via hindlimb suspension (HS). Wistar rats were divided into four groups: (1) control (C), (2) control + metformin for 10 days (C+Met), (3) HS for 7 days (HS), (4) metformin treatment for 7 days before HS and during the first 3 days of 1-week HS (HS+Met). Anabolic and catabolic markers were assessed using WB and RT-PCR. Treatment with metformin partly prevented an HS-induced decrease in rat soleus weight and size of slow-twitch fibers. Metformin prevented HS-related slow-to-fast fiber transformation. Absolute soleus muscle force in the HS+Met group was increased vs. the HS group. GSK-3β (Ser9) phosphorylation was significantly increased in the HS+Met group vs. the HS group. Metformin pre-treatment partly prevented HS-induced decrease in 18S+28S rRNA content and attenuated upregulation of calpain-1 and ubiquitin. Thus, pre-treatment of rats with metformin can ameliorate disuse-induced reductions in soleus muscle weight, the diameter of slow-type fibers, and absolute muscle strength.

The APOBEC3 (A3) proteins are host antiviral cellular proteins that hypermutate the viral genome of diverse viral families. In retroviruses, this process requires A3 packaging into viral particles1–4. The lentiviruses encode a protein, Vif, that antagonizes A3 family members by targeting them for degradation. Diversification of A3 allows host escape from Vif whereas adaptations in Vif enable cross-species transmission of primate lentiviruses. How this ‘molecular arms race’ plays out at the structural level is unknown. Here, we report the cryogenic electron microscopy structure of human APOBEC3G (A3G) bound to HIV-1 Vif, and the hijacked cellular proteins that promote ubiquitin-mediated proteolysis. A small surface explains the molecular arms race, including a cross-species transmission event that led to the birth of HIV-1. Unexpectedly, we find that RNA is a molecular glue for the Vif–A3G interaction, enabling Vif to repress A3G by ubiquitin-dependent and -independent mechanisms. Our results suggest a model in which Vif antagonizes A3G by intercepting it in its most dangerous form for the virus—when bound to RNA and on the pathway to packaging—to prevent viral restriction. By engaging essential surfaces required for restriction, Vif exploits a vulnerability in A3G, suggesting a general mechanism by which RNA binding helps to position key residues necessary for viral antagonism of a host antiviral gene.

Serine/threonine-protein kinase 16 (STK16) is a novel member of the Numb-associated family of protein kinases with an atypical kinase domain. In this study, we aimed to investigate the involvement of STK16 in sleep–wake mechanisms. We confirmed the expression of Stk16 in the murine hypothalamus, the sleep–wake center, and found considerable changes in STK16 protein levels in the anterior hypothalamus during the light–dark cycle. We found that the coexistence of the potassium channel tetramerization domain containing 17 (KCTD17), an STK16 interactor, caused STK16 degradation. In contrast, the proteasome inhibitor MG132 inhibited the degradation of STK16. In addition, polyubiquitinated STK16 was observed, suggesting that KCTD17 acts as an adapter for E3 ligase to recognize STK16 as a substrate, leading to STK16 degradation via the ubiquitin–proteasome system. The vast changes in STK16 in the anterior hypothalamus, a mammalian sleep center, as well as the reported sleep abnormalities in the ubiquitin B knockout mice and the Drosophila with the inhibition of the KCTD17 homolog or its E3 ligase cullin-3, suggest that STK16 plays a major role in sleep–wake regulation.

Cullin-RING E3 ubiquitin ligases (CRLs) spatiotemporally regulate the proteasomal degradation of numerous cellular proteins involved in cell cycle control, DNA replication, and maintenance of genome stability. Activation of CRLs is controlled via neddylation by NEDD8-activating, -conjugating, and -attaching enzymes to the C-terminus of scaffold cullins (CULs), whereas the COP9 signalosome (CSN) inactivates CRLs via deneddylation. Here, we show that the deneddylation rate of each CUL is differentially modulated. Dose- or time-dependent treatment with pevonedistat, a small molecule inhibitor of NEDD8-activating enzyme (NAE), rapidly inhibits neddylation in most CULs, including CUL1, CUL3, CUL4A/B, and CUL5, whereas the deneddylation of CUL2 is slowly increased. We revealed that the different deneddylation speeds of each CUL depend on its binding strength with CSN5, the catalytic core of the CSN complex. Immunoprecipitation analysis revealed that CUL2 has a lower binding affinity for CSN5 than other CULs. Consistently, released cells treated with CSN5 inhibitor showed that CUL2 was slowly converted to the deneddylated form compared to the rapid deneddylation of other CULs. These findings provide mechanistic insights into the different dynamics of CULs in neddylation-deneddylation conversion.

mRNAs translation to proteins constitutes an important step of cellular gene expression that is highly regulated in response to different extracellular stimuli and stress situations. The fine control of protein synthesis is carried out both qualitatively and quantitatively, depending on the cellular demand at each moment. Post-translational modifications, in turn regulated by intracellular signaling pathways, play a key role in translation regulation. Among them, ubiquitination, whose role is becoming increasingly important in the control of translation, determines a correct balance between protein synthesis and degradation. In this review we focus on the role of ubiquitination (both degradative K48-linkage type and non-degradative K63-linkage type and monoubiquitination) in eukaryotic translation, both at the pre-translational level during the biogenesis/degradation of the components of translational machinery as well as at the co-translational level under stressful conditions. We also discuss other ubiquitin-dependent regulatory mechanisms of mRNA protection and resumption of translation after stress removal, where the ubiquitination of ribosomal proteins and associated regulatory proteins play an important role in the global rhythm of translation.

The covalent attachment of ubiquitin and ubiquitin-like polypeptides to cellular and viral proteins regulates numerous processes that enable virus infection, viral genome replication, and the spread of viruses to new hosts. The importance of these protein modifications in the regulation of the life cycle of herpesviruses is underscored by the discovery that all known members of this virus family encode at least one protease that specifically recognizes and disassembles ubiquitin conjugates. The structural and functional characterization of the viral enzymes and the identification of their viral and cellular substrates is providing valuable insights into the biology of viral infection and increasing evidence suggests that the viral deconjugases may also play a role in malignant transformation.

The reversible attachment of ubiquitin (Ub) and ubiquitin like modifiers (Ubls) to proteins are crucial post-translational modifications (PTMs) for many cellular processes. Not only do cells possess hundreds of ligases to mediate substrate specific modification with Ub and Ubls, but they also have a repertoire of more than 100 dedicated enzymes for the specific removal of ubiquitin (Deubiquitinases or DUBs) and Ubl modifications (Ubl-specific proteases or ULPs). Over the past two decades, there has been significant progress in our understanding of how DUBs and ULPs function at a molecular level and many novel DUBs and ULPs, including several new DUB classes, have been identified. Here, the development of chemical tools that can bind and trap active DUBs has played a key role. Since the introduction of the first activity-based probe for DUBs in 1986, several innovations have led to the development of more sophisticated tools to study DUBs and ULPs. In this review we discuss how chemical biology has led to the development of activity-based probes and substrates that have been invaluable to the study of DUBs and ULPs. We summarise our currently available toolbox, highlight the main achievements and give an outlook of how these tools may be applied to gain a better understanding of the regulatory mechanisms of DUBs and ULPs.

Post-translational modification by Small Ubiquitin-like Modifier (SUMO) proteins regulates numerous cellular processes. This modification involves the covalent and reversible attachment of SUMO to target proteins through an isopeptide bond, using a cascade of E1, E2 and E3 SUMOylation enzymes. Most functions of SUMO depend on the establishment of non-covalent protein-protein interactions between SUMOylated substrates and their binding partners. The vast majority of these interactions involve a conserved surface in the SUMO protein and a SUMO interacting motif (SIM), a short stretch of hydrophobic amino acids and an acidic region, in the interactor protein. Despite single SUMO-SIM interactions are relatively weak, they can have a huge impact at different levels, altering the activity, localization and stability of proteins, triggering the formation of macromolecular assemblies or inducing phase separation. Moreover, SUMO-SIM interactions are ubiquitous in most enzymes of the SUMO pathway, and play essential roles in SUMO conjugation and deconjugation. Here, we analyze the role of SUMO-SIM contacts in SUMO enzymes and targets and discuss how this humble interaction participates in SUMOylation reactions and mediates the outcome of this essential post-translational modification.

Ubiquitination is an important post-translational modification that regulates multiple cellular activities in plants including environmental stress responses. In addition to activity of ubiquitin ligases, the activity of deubiquitinating enzymes (DUBs) is critical for modulating the optimal ubiquitination status of target proteins in response to environmental stimuli. However, while several ubiquitin ligases have been isolated to date, little is known about the DUBs involved in plant stress responses. Here, we report that two DUBs, UBP12 and UBP13, function in response to disrupted carbon (C)/nitrogen (N)-nutrient stress conditions in Arabidopsis. Knockdown of UBP12 and UBP13 expression resulted in hypersensitivity to high C/low N-nutrient stress conditions, whereas overexpression of UBP13 reduced the sensitivity. Additionally, UBP13 physically interacted with and deubiquitinated the ubiquitin ligase ATL31, a key regulator of plant resistance to high C/low N-nutrient stress conditions. Genetic analysis showed that the loss of ATL31 and its homolog ATL6 suppressed the high C/low N-hyposensitivity of UBP13-overexpressing plants, suggesting that ATL31 is epistatic to UBP12 and UBP13. Taken together, our results suggest that the DUBs UBP12 and UBP13 function together with the ubiquitin ligase ATL31 to mediate C/N-nutrient stress responses in plants.

Stress is unavoidable and essential to cellular and organismal evolution and failure to adapt or restore homeostasis can lead to severe diseases or even death. At the cellular level, stress drives a plethora of molecular changes, of which variations in the profile of protein post-translational modifications plays a key role in mediating the adaptative response of the genome and proteome to stress. In this context, post-translational modification of proteins by ubiquitin-like modifiers, (Ubl), notably SUMO, is an essential stress response mechanism. In this review, aiming to draw universal concepts of the Ubls stress response, we will decipher how stress alters the expression level, activity, specificity and/or localization of the proteins involved in the conjugation pathways of the various type-I Ubls, and how this result in the modification of particular Ubl targets that will translate an adaptive physiological stress response and allow cells to restore homeostasis.

Mitochondria are involved in various cellular processes, such as energy production, inflammatory responses and cell death. Mitochondrial dysfunction is associated with many age-related diseases, including neurological disorders and heart failure. Mitochondrial quality is strictly maintained by mitochondrial dynamics linked to an adequate supply of phospholipids and other substances from the endoplasmic reticulum (ER). The outer mitochondrial membrane-localized E3 ubiquitin ligase MITOL/MARCHF5 is responsible for mitochondrial quality control through the regulation of mitochondrial dynamics, formation of mitochondria-ER contacts and mitophagy. MITOL deficiency has been shown to impair mitochondrial function, cause an excessive inflammatory response and increase vulnerability to stress, resulting in the exacerbation of the disease. In this study, we overview the ubiquitin-mediated regulation of mitochondrial function by MITOL and the relationship between MITOL and diseases.

Cyclin F, unlike canonical and transcriptional cyclins, does not bind or activate any cyclin-dependent kinases. Instead, it harbors an F-box motif and primarily functions as the substrate recognition subunit of the Skp1-Cul1-F-box E3 ubiquitin ligase complex, SCF

Histone deacetylase 4/5 (HDAC4/5) are essential for regulating metabolic gene expression; AMPKα2 regulates HDAC4/5 activity and the expression of MuRF1 during exercise. In this study, we used wild-type and AMPKα2−/− mice to explore the potential regulatory relationship between AMPKα2 and HDAC4/5 expression during exercise. Firstly, we fed C57BL/6J mice with high-fat diet for 8 weeks to assess the effects of high-fat diet on skeletal muscle metabolism and HDAC4/5 expression. We then performed a 6-week treadmill exercise on both wild-type and AMPKα2−/− mice. After exercise, the expressions of HDAC4/5 were examined in both gastrocnemius and soleus. The citrate synthase activity and proteins involved in skeletal muscle oxidative process were assessed. To determine the relationship of HDAC4/5 and skeletal muscle oxidative capacity, citrate synthase activity was assessed after silencing HDAC4/5. Moreover, HDAC5 ubiquitination and the association of MuRF1 to HDAC5 were also investigated. Our results showed that 6-week exercise increased the skeletal muscle oxidative capacity and decreased HDAC4/5 expression only in soleus. HDAC5 silencing increased C2C12 cell oxidative capacity. Proteasome inhibition by MG132 abolished exercise-induced HDAC5 degradation mediated by MuRF1–ubiquitin–proteasome system. However, the ubiquitin–proteasome system (UPS) did not dominantly account for exercise-induced HDAC4 degradation. Exercise upregulated MuRF1–HDAC5 association in wild-type mice but not in AMPKα2−/− mice. Our results revealed that 6-week exercise increased the skeletal muscle oxidative capacity and promoted HDAC5 degradation in soleus through the UPS, MuRF1-mediated HDAC5 ubiquitination. Although AMPKα2 played a partial role in regulating MuRF1 expression and HDAC5 ubiquitination, exercise-induced HDAC5 degradation did not fully depend on AMPKα2.

The receptor tyrosine kinase EPHB2 (EPH receptor B2) is highly expressed in many human cancer types, especially in gastrointestinal cancers, such as colorectal cancer. Several coding mutations of the EPHB2 gene have been identified in many cancer types, suggesting that EPHB2 plays a critical role in carcinogenesis. However, the exact functional mechanism of EPHB2 in carcinogenesis remains unknown. In this study, we find that EPHB2 is required for TNF-induced signaling activation and proinflammatory cytokine production in colorectal epithelial cells. Mechanistically, after TNF stimulation, EPHB2 is ubiquitinated by its E3 ligase RNF186. Then, ubiquitinated EPHB2 recruits and further phosphorylates TAB2 at nine tyrosine sites, which is a critical step for the binding between TAB2 and TAK1. Due to defects in TNF signaling in RNF186-knockout colorectal epithelial cells, the phenotype of colitis-propelled colorectal cancer model in RNF186-knockout mice is significantly reduced compared with that in wild-type control mice. Moreover, we find that a genetic mutation in EPHB2 identified in a family with colorectal cancer is a gain-of-function mutation that promoted TNF signaling activation compared with wild-type EPHB2. We provide evidence that the EPHB2-RNF186-TAB2-TAK1 signaling cascade plays an essential role in TNF-mediated signal transduction in colorectal epithelial cells and the carcinogenesis of colorectal cancer, which may provide potential targets for the treatment of colorectal cancer.

The carboxy-terminus of Hsp70-interacting protein (CHIP) is a ubiquitin ligase and co-chaperone belonging to Ubox family that plays a crucial role in the maintenance of cellular homeostasis by switching the equilibrium of the folding-refolding mechanism towards the proteasomal or lysosomal degradation pathway. It links molecular chaperones viz. HSC70, HSP70 and HSP90 with ubiquitin proteasome system (UPS), acts as a quality control system. CHIP contains charged domain in between N-terminal TPR and C-terminal Ubox domain. TPR domain interacts with the aberrant client proteins via chaperones while Ubox domain facilitates the ubiquitin transfer to the client proteins for ubiquitination. Thus, CHIP is a classic molecule that executes ubiquitination for degradation of client proteins. Further, CHIP has been found to be indulged in cellular differentiation, proliferation, metastasis and tumorigenesis. Additionally, CHIP can play its dual role as a tumor suppressor as well as an oncogene in numerous malignancies, thus acts as a double agent. Here, in this review, we have reported almost all substrates of CHIP established till date and classified them according to the hallmarks of cancer. In addition, we discussed about its architectural alignment, tissue specific expression, sub-cellular localization, folding-refolding mechanisms of client proteins, E4 ligase activity, normal physiological roles, involvement in various diseases and tumor biology. Further, we aim to discuss its importance in HSP90 inhibitors mediated cancer therapy. Thus, this report concludes that CHIP may be a promising and worthy drug target towards pharmaceutical industry for drug development.

Coronaviruses have been responsible for multiple challenging global pandemics, including coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Papain-like protease (PLpro), one of two cysteine proteases responsible for the maturation and infectivity of SARS-CoV-2, processes and liberates functional proteins from the viral polyproteins and cleaves ubiquitin and ISG15 modifications to inhibit innate immune sensing. Consequently, PLpro is an attractive target for developing COVID-19 therapies. PLpro contains a zinc-finger domain important for substrate binding and structural stability. However, the impact of metal ions on the activity and biophysical properties of SARS-CoV-2 PLpro has not been comprehensively studied. Here, we assessed the impacts of metal ions on the catalytic activity of PLpro. Zinc had the largest inhibitory effect on PLpro, followed by manganese. Calcium, magnesium, and iron had smaller or no effects on PLpro activity. EDTA at a concentration of 0.5 mM was essential for PLpro activity, likely by chelating trace metals that inhibit PLpro. IC50 values for ZnCl2, ZnSO4, and MnCl2 of 0.42 ± 0.02 mM, 0.35 ± 0.01 mM, and 2.6 ± 0.3 mM were obtained in the presence of 0.5 mM EDTA; in the absence of EDTA, the estimated IC50 of ZnCl2 was 14 µM. Tryptophan intrinsic fluorescence analysis confirmed the binding of zinc and manganese to PLpro, and differential scanning calorimetry revealed that zinc but not manganese reduced ΔHcal of PLpro. The results of this study provide a reference for further work targeting PLpro to prevent and treat COVID-19.

Infection with Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), remains a significant global epidemic. Host resistance to Mtb depends on both adaptive and innate immunity mechanisms, including development of antigen-specific CD4 and CD8 T cells, production of inflammatory cytokines, bacterial phagocytosis and destruction within phagolysosomes, host cell apoptosis, and autophagy. A key regulatory mechanism in innate immunity is the attachment of the small protein ubiquitin to protein and lipid targets by the enzymatic activity of ubiquitin ligases. Here, we summarize the latest advances on the role of ubiquitination and ubiquitin ligases in host immunity against Mtb, with a focus on innate immunity signaling, inflammation, and antimicrobial autophagy. Understanding how ubiquitin ligases mediate immunity to Mtb, and the specific substrates of distinct ubiquitin ligases in the context of Mtb infection, could facilitate development of new host-directed antimicrobials.

Cancer cells rely on glycolysis to generate ATP for survival. However, inhibiting glycolysis is insufficient for the eradication of cancer cells because glycolysis-suppressed cells undergo metabolic reprogramming toward mitochondrial oxidative phosphorylation. We previously described that upon glycolytic suppression in pancreatic cancer cells, intracellular glycometabolism is shifted toward mitochondrial oxidative phosphorylation in an autophagy-dependent manner for cellular survival. Here, we hypothesized that mitophagy, which selectively degrades mitochondria via autophagy, is involved in mitochondrial activation under metabolic reprogramming. We revealed that glycolytic suppression notably increased mitochondrial membrane potential and mitophagy in a pancreatic cancer cell model (PANC-1). PTEN-induced kinase 1 (PINK1), a ubiquitin kinase that regulates mitophagy in healthy cells, regulated mitochondrial activation through mitophagy by glycolytic suppression. However, Parkin, a ubiquitin ligase regulated by PINK1 in healthy cells to induce mitophagy, was not involved in the PINK1-dependent mitophagy of the cancer glycometabolism. These results imply that cancer cells and healthy cells have different regulatory pieces of machinery for mitophagy, and inhibition of cancer-specific mechanisms may be a potential strategy for cancer therapy targeting metabolic reprogramming.

Members of the HECT family of E3 ubiquitin ligases have emerged as prominent regulators of PTEN function, subcellular localization and levels. In turn this unfolding regulatory network is allowing for the identification of genes directly involved in both tumorigenesis at large and cancer susceptibility syndromes. While the complexity of this regulatory network is still being unraveled, these new findings are paving the way for novel therapeutic modalities for cancer prevention and therapy as well as for other diseases. Here we will review the signal transduction and therapeutic implications of the cross-talk between HECT family members and PTEN.

Proteins modified in a controlled manner with artificial moieties such as fluorophores or affinity tags have been shown to be a powerful tool for functional or structural analysis of proteins. A reliable way to prepare proteins with a well-defined modification is protein synthesis. Although many successful syntheses have been reported, the poor aqueous solubility of synthetic intermediates causes difficulty in the chemical synthesis of proteins. Here we describe a solubilizing strategy for poorly soluble peptides which uses chemoselective incorporation of a hydrophilic tag onto a hydrazide in a peptide. We found that a hydrophilic tag possessing a dialkoxybenzaldehyde moiety can react with peptide hydrazides through reductive N-alkylation. No protecting groups are required for this reaction, and peptides modified in this way show enhanced solubility and consequently good peak separation during HPLC purification. The tag can be removed subsequently by treatment with trifluoroacetic acid to generate a free hydrazide, which can be converted in a one-pot reaction to a thioester for further modification. This method was validated by synthesis of a Lys

Small ubiquitin-like modifier (SUMO) plays a key regulatory role in cardiovascular diseases, such as cardiac hypertrophy, hypertension, atherosclerosis, and cardiac ischemia-reperfusion injury. As a multifunctional posttranslational modification molecule in eukaryotic cells, SUMOylation is essentially associated with the regulation of mitochondrial dynamics, especially mitophagy, which is involved in the progression and development of cardiovascular diseases. SUMOylation targeting mitochondrial-associated proteins is admittedly considered to regulate mitophagy activation and mitochondrial functions and dynamics, including mitochondrial fusion and fission. SUMOylation triggers mitochondrial fusion to promote mitochondrial dysfunction by modifying Fis1, OPA1, MFN1/2, and DRP1. The interaction between SUMO and DRP1 induces SUMOylation and inhibits lysosomal degradation of DRP1, which is further involved in the regulation of mitochondrial fission. Both SUMOylation and deSUMOylation contribute to the initiation and activation of mitophagy by regulating the conjugation of MFN1/2 SERCA2a, HIF1α, and PINK1. SUMOylation mediated by the SUMO molecule has attracted much attention due to its dual roles in the development of cardiovascular diseases. In this review, we systemically summarize the current understanding underlying the expression, regulation, and structure of SUMO molecules; explore the biochemical functions of SUMOylation in the initiation and activation of mitophagy; discuss the biological roles and mechanisms of SUMOylation in cardiovascular diseases; and further provide a wider explanation of SUMOylation and deSUMOylation research to provide a possible therapeutic strategy for cardiovascular diseases. Considering the precise functions and exact mechanisms of SUMOylation in mitochondrial dysfunction and mitophagy will provide evidence for future experimental research and may serve as an effective approach in the development of novel therapeutic strategies for cardiovascular diseases. Regulation and effect of SUMOylation in cardiovascular diseases via mitophagy. SUMOylation is involved in multiple cardiovascular diseases, including cardiac hypertrophy, hypertension, atherosclerosis, and cardiac ischemia-reperfusion injury. Since it is expressed in multiple cells associated with cardiovascular disease, SUMOylation can be regulated by numerous ligases, including the SENP family proteins PIAS1, PIASy/4, UBC9, and MAPL. SUMOylation regulates the activation and degradation of PINK1, SERCA2a, PPARγ, ERK5, and DRP1 to mediate mitochondrial dynamics, especially mitophagy activation. Mitophagy activation regulated by SUMOylation further promotes or inhibits ventricular diastolic dysfunction, perfusion injury, ventricular remodelling and ventricular noncompaction, which contribute to the development of cardiovascular diseases.

Intron diversity facilitates regulated gene expression and alternative splicing. Spliceosomes excise introns after recognizing their splicing signals: the 5′-splice site (5′ss), branchpoint (BP) and 3′-splice site (3′ss). The latter two signals are recognized by U2 small nuclear ribonucleoprotein (snRNP) and its accessory factors (U2AFs), but longer spacings between them result in weaker splicing. Here, we show that excision of introns with a BP-distant 3′ss (e.g. rap1 intron 2) requires the ubiquitin-fold-activated splicing regulator Sde2 in Schizosaccharomyces pombe. By monitoring splicing-specific ura4 reporters in a collection of S. pombe mutants, Cay1 and Tls1 were identified as additional regulators of this process. The role of Sde2, Cay1 and Tls1 was further confirmed by increasing BP–3′ss spacings in a canonical tho5 intron. We also examined BP-distant exons spliced independently of these factors and observed that RNA secondary structures possibly bridged the gap between the two signals. These proteins may guide the 3′ss towards the spliceosome's catalytic centre by folding the RNA between the BP and 3′ss. Orthologues of Sde2, Cay1 and Tls1, although missing in the intron-poor Saccharomyces cerevisiae, are present in intron-rich eukaryotes, including humans. This type of intron-specific pre-mRNA splicing appears to have evolved for regulated gene expression and alternative splicing of key heterochromatin factors.

Dietary supplementation with branched-chain amino acids (BCAAs) to lactating sows has been reported to enhance their milk production, but the underlying mechanisms remain largely unknown. This study was conducted with porcine mammary epithelial cells (PMECs) to test the hypothesis that individual BCAAs or their mixture stimulates protein synthesis and inhibit proteolysis in PMECs. Cells were cultured at 37 °C in customized Dulbecco's modified Eagle medium containing 5 mmol/L D-glucose, 1 mmol/L L-phenylalanine, L-[ring-2,4

Parkinson's disease is a common incurable neurodegenerative disease. The identification of genetic variants via genome-wide association studies has considerably advanced our understanding of the Parkinson's disease genetic risk. Understanding the functional significance of the risk loci is now a critical step towards translating these genetic advances into an enhanced biological understanding of the disease. Impaired mitophagy is a key causative pathway in familial Parkinson's disease, but its relevance to idiopathic Parkinson's disease is unclear. We used a mitophagy screening assay to evaluate the functional significance of risk genes identified through genome-wide association studies. We identified two new regulators of PINK1-dependent mitophagy initiation, KAT8 and KANSL1, previously shown to modulate lysine acetylation. These findings suggest PINK1-mitophagy is a contributing factor to idiopathic Parkinson's disease. KANSL1 is located on chromosome 17q21 where the risk associated gene has long been considered to be MAPT. While our data do not exclude a possible association between the MAPT gene and Parkinson's disease, they provide strong evidence that KANSL1 plays a crucial role in the disease. Finally, these results enrich our understanding of physiological events regulating mitophagy and establish a novel pathway for drug targeting in neurodegeneration. ispartof: BRAIN vol:145 issue:12 pages:4349-4367 ispartof: location:England status: published

Celastrol (CEL), a pentacyclic triterpene compound, has been proven to have a definite antipulmonary fibrosis effect. However, its direct targets for antipulmonary fibrosis remain unknown. In this study, we designed and synthesized a series of celastrol-based probes to identify the direct targets in human pulmonary fibroblasts using an activity-based protein profiling strategy. Among many fished targets, we identified a key protein, cullin-associated and neddylation-dissociated 1 (CAND1), which was involved in fibroblast-myofibroblast transformation (FMT). More importantly, we found that the inhibitory effect of celastrol on FMT is dependent on CAND1, through improving the interactions between CAND1 and Cullin1 to promote the activity of Skp1/Cullin1/F-box ubiquitin ligases. In silico studies and cysteine mutation experiments further demonstrated that Cys264 of CAND1 is the site for conjugation of celastrol. This reveals a new mechanism of celastrol against pulmonary fibrosis and may provide a novel therapeutic option for antipulmonary fibrosis.

Cancer cells are dependent on protein quality-control mechanisms, including protein chaperones, the ubiquitin-proteasome system, and autophagy. The p62 receptor is a classical, ubiquitously expressed receptor, involved in many signal transduction pathways. Upregulation and/or reduced degradation of p62 have been implicated in tumor formation and resistance to therapy. PTX80 is a first-in-class novel inhibitor of protein degradation, developed by Pi Therapeutics for the treatment of cancer. PTX80 binds to p62, inducing a decrease in soluble p62 and formation of insoluble p62 aggregates, and failure of polyubiquitinated proteins to colocalize with p62. PTX80 induces proteotoxic stress and activation of unfolded protein response, which, in turn, leads to apoptosis. Targeting p62, which is a major protein degradation hub, may serve as a novel and beneficial strategy for the treatment of cancer.

Tick-borne orthonairoviruses have been characterized as a global health threat to humans and animals. Tacheng Tick virus 1 (TcTV-1) from this family was provided as evidence that is associated with the febrile illness syndrome. Here, we first identify and demonstrate that the ovarian tumor (OTU) domain of TcTV-1 has remarkable deubiquitinating activity both in vitro and in vivo. By solving the crystal structure of TcTV-1 OTU (tcOTU) domain and comparing it to that of human deubiquitinating enzymes, we found that overall structures of tcOTU and human OTU family are similar, but the residues involved in the catalytic pocket vary widely. Based on the tcOTU domain we screened 5090 bioactive compounds and found mecobalamin had a good effect on suppressing the deubiquitinating activity. The structural model of tcOTU and mecobalamin suggests that mecobalamin occupies the site of the substrate Ub, by blocking the substrate binding to the enzyme. Thus, our results showed OTU domain of TcTV-1 has a robust deubiquitinating activity and mecobalamin or its derivatives might be promising candidates for the treatment or prevention of disease caused by the TcTV-1 virus.

The ubiquitin proteasome system (UPS) is a primary mechanism through which proteins are degraded in cells. UPS activity in the dorsal hippocampus (DH) is necessary for multiple types of memory, including object memory, in male rodents. However, sex differences in DH UPS activation after fear conditioning suggest that other forms of learning may also differentially regulate DH UPS activity in males and females. Here, we examined markers of UPS activity in the synaptic and cytoplasmic fractions of DH and medial prefrontal cortex (mPFC) tissue collected 1 h following object training. In males, training increased phosphorylation of proteasomal subunit Rpt6, 20S proteasome activity, and the amount of PSD-95 in the DH synaptic fraction, as well as proteasome activity in the mPFC synaptic fraction. In females, training did not affect measures of UPS or synaptic activity in the DH synaptic fraction or in either mPFC fraction but increased Rpt6 phosphorylation in the DH cytoplasmic fraction. Overall, training-induced UPS activity was greater in males than in females, greater in the DH than in the mPFC, and greater in synaptic fractions than in cytosol. These data suggest that object training drives sex-specific alterations in UPS activity across brain regions and subcellular compartments important for memory.

Abstract: Impairment in the function of insulin-producing pancreatic β-cells is a hallmark of both type 1 and 2 diabetes (T1D/T2D). Despite over a century of effort, there is still no precise treatment regimen available for acute diabetes. Enhancing the endogenous β-cells either by protecting them from apoptosis or dedifferentiation is a classic alternative to retaining the β-cell pool. Recent reports have acknowledged the protein homeostasis mediated by the ubiquitin-proteasome system as one of the essential components in maintaining the β-cell pool. Degradation of the targeted substrate by the proteasome is majorly regulated by the ubiquitination status of the targeted protein dictated by E3 ligases and deubiquitinase enzymes. Imbalance in the function of these enzymes results in the malfunction of β-cells and, subsequently, hyperglycemia. Ubiquitination involves the covalent attachment of one or more ubiquitin moieties to the target protein by E3 ubiquitin ligases and deubiquitinases (DUBs) are the enzymes that antagonize the action of E3 ligases. Knowing different E3 ligases and deubiquitinases in the process of differentiation and dedifferentiation of β-cells probably paves the way for designing novel modulators that enhance either the differentiation or abate the dedifferentiation process. In this review, we will discuss the importance of the balanced ubiquitination process, an understanding of which would facilitate the restraining of β-cells from exhaustion.

The extracellular ubiquitin–proteasome system is involved in sperm binding to and/or penetration of the vitelline coat (VC), a proteinaceous egg coat, during fertilization of the ascidian (Urochordata) Halocynthia roretzi. It is also known that the sperm receptor on the VC, HrVC70, is ubiquitinated and degraded by the sperm proteasome during the sperm penetration of the VC and that a 700-kDa ubiquitin-conjugating enzyme complex is released upon sperm activation on the VC, which is designated the “sperm reaction”. However, the de novo function of ubiquitin-activating enzyme (UBA/E1) during fertilization is poorly understood. Here, we show that PYR-41, a UBA inhibitor, strongly inhibited the fertilization of H. roretzi. cDNA cloning of UBA1 and UBA6 from H. roretzi gonads was carried out, and their 3D protein structures were predicted to be very similar to those of human UBA1 and UBA6, respectively, based on AlphaFold2. These two genes were transcribed in the ovary and testis and other organs, among which the expression of both was highest in the ovary. Immunocytochemistry showed that these enzymes are localized on the sperm head around a mitochondrial region and the follicle cells surrounding the VC. These results led us to propose that HrUBA1, HrUBA6, or both in the sperm head mitochondrial region and follicle cells may be involved in the ubiquitination of HrVC70, which is responsible for the fertilization of H. roretzi.

Ubiquitin-related post translation modification (PTM) plays an important role in cellular signalling. TRABID is a deubiquitylase that preferentially hydrolyses Lys29 and 33 ubiquitin chains and is known to affect inflammation and consequently, neurodegeneration as the latter is considered functionally linked to the former. However, sex differences are widely observed in both inflammation as well as neurodegeneration phenotypes. In this study, I investigated sex dimorphism, in ubiquitination at the intersection of inflammation and neurodegeneration. I demonstrated Drosophila TRABID has similar enzymatic activity as human TRABID and generated an enzymatically inactive TRABID (trbdC518A) in Drosophila flies. I discovered that trbdC518A has a sexually dimorphic impact in the insect’s locomotor activity, sleep pattern, lifespan, cerebral cells count, brain volume and NF-κB signalling. When blocking female development in astrocytes or immunocompetent cells, sex dimorphisms in locomotor activity and NF-κB signalling are partially eliminated. Further evidence ubiquitinomics and proteomics of fly heads, provided insight into the sex-specific impact of trbdC518A.

Cleistogamy, self-pollination within closed flowers, can help maintain seed purity, accelerate breeding speed, and aid in the development of ornamental flowers. However, the mechanism underlying petal closing/opening behavior remains elusive. Here, we found that a Brassica napus petal closing/opening behavior was inherited in a Mendelian manner. Fine mapping and positional cloning experiments revealed that the Mendelian factor originated from a short (29.8 kb) inversion mediated by BnDTH9 miniature inverted-repeat transposable elements (MITEs) on chromosome C03. This inversion led to tissue-specific gene promoter exchange between BnaC03.FBA (BnaC03G0156800ZS encoding an F-Box-associated domain-containing protein) and BnaC03.EFO1 (BnaC03G0157400ZS encoding an EARLY FLOWERING BY OVEREXPRESSION 1 protein) positioned near the respective inversion breakpoints. Our genetic transformation work demonstrated that the cleistogamy originated from high tissue-specific expression of the BnaC03.FBA gene caused by promoter changes due to the MITE-mediated inversion. BnaC03.FBA is involved in the formation of an SCF (Skp1–Cullin–F-box) complex, which participates in ubiquitin-mediated protein targeting for degradation through the ubiquitin 26S-proteasome system. Our results shed light on a molecular model of petal-closing behavior.

The gas phase is an idealized laboratory for the study of protein structure, from which it is possible to examine stable and transient forms of mass-selected ions in the absence of bulk solvent. With ion mobility-mass spectrometry (IM-MS) apparatus built to operate at both cryogenic and elevated temperatures, we have examined conformational transitions that occur to the monomeric proteins: ubiquitin, lysozyme, and α-synuclein as a function of temperature and in source activation. We rationalize the experimental observations with a temperature-dependent framework model and comparison to known conformers. Data from ubiquitin show unfolding transitions that proceed through diverse and highly elongated intermediate states, which converge to more compact structures. These findings contrast with data obtained from lysozyme─a protein where (un)-folding plasticity is restricted by four disulfide linkages, although this is alleviated in its reduced form. For structured proteins, collision activation of the protein ions in-source enables subsequent "freezing" or thermal annealing of unfolding intermediates, whereas disordered proteins restructure substantially at 250 K even without activation, indicating that cold denaturation can occur without solvent. These data are presented in the context of a toy model framework that describes the relative occupancy of the available conformational space.

The response to abiotic and biotic stresses in plants and crops is considered a multifaceted process. Due to their sessile nature, plants have evolved unique mechanisms to ensure that developmental plasticity remains during their life cycle. Among these mechanisms, post-translational modifications (PTMs) are crucial components of adaptive responses in plants and transduce environmental stimuli into cellular signalling through the modulation of proteins. SUMOylation is an emerging PTM that has received recent attention due to its dynamic role in protein modification and has quickly been considered a significant component of adaptive mechanisms in plants during stress with great potential for agricultural improvement programs. In the present review, we outline the concept that small ubiquitin-like modifier (SUMO)-mediated response in plants and crops to abiotic and biotic stresses is a multifaceted process with each component of the SUMO cycle facilitating tolerance to several different environmental stresses. We also highlight the clear increase in SUMO genes in crops when compared with Arabidopsis thaliana. The SUMO system is understudied in crops, given the importance of SUMO for stress responses, and for some SUMO genes, the apparent expansion provides new avenues to discover SUMO-conjugated targets that could regulate beneficial agronomical traits.

Plants must cope with an ever-changing environment, including concurrent biotic and abiotic stresses. The ubiquitin–proteasome system (UPS) is intricately involved in regulating signaling events that facilitate cellular changes required to mitigate the detrimental effects of environmental stress. A key component of the UPS are ubiquitin ligases (or E3s) that catalyze the attachment of ubiquitin molecules to select substrate proteins, which are then recognized by the 26S proteasome for degradation. With the identification of substrate proteins, a growing number of E3s are shown to differentially regulate responses to abiotic as well as bioitic stresses. The review discusses select E3s to illustrate the role of ubiquitin ligases as negative and/or positive regulators of responses to both biotic and abiotic stresses.

The majority of human proteins operate as multimeric complexes with defined compositions and distinct architectures. How the assembly of these complexes is surveyed and how defective complexes are recognized is just beginning to emerge. In eukaryotes, over 600 E3 ubiquitin ligases form part of the ubiquitin-proteasome system (UPS) which detects structural characteristics in its target proteins and selectively induces their degradation. The UPS has recently been shown to oversee key quality control steps during the assembly of protein complexes. We review recent findings on how E3 ubiquitin ligases regulate protein complex assembly and highlight unanswered questions relating to their mechanism of action.

Most research in the field of ubiquitination has focused on E3 ubiquitin ligases because they are the specificity determinants of the ubiquitination process. Nevertheless, E2s are responsible for the catalysis during ubiquitin transfer, and are therefore, at the heart of the ubiquitination process. Arabidopsis has 37 ubiquitin E2s with additional ones mediating the attachment of ubiquitin-like proteins (e.g. SUMO, Nedd8 and ATG8). Importantly, E2s largely determine the type of ubiquitin chain built, and therefore, the type of signal that decides over the fate of the modified protein, such as degradation by the proteasome (Lys48-linked ubiquitin chains) or relocalization (Lys63-linked ubiquitin chains). Moreover, new regulatory layers impinging on E2s activity, including post-translational modifications or cofactors, are emerging that highlight the importance of E2s.

The proteasome plays vital roles in eukaryotic cells by orchestrating the regulated degradation of large repertoires of substrates involved in numerous biological processes. Proteasome dysfunction is associated with a wide variety of human pathologies and in plants severely affects growth, development and responses to stress. The activity of E3 ubiquitin ligases marks proteins fated for degradation with chains of the post-translational modifier, ubiquitin. Proteasomal processing of ubiquitinated substrates involves ubiquitin chain recognition, deubiquitination, ATP-mediated unfolding and translocation, and proteolytic digestion. This complex series of steps is made possible not only by the many specialised subunits of the 1.5 MDa proteasome complex but also by a range of accessory proteins that are recruited to the proteasome. A surprising class of accessory proteins are members of the HECT-type family of ubiquitin ligases that utilise a unique mechanism for post-translational attachment of ubiquitin to their substrates. So why do proteasomes that already contain all the necessary machinery to recognise ubiquitinated substrates, harbour HECT ligase activity? It is now clear that some ubiquitin ligases physically relay their substrates to proteasome-associated HECT ligases, which prevent substrate stalling at the proteasome. Moreover, HECT ligases ubiquitinate proteasome subunits, thereby modifying the proteasome’s ability to recognise substrates. They may therefore enable proteasomes to be both non-specific and extraordinarily selective in a complex substrate environment. Understanding the relationship between the proteasome and accessory HECT ligases will reveal how the proteasome controls so many diverse plant developmental and stress responses.

Proper regulation of protein homeostasis (proteostasis) is essential for all organisms to survive. A diverse range of post-translational modifications (PTMs) allow precise control of protein abundance, function and cellular localisation. In eukaryotic cells, ubiquitination is a widespread, essential PTM that regulates most, if not all cellular processes. Ubiquitin is added to target proteins via a well-defined enzymatic cascade involving a range of conjugating enzymes and ligases, while its removal is catalysed by a class of enzymes known as deubiquitinases (DUBs). Many human diseases have now been linked to DUB dysfunction, demonstrating the importance of these enzymes in maintaining cellular function. These findings have led to a recent explosion in studying the structure, molecular mechanisms and physiology of DUBs in mammalian systems. Plant DUBs have however remained relatively understudied, with many DUBs identified but their substrates, binding partners and the cellular pathways they regulate only now beginning to emerge. This review focuses on the most recent findings in plant DUB biology, particularly on newly identified DUB substrates and how these offer clues to the wide-ranging roles that DUBs play in the cell. Furthermore, the future outlook on how new technologies in mammalian systems can accelerate the plant DUB field forward is discussed.

Endoplasmic reticulum-associated degradation (ERAD) plays important roles in plant development, hormone signaling, and plant-environment stress interactions by promoting the clearance of certain proteins or soluble misfolded proteins through the ubiquitin–proteasome system. Selective autophagy is involved in the autophagic degradation of protein aggregates mediated by specific selective autophagy receptors. These two major degradation routes co-operate with each other to relieve the cytotoxicity caused by ER stress. In this review, we analyze ERAD and different types of autophagy, including nonselective macroautophagy and ubiquitin-dependent and ubiquitin-independent selective autophagy in plants, and specifically summarize the selective autophagy receptors characterized in plants. In addition to being a part of selective autophagy, ERAD components also serve as their cargos. Moreover, an ubiquitinated substrate can be delivered to two distinguishable degradation systems, while the underlying determinants remain elusive. These excellent findings suggest an interdependent but complicated relationship between ERAD and selective autophagy. According to this point, we propose several key issues that need to be addressed in the future.

Nuclear magnetization storage, once limited by longitudinal and transverse relaxation lifetimes

Although nanopores can be used for single-molecule sequencing of nucleic acids using low-cost portable devices, the characterization of proteins and their modifications has yet to be established. Here, we show that hydrophilic or glycosylated peptides translocate too quickly across FraC nanopores to be recognized. However, high ionic strengths (i.e., 3 M LiCl) and low pH (i.e., pH 3) together with using a nanopore with a phenylalanine at its constriction allows the recognition of hydrophilic peptides, and to distinguish between mono- and diglycosylated peptides. Using these conditions, we devise a nanopore method to detect, characterize, and quantify post-translational modifications in generic proteins, which is one of the pressing challenges in proteomic analysis. ispartof: NANO LETTERS vol:22 issue:13 pages:5357-5364 ispartof: location:United States status: published

Small cell lung cancers (SCLC) are highly aggressive, and currently there are no available targeted therapies. To identify clinically actionable drug combinations, we analyzed our previously reported chemogenomics screens and identified a synergistically cytotoxic combination of the topoisomerase I (TOP1) inhibitor topotecan and cycle-dependent kinase 7 (CDK7) inhibitor THZ1. Topotecan causes cell death by generating TOP1-induced DNA breaks and DNA-protein cross-links (TOP1-DPC) that require proteolysis by the ubiquitin-proteasome pathway for their repair. We find that inhibition of the transcriptional kinase CDK7 by THZ1 induces ubiquitin-mediated proteasomal degradation of RNA polymerase II and prevents the proteasomal degradation of TOP1-DPCs. We provide a mechanistic basis for combinatorial targeting of transcription using selective inhibitors of CDK7 and TOP1 in clinical trials to advance SCLC therapeutics.

Functional studies of the ubiquitin-26S proteasome system (UPS) have demonstrated that virtually all aspects of the plant's life involve UPS-mediated turnover of abnormal or short-lived proteins. However, the role of the UPS during development, including in seeds and fruits, remains to be determined in detail, although mutants of several of its core elements are known to be embryonically lethal. Unfortunately, early termination of embryogenesis limits the possibility to characterize the activities of the UPS in reproductive organs. Given both the economic and the societal impact of reproductive production, such studies are indispensable. Here, we systematically compared expression of multiple 26S proteasome subunits along with the dynamics of proteasome activity and total protein ubiquitylation in seedlings, developing siliques, and embryos of Arabidopsis thaliana. Since autophagy plays the second largest role in maintaining proteome stability, we parallelly studied three rate-limiting enzymes that are involved in autophagy flux. Our experiments unexpectedly discovered that, in contrast to the activities in seedlings, both protein and transcript levels of six selected 26S proteasome subunits gradually decline in immature siliques or embryos toward maturation while the autophagy flux rises despite the nutrient-rich condition. We also discovered a reciprocal turnover pathway between the proteasome and autophagy. While the autophagy flux is suppressed in seedlings by UPS-mediated degradation of its three key enzymes, transcriptional reprogramming dampens this process in siliques, which in turn stimulates a bulk autophagic degradation of proteasomes. Collectively, our study of the developmental changes of the UPS and autophagy activities suggests that they relay the proteome homeostasis regulation in early silique and/or seed development, highlighting their interactions during development.

Small ubiquitin-related modifier (SUMO)-mediated post-translational protein modification is widely conserved among eukaryotes. SUMOylation refers to the covalent attachment of SUMO to target proteins that alters their function, location, and protein-protein interactions when plants are under abiotic stress. We identified 37 genes in the apple genome that encoded members of the SUMOylation pathway. In addition, RNA-Seq data shows their expression levels between different tissues. We can find that there are mainly expressed genes between each component to ensure that the entire pathway works in the plant. We found that the expression levels of 12 genes were significantly changed under NaCl and ABA treatment through qRT-PCR. MdSIZ1a strongly expression responded to NaCl and ABA treatment. Subsequently, MdSIZ1a was cloned and transformed into apple callus, further verifying the important role of the SUMOylation pathway under stress conditions. The interaction between MdSIZ1a and MdSCEa was verified by yeast two-hybrid, confirming that MdSIZ1a acts as bridge enzyme on MdSCEa and target substrates. Finally, we predicted and analyzed the functional interaction network of E3 ligase to shed light on protein interactions and gene regulatory networks associated with DNA damage repair under abiotic stress in apples.

Proteins that regulate the cell cycle are accumulated and degraded in a coordinated manner during the transition from one cell cycle phase to the next. The rapid loss of a critical protein, for example, to allow the cell to move from G1/G0 to S phase, is often regulated by its ubiquitination and subsequent proteasomal degradation. Protein ubiquitination is mediated by a series of three ligases, of which the E3 ligases provide the specificity for a particular protein substrate. One such E3 ligase is SCF(Skp1/Cks1), which has a substrate recruiting subunit called S-phase kinase-associated protein 2 (Skp2). Skp2 regulates cell proliferation, apoptosis, and differentiation, can act as an oncogene, and is overexpressed in human cancer. A primary target of Skp2 is the cyclin-dependent kinase inhibitor p27 (CDKN1b) that regulates the cell cycle at several points. The RB1 tumour suppressor gene regulates Skp2 activity by two mechanisms: by controlling its mRNA expression, and by an effect on Skp2’s enzymatic activity. For the latter, the RB1 protein (pRb) directly binds to the substrate-binding site on Skp2, preventing protein substrates from being ubiquitinated and degraded. Inactivating mutations in RB1 are common in human cancer, becoming more frequent in aggressive, metastatic, and drug-resistant tumours. Hence, RB1 mutation leads to the loss of pRb, an unrestrained increase in Skp2 activity, the unregulated decrease in p27, and the loss of cell cycle control. Because RB1 mutations lead to the loss of a functional protein, its direct targeting is not possible. This perspective will discuss evidence validating Skp2 as a therapeutic target in RB1-deficient cancer.

Ubiquitination-dependent histone crosstalk plays critical roles in chromatin-associated processes and is highly associated with human diseases. Mechanism studies of the crosstalk have been of the central focus. Here our study on the crosstalk between H2BK34ub and Dot1L-catalyzed H3K79me suggests a novel mechanism of ubiquitination-induced nucleosome distortion to stimulate the activity of an enzyme. We determined the cryo-electron microscopy structures of Dot1L-H2BK34ub nucleosome complex and the H2BK34ub nucleosome alone. The structures reveal that H2BK34ub induces an almost identical orientation and binding pattern of Dot1L on nucleosome as H2BK120ub, which positions Dot1L for the productive conformation through direct ubiquitin-enzyme contacts. However, H2BK34-anchored ubiquitin does not directly interact with Dot1L as occurs in the case of H2BK120ub, but rather induces DNA and histone distortion around the modified site. Our findings establish the structural framework for understanding the H2BK34ub-H3K79me trans-crosstalk and highlight the diversity of mechanisms for histone ubiquitination to activate chromatin-modifying enzymes.

The family of deubiquitinases (DUBs) comprises ∼100 enzymes that cleave ubiquitin from substrate proteins and thereby regulate key aspects of human physiology. DUBs have recently emerged as disease-relevant and chemically tractable, although currently there are no approved DUB-targeting drugs and most preclinical small molecules are low-potency and/or multitargeted. We paired a novel capillary electrophoresis microchip containing an integrated, "on-chip" C18 bed (SPE-ZipChip) with a TMT version of our recently described PRM-LIVE acquisition scheme on a timsTOF Pro mass spectrometer to facilitate rapid activity-based protein profiling of DUB inhibitors. We demonstrate the ability of the SPE-ZipChip to improve proteome coverage of complex samples as well as the quantitation integrity of CE-PRM-LIVE for TMT labeled samples. These technologies provide a platform to accurately quantify competitive binding of covalent and reversible inhibitors in a multiplexed assay that spans 49 endogenous DUBs in less than 15 min.

Methods to direct the degradation of protein targets with proximity-inducing molecules that coopt the cellular degradation machinery are advancing in leaps and bounds, and diverse modalities are emerging. The most used and well-studied approach is to hijack E3 ligases of the ubiquitin–proteasome system. E3 ligases use specific molecular recognition to determine which proteins in the cell are ubiquitinated and degraded. This review focuses on the structural determinants of E3 ligase recruitment of natural substrates and neo-substrates obtained through monovalent molecular glues and bivalent proteolysis-targeting chimeras. We use structures to illustrate the different types of substrate recognition and assess the basis for neo-protein–protein interactions in ternary complex structures. The emerging structural and mechanistic complexity is reflective of the diverse physiological roles of protein ubiquitination. This molecular insight is also guiding the application of structure-based design approaches to the development of new and existing degraders as chemical tools and therapeutics.