Uncovering DUB Selectivity through an Ion Mobility-Based Assessment of Ubiquitin Chain Isomers.

Ubiquitination is a reversible post-translational modification that maintains cellular homeostasis and regulates protein turnover. Deubiquitinases (DUBs) are a large family of proteases that catalyze the removal of ubiquitin (Ub) along with the dismantling and editing of Ub chains. Assessing the activity and selectivity of DUBs is critical for defining physiological functions. Despite numerous methods for evaluating DUB activity, none are capable of assessing activity and selectivity in the context of multicomponent mixtures of native unlabeled Ub conjugates. Here, we report an ion mobility (IM)-based approach for measuring DUB selectivity in the context of unlabeled mixtures of Ub chains. We show that IM-mass spectrometry (IM-MS) can be used to assess the selectivity of DUBs in a time-dependent manner. Moreover, using the branched Ub chain selective DUB UCH37/UCHL5 along with a mixture of Ub trimers, a strong preference for branched Ub trimers bearing K6 and K48 linkages is revealed. Our results demonstrate that IM-MS is a powerful method for evaluating DUB selectivity under conditions more physiologically relevant than single-component mixtures.

Quantitative Assessment of Arsenite-Induced Perturbation of Ubiquitinated Proteome.

Arsenic contamination in food and groundwater constitutes a public health concern for more than 200 million people worldwide. Individuals chronically exposed to arsenic through drinking and ingestion exhibit a higher risk of developing cancers and cardiovascular diseases. Nevertheless, the underlying mechanisms of arsenic toxicity are not fully understood. Arsenite is known to bind to and deactivate RING finger E3 ubiquitin ligases; thus, we reason that a systematic interrogation about how arsenite exposure modulates global protein ubiquitination may reveal novel molecular targets for arsenic toxicity. By employing liquid chromatography-tandem mass spectrometry, in combination with stable isotope labeling by amino acids in cell culture (SILAC) and immunoprecipitation of di-glycine-conjugated lysine-containing tryptic peptides, we assessed the alterations in protein ubiquitination in GM00637 human skin fibroblast cells upon arsenite exposure at the entire proteome level. We observed that arsenite exposure led to altered ubiquitination of many proteins, where the alterations in a large majority of ubiquitination events are negatively correlated with changes in expression of the corresponding proteins, suggesting their modulation by the ubiquitin-proteasomal pathway. Moreover, we observed that arsenite exposure confers diminished ubiquitination of a rate-limiting enzyme in cholesterol biosynthesis, HMGCR, at Lys248. We also revealed that TRC8 is the major E3 ubiquitin ligase for HMGCR ubiquitination in HEK293T cells, and the arsenite-induced diminution of HMGCR ubiquitination is abrogated upon genetic depletion of TRC8. In summary, we systematically characterized arsenite-induced perturbations in a ubiquitinated proteome in human cells and found that the arsenite-elicited attenuation of HMGCR ubiquitination in HEK293T cells involves TRC8.

Grass Carp Reovirus VP56 Allies VP4, Recruits, Blocks, and Degrades RIG-I to More Effectively Attenuate IFN Responses and Facilitate Viral Evasion.

Grass carp reovirus (GCRV), the most virulent aquareovirus, causes epidemic hemorrhagic disease and tremendous economic loss in freshwater aquaculture industry. VP56, a putative fibrin inlaying the outer surface of GCRV-II and GCRV-III, is involved in cell attachment. In the present study, we found that VP56 localizes at the early endosome, lysosome, and endoplasmic reticulum, recruits the cytoplasmic viral RNA sensor retinoic acid-inducible gene I (RIG-I) and binds to it. The interaction between VP56 and RIG-I was detected by endogenous coimmunoprecipitation (co-IP), glutathione S-transferase (GST) pulldown, and subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) and was then confirmed by traditional co-IPs and a novel far-red mNeptune-based bimolecular fluorescence complementation system. VP56 binds to the helicase domain of RIG-I. VP56 enhances K48-linked ubiquitination of RIG-I to degrade it by the proteasomal pathway. Thus, VP56 impedes the initial immune function of RIG-I by dual mechanisms (blockade and degradation) and attenuates signaling from RIG-I recognizing viral RNA, subsequently weakening downstream signaling transduction and interferon (IFN) responses. Accordingly, host antiviral effectors are reduced, and cytopathic effects are increased. These findings were corroborated by RNA sequencing (RNA-seq) and VP56 knockdown. Finally, we found that VP56 and the major outer capsid protein VP4 bind together in the cytosol to enhance the degradation of RIG-I and more efficiently facilitate viral replication. Collectively, the results indicated that VP56 allies VP4, recruits, blocks, and degrades RIG-I, thereby attenuating IFNs and antiviral effectors to facilitate viral evasion more effectively. This study reveals a virus attacking target and an escaping strategy from host antiviral immunity for GCRV and will help understand mechanisms of infection of reoviruses. IMPORTANCE Grass carp reovirus (GCRV) fibrin VP56 and major outer capsid protein VP4 inlay and locate on the outer surface of GCRV-II and GCRV-III, which causes tremendous loss in grass carp and black carp industries. Fibrin is involved in cell attachment and plays an important role in reovirus infection. The present study identified the interaction proteins of VP56 and found that VP56 and VP4 bind to the different domains of the viral RNA sensor retinoic acid-inducible gene I (RIG-I) in grass carp to block RIG-I sensing of viral RNA and induce RIG-I degradation by the proteasomal pathway to attenuate signaling transduction, thereby suppressing interferons (IFNs) and antiviral effectors, facilitating viral replication. VP56 and VP4 bind together in the cytosol to more efficiently facilitate viral evasion. This study reveals a virus attacking a target and an escaping strategy from host antiviral immunity for GCRV and will be helpful in understanding the mechanisms of infection of reoviruses.

MicroRNA-205-5p targets E2F1 to promote autophagy and inhibit pulmonary fibrosis in silicosis through impairing SKP2-mediated Beclin1 ubiquitination.

Silicosis is an occupational disease characterized by extensive pulmonary fibrosis, and the underlying pathological process remains uncertain. Herein, we explored the molecular mechanism by which microRNA-205-5p (miR-205-5p) affects the autophagy of alveolar macrophages (AMs) and pulmonary fibrosis in mice with silicosis through the E2F transcription factor 1 (E2F1)/S-phase kinase-associated protein 2 (SKP2)/Beclin1 axis. Alveolar macrophages (MH-S cells) were exposed to crystalline silica (CS) to develop an in vitro model, and mice were treated with CS to establish an in vivo model. Decreased Beclin1 and increased SKP2 and E2F1 were identified in mice with silicosis. We silenced or overexpressed miR-205-5p, E2F1, SKP2 and Beclin1 to investigate their potential roles in pulmonary fibrosis in vivo and autophagy in vitro. Recombinant adenovirus mRFP-GFP-LC3 was transduced into the MH-S cells to assay autophagic flow. Knocking down Beclin1 promoted pulmonary fibrosis and suppressed the autophagy. Co-immunoprecipitation and ubiquitination assays suggested that SKP2 induced K48-linked ubiquitination of Beclin1. Furthermore, chromatin immunoprecipitation-PCR revealed the site where E2F1 bound to the SKP2 promoter between 1638 bp and 1645 bp. As shown by dual-luciferase reporter gene assay, the transfection with miR-205-5p mimic inhibited the luciferase activity of the wild-type E2F1 3'untranslated region, suggesting that miR-205-5p targeted E2F1. Additionally, miR-205-5p overexpression increased autophagy and reduced the pulmonary fibrosis, while overexpression of E2F1 or SKP2 or inhibition of Beclin1 could annul this effect. The current study elucidated that miR-205-5p targeted E2F1, thereby inhibiting SKP2-mediated Beclin1 ubiquitination to promote macrophage autophagy and inhibit pulmonary fibrosis in mice with silicosis.

Small molecules as tools for functional assessment of deubiquitinating enzyme function.

Deubiquitinating enzymes (DUBs) are a largely understudied and untapped resource in the toolkit of protein degradation functionalities. They comprise a large repertoire of enzymes that remove ubiquitin from substrates in a variety of cellular and pathophysiological contexts, and have enormous potential for research and clinical use. It is only within the last 5 years that potent, selective, and well-characterized small-molecule inhibitors of DUBs have been described. These compounds are now being used to study the biological roles of DUBs. Here, we describe downstream applications of small-molecule inhibitors for studying DUBs and provide a framework for future studies. We highlight recent examples of using these inhibitors to confirm and explore the role of these enzymes in both normal and pathological contexts. These studies represent the first steps in the burgeoning field of pharmacological and chemoproteomic studies of DUBs, which will be critical for the continued advancement of DUB field.

Assessment of ESCRT Protein CHMP5 Activity on Client Protein Ubiquitination by Immunoprecipitation and Western Blotting.

The charged multivesicular body protein-5 (CHMP5) is a member of the endosomal-sorting complex required for transport (ESCRT) that controls membrane-scission events in eukaryotic cells. Recent studies have revealed novel functions of CHMP5 beyond its role in the ESCRT machinery, notably as a critical nonenzymatic regulator of the ubiquitination and subsequent degradation of proteins in immune cells. Here we describe an immunoprecipitation and western blot methodology for assessing CHMP5 activity on client protein ubiquitination in T lymphocytes.

Assessment of Ubiquitin Chain Topology by Targeted Mass Spectrometry.

Protein homeostasis is essential for the survival of cells. It is closely related to the functioning of the ubiquitin-proteasome system, which utilizes the small protein ubiquitin as a posttranslational modifier (PTM). Clinically, the modification is of great importance as its disruption is the cause of many diseases. Unlike other PTMs, ubiquitin can encode several cellular signals by being attached as a single molecule or as a chain of several ubiquitins in various conformations. Thus, ubiquitin signaling is dependent not only on the site of attachment but also on the chain type, the so-called ubiquitin chain topology.The most reliable quantification method for the chain topology uses a bottom-up targeted mass spectrometry-based proteomics technique. While similar to other targeted proteomics techniques, the measurement of ubiquitination chain topology is complicated. First, the ubiquitin chains in the sample have to be biochemically stabilized. Second, the selection of peptides for the analysis is restricted to a given set harboring the PTMs and does not allow for optimization for amenability to mass spectrometry-based quantification. Instead, the topology-characteristic peptides are fixed. We here present such a methodology, including notes for a successful application.

Induction of Cbl-dependent epidermal growth factor receptor degradation in Ling Zhi-8 suppressed lung cancer.

Activating mutation of epidermal growth factor receptor (EGFR) is correlated with malignant lung tumor. In our study, we demonstrated that recombinant LZ-8 (rLZ-8), a medicinal mushroom Ganoderma lucidum protein, induced cell cycle arrest and apoptosis by downregulating the expression of wild-type and mutated EGFR and inhibiting EGFR downstream effectors, AKT and ERK1/2 in lung cancer cells. We showed that rLZ-8 effectively inhibited lung cancer progression and suppressed EGFR expression of lung tumor lesions in mouse model. Functional studies revealed that rLZ-8 reduced the amount of EGFR in cell membranes by altering EGFR localization to enhance the EGF-induced degradation of EGFR. Mechanistically, we demonstrated that rLZ-8 bound to EGFR to induce EGFR autophosphorylation at tyrosine1045 and trigger ubiquitination by inducing the formation of EGFR/Cbl complexes, resulting in the degradation of EGFR; however, Cbl-shRNA abolished rLZ-8-induced EGFR degradation. We provide the first evidence showing that rLZ-8 inhibits growth and induces apoptosis of lung cancer cells by promoting EGFR degradation. The current findings therefore suggest a novel anti-cancer function of rLZ-8 that targeting EGFR overexpression or mutation as well as EGFR-dependent processes in cancer cells.

Assessment of adherence to the guidelines for the management of nausea and vomiting induced by chemotherapy / Avaliação da aderência à diretriz de cuidados para náuseas e vômitos induzidos por quimioterapia

ABSTRACT Objective: To assess adherence of the prescribing physicians in a private cancer care center to the American Society of Clinical Oncology guideline for antiemetic prophylaxis, in the first cycle of antineoplastic chemotherapy. Methods: A total of 139 chemotherapy regimens, of 105 patients, were evaluated retrospectively from 2011 to 2013. Results: We observed 78% of non-adherence to the guideline rate. The main disagreements with the directive were the prescription of higher doses of dexamethasone and excessive use of 5-HT3 antagonist for low risk emetogenic chemotherapy regimens. On univariate analysis, hematological malignancies (p=0.005), the use of two or more chemotherapy (p=0.05) and high emetogenic risk regimes (p=0.012) were factors statistically associated with greater adherence to guidelines. Treatment based on paclitaxel was the only significant risk factor for non-adherence (p=0.02). By multivariate analysis, the chemotherapy of high emetogenic risk most correlated with adherence to guideline (p=0.05). Conclusion: We concluded that the adherence to guidelines is greater if the chemotherapy regime has high emetogenic risk. Educational efforts should focus more intensely on the management of chemotherapy regimens with low and moderate emetogenic potential. Perhaps the development of a computer generated reminder may improve the adherence to guidelines. .

RESUMO Objetivo: Avaliar a adesão dos médicos prescritores, de um centro privado especializado em oncologia, à diretriz de antiêmese profilática da American Society of Clinical Oncology, no primeiro ciclo de quimioterapia antineoplásica. Métodos: Foram avaliados retrospectivamente 139 esquemas de quimioterapia, de 105 pacientes, tratados no período de 2011 a 2013. Resultados: Foram observados 78% de taxa de não adesão à diretriz. As principais discordâncias com a diretriz foram prescrição de doses mais elevadas de dexametasona e uso excessivo de antagonista 5-HT3 para regimes de quimioterapia de risco emetogênico baixo. Pela análise univariada, malignidades hematológicas (p=0,005), uso de dois ou mais quimioterápicos (p=0,05) e regimes de alto risco emetogênico (p=0,012) foram fatores estatisticamente associados a maior adesão à diretriz. O tratamento baseado em paclitaxel foi o único fator estatisticamente significativo para a não adesão (p=0,02). Pela análise multivariada, a quimioterapia de alto risco emetogênico apresentou maior correlação com a adesão à diretriz (p=0,05). Conclusão: Houve maior aderência para a quimioterapia de alto risco emetogênico. Esforços educacionais devem se concentrar mais intensamente na gestão de regimes de quimioterapia com potencial emetogênico baixo e moderado. Talvez o desenvolvimento de lembretes gerados por sistemas informatizados possa melhorar a aderência à diretriz. .

MG132 ameliorates kidney lesions by inhibiting the degradation of Smad7 in streptozotocin-induced diabetic nephropathy.

BACKGROUND: Smad7 is the main negative regulatory protein in the transforming growth factor-ß (TGF-ß) downstream signaling pathway, which plays an important role in diabetic nephropathy (DN) and may be related to the ubiquitin proteasome pathway (UPP). AIM: We investigated the role of UPP in regulating TGF-ß/SMAD signaling and explored the therapeutic effect of the ubiquitin proteasome inhibitor MG132 on DN. METHODS: Wistar rats were randomly divided into a diabetes group and a normal control group. Rats in the diabetes group were injected intraperitoneally with streptozotocin. Diabetic rats were then randomly divided into a diabetic nephropathy group (DN group), an MG132 high concentration (MH) group, and an MG132 low concentration (ML) group. After 8 weeks of treatment, 24-hour urinary microalbumin (UAlb), urinary protein/urinary creatinine (Up/Ucr) values, ALT, AST, Bcr, kidney damage, TGF-ß, Smad7, fibronectin (FN), and Smurf2 were detected. RESULTS: The body mass and Smad7 protein expression decreased in DN group, but kidney weight, kidney weight index, UAlb, Up/Ucr, FN and Smurf2 mRNA expression, and TGF-ß protein expression increased. However, these changes diminished following treatment with MG132, and a more pronounced effect was evident in MH group compared to ML group. CONCLUSION: MG132 alleviates kidney damage by inhibiting Smad7 ubiquitin degradation and TGF-ß activation in DN.

The repertoires of ubiquitinating and deubiquitinating enzymes in eukaryotic genomes.

Reversible protein ubiquitination regulates virtually all known cellular activities. Here, we present a quantitatively evaluated and broadly applicable method to predict eukaryotic ubiquitinating enzymes (UBE) and deubiquitinating enzymes (DUB) and its application to 50 distinct genomes belonging to four of the five major phylogenetic supergroups of eukaryotes: unikonts (including metazoans, fungi, choanozoa, and amoebozoa), excavates, chromalveolates, and plants. Our method relies on a collection of profile hidden Markov models, and we demonstrate its superior performance (coverage and classification accuracy >99%) by identifying approximately 25% and approximately 35% additional UBE and DUB genes in yeast and human, which had not been reported before. In yeast, we predict 85 UBE and 24 DUB genes, for 814 UBE and 107 DUB genes in the human genome. Most UBE and DUB families are present in all eukaryotic lineages, with plants and animals harboring massively enlarged repertoires of ubiquitin ligases. Unicellular organisms, on the other hand, typically harbor less than 300 UBEs and less than 40 DUBs per genome. Ninety-one UBE/DUB genes are orthologous across all four eukaryotic supergroups, and these likely represent a primordial core of enzymes of the ubiquitination system probably dating back to the first eukaryotes approximately 2 billion years ago. Our genome-wide predictions are available through the Database of Ubiquitinating and Deubiquitinating Enzymes (www.DUDE-db.org), where users can also perform advanced sequence and phylogenetic analyses and submit their own predictions.

CBFß stabilizes HIV Vif to counteract APOBEC3 at the expense of RUNX1 target gene expression.

The HIV-1 accessory protein Vif hijacks a cellular Cullin-RING ubiquitin ligase, CRL5, to promote degradation of the APOBEC3 (A3) family of restriction factors. Recently, the cellular transcription cofactor CBFß was shown to form a complex with CRL5-Vif and to be essential for A3 degradation and viral infectivity. We now demonstrate that CBFß is required for assembling a well-ordered CRL5-Vif complex by inhibiting Vif oligomerization and by activating CRL5-Vif via direct interaction. The CRL5-Vif-CBFß holoenzyme forms a well-defined heterohexamer, indicating that Vif simultaneously hijacks CRL5 and CBFß. Heterodimers of CBFß and RUNX transcription factors contribute toward the regulation of genes, including those with immune system functions. We show that binding of Vif to CBFß is mutually exclusive with RUNX heterodimerization and impacts the expression of genes whose regulatory domains are associated with RUNX1. Our results provide a mechanism by which a pathogen with limited coding capacity uses one factor to hijack multiple host pathways.

Assessment of FANCD2 nuclear foci formation in paraffin-embedded tumors: a potential patient-enrichment strategy for treatment with DNA interstrand crosslinking agents.

A major mechanism of DNA repair related to homologous recombination is the Fanconi anemia (FA) pathway. FA genes collaborate with BRCA genes to form foci of DNA repair on chromatin after DNA damage or during the S phase of the cell cycle. Our goal was to develop a method capable of evaluating the functional status of the pathway in patients' tumor tissue, which could also be practically incorporated into large-scale screening. To develop this method, we first used Western immunoblot to detect FANCD2 protein monoubiquitination in fresh tumor specimens of patients with ovarian cancer undergoing surgery and stained formalin-fixed paraffin-embedded tumor tissue simultaneously with 4',6-diamidino-2-phenylindole, FANCD2, and Ki67 antibodies, eventually extending this method to other solid tumors. This triple stain permitted evaluation of the presence, or lack thereof, of FANCD2 subnuclear repair foci in proliferating cells by immunofluorescence microscopy. Overall, we evaluated 156 formalin-fixed paraffin-embedded tumor samples using the FA triple-staining immunofluorescence method. The ratios of FANCD2 foci-negative tumors in ovarian, lung, and breast tumor samples were 21%, 20%, and 29.4%, respectively. Our studies have led to the development of a suitable method for screening, capable of identifying tumors with somatic functional defects in the FA pathway. The use of paraffin-embedded tissues renders the reported method suitable for large-scale screening to select patients for treatment with DNA interstrand crosslinking agents, poly ADP-ribose polymerase inhibitors, or their combination.

Systematic and quantitative assessment of the ubiquitin-modified proteome.

Despite the diverse biological pathways known to be regulated by ubiquitylation, global identification of substrates that are targeted for ubiquitylation has remained a challenge. To globally characterize the human ubiquitin-modified proteome (ubiquitinome), we utilized a monoclonal antibody that recognizes diglycine (diGly)-containing isopeptides following trypsin digestion. We identify ~19,000 diGly-modified lysine residues within ~5000 proteins. Using quantitative proteomics we monitored temporal changes in diGly site abundance in response to both proteasomal and translational inhibition, indicating both a dependence on ongoing translation to observe alterations in site abundance and distinct dynamics of individual modified lysines in response to proteasome inhibition. Further, we demonstrate that quantitative diGly proteomics can be utilized to identify substrates for cullin-RING ubiquitin ligases. Interrogation of the ubiquitinome allows for not only a quantitative assessment of alterations in protein homeostasis fidelity, but also identification of substrates for individual ubiquitin pathway enzymes.

E2s: structurally economical and functionally replete.

Ubiquitination is a post-translational modification pathway involved in myriad cellular regulation and disease pathways. The Ub (ubiquitin) transfer cascade requires three enzyme activities: a Ub-activating (E1) enzyme, a Ub-conjugating (E2) enzyme, and a Ub ligase (E3). Because the E2 is responsible both for E3 selection and substrate modification, E2s function at the heart of the Ub transfer pathway and are responsible for much of the diversity of Ub cellular signalling. There are currently over 90 three-dimensional structures for E2s, both alone and in complex with protein binding partners, providing a wealth of information regarding how E2s are recognized by a wide variety of proteins. In the present review, we describe the prototypical E2-E3 interface and discuss limitations of current methods to identify cognate E2-E3 partners. We present non-canonical E2-protein interactions and highlight the economy of E2s in their ability to facilitate many protein-protein interactions at nearly every surface on their relatively small and compact catalytic domain. Lastly, we compare the structures of conjugated E2~Ub species, their unique protein interactions and the mechanistic insights provided by species that are poised to transfer Ub.

Ubiquitin-mediated regulation of TNFR1 signaling.

Ubiquitin ligase enzymes promote substrate protein ubiquitination, a post-translational modification whereby the 76-amino acid protein ubiquitin is covalently bound to substrate proteins. Ubiquitination may target substrates for proteasomal degradation or regulate substrate function in a degradation-independent manner. Ubiquitination is reversible, and this is achieved by de-ubiquitinase enzymes [Jackson PK, Eldridge AG, Freed E, et al. The lore of the RINGs: substrate recognition and catalysis by ubiquitin ligases. Trends Cell Biol 2000;10(October (10)):429-39]. The first identified target of ubiquitination in the Tumor Necrosis Factor Receptor 1 (TNFR1) signaling cascade was Inhibitor of NF-kappaB (I-kappaB), which sequesters Nuclear Factors at kappa-chain promoters in B-cells (NF-kappaB) transcription factors in the cytosol. Following TNF-alpha stimulation, I-kappaB is ubiquitinated and subsequently degraded by the proteasome, permitting NF-kappaB transcriptional activity [Glickman MH, Ciechanover A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 2002;82(April (2)):373-428]. Since this seminal finding, it is now evident that nearly every step of TNFR1 signaling is regulated by ubiquitination. In this review, we will summarize the ubiquitin/proteasome system and discuss the ubiquitin-mediated regulation of TNFR1 signaling.