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1.
Nat Commun ; 12(1): 374, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33446636

RESUMO

During autophagy the enzyme Atg3 catalyzes the covalent conjugation of LC3 to the amino group of phosphatidylethanolamine (PE) lipids, which is one of the key steps in autophagosome formation. Here, we have demonstrated that an N-terminal conserved region of human Atg3 (hAtg3) communicates information from the N-terminal membrane curvature-sensitive amphipathic helix (AH), which presumably targets the enzyme to the tip of phagophore, to the C-terminally located catalytic core for LC3-PE conjugation. Mutations in the putative communication region greatly reduce or abolish the ability of hAtg3 to catalyze this conjugation in vitro and in vivo, and alter the membrane-bound conformation of the wild-type protein, as reported by NMR. Collectively, our results demonstrate that the N-terminal conserved region of hAtg3 works in concert with its geometry-selective AH to promote LC3-PE conjugation only on the target membrane, and substantiate the concept that highly curved membranes drive spatial regulation of the autophagosome biogenesis during autophagy.


Assuntos
Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Membrana Celular/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas Relacionadas à Autofagia/genética , Biocatálise , Membrana Celular/genética , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Enzimas de Conjugação de Ubiquitina/genética
2.
Biomed Pharmacother ; 134: 111160, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33370630

RESUMO

Natural products are an important source of new drugs. Some of them may be used directly in clinical settings without further structural modification. One of these directly used natural products is puerarin (Pue), which protects cardiomyocytes against oxidative stress and high glucose stress. Although Pue has been used in clinics for many years, its direct binding targets involved in the protection of cardiomyocytes are not yet fully understood. Here, we reported that Pue could prevent cardiomyocytes from apoptosis under H2O2 and high glucose conditions. Based on affinity-based protein profiling methods, we synthesized an active Pue probe (Pue-DA) with a photosensitive crosslinker to initiate a biological orthogonal reaction. Because of the steric hindrance of Pue-DA, two conformational isomers (syn and anti) unequivocally existed in the probe, and these transformed into one isomer when the probe was heated at 60 °C. We confirmed that the alkylation was on the 7-position phenol group of Pue. Mass spectroscopy revealed that Pue-DA can bind with three proteins, namely CHAF1B, UBE2C, and UBE2T. Finally, cellular thermal shift assay showed that Pue has the ability to stabilize CHAF1B stabilization. The knock-down of CHAF1B reduced the protective effect of Pue on cardiomyocytes. In conclusion, Pue protects cardiomyocytes from apoptosis through binding with CHAF1B.


Assuntos
Antioxidantes/farmacologia , Fator 1 de Modelagem da Cromatina/metabolismo , Isoflavonas/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteômica , Apoptose/efeitos dos fármacos , Linhagem Celular , Fator 1 de Modelagem da Cromatina/genética , Glucose/toxicidade , Humanos , Peróxido de Hidrogênio/toxicidade , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Transdução de Sinais , Enzimas de Conjugação de Ubiquitina/metabolismo
3.
FASEB J ; 35(1): e21197, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33368679

RESUMO

SARS-CoV and SARS-CoV-2 encode four structural and accessory proteins (spike, envelope, membrane and nucleocapsid proteins) and two polyproteins (pp1a and pp1ab). The polyproteins are further cleaved by 3C-like cysteine protease (3CLpro ) and papain-like protease (PLpro ) into 16 nonstructural proteins (nsps). PLpro is released from nsp3 through autocleavage, and then it cleaves the sites between nsp1/2, between nsp2/3 and between nsp3/4 with recognition motif of LXGG, and the sites in the C-terminus of ubiquitin and of protein interferon-stimulated gene 15 (ISG15) with recognition motif of RLRGG. Alone or together with SARS unique domain (SUD), PLpro can stabilize an E3 ubiquitin ligase, the ring-finger, and CHY zinc-finger domain-containing 1 (RCHY1), through domain interaction, and thus, promote RCHY1 to ubiquitinate its target proteins including p53. However, a dilemma appears in terms of PLpro roles. On the one hand, the ubiquitination of p53 is good for SARS-CoV because the ubiquitinated p53 cannot inhibit SARS-CoV replication. On the other hand, the ubiquitination of NF-κB inhibitor (IκBα), TNF receptor-associated factors (TRAFs), and stimulator of interferon gene (STING), and the ISGylation of targeted proteins are bad for SARS-CoV because these ubiquitination and ISGylation initiate the innate immune response and antiviral state. This mini-review analyzes the dilemma and provides a snapshot on how the viral PLpro smartly manages its roles to avoid its simultaneously contradictory actions, which could shed lights on possible strategies to deal with SARS-CoV-2 infections.


Assuntos
/virologia , Vírus da SARS/fisiologia , Síndrome Respiratória Aguda Grave/virologia , /imunologia , /genética , Genes Virais , Interações Hospedeiro-Patógeno , Humanos , Terapia de Alvo Molecular , NF-kappa B/metabolismo , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Vírus da SARS/genética , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/terapia , Especificidade por Substrato , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Virais/metabolismo , Replicação Viral
4.
Anticancer Res ; 40(11): 6305-6317, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33109568

RESUMO

BACKGROUND/AIM: Neoadjuvant concurrent chemoradiotherapy (CCRT) is the standard therapeutic strategy for rectal cancer. However, 15-20% of patients undergoing neoadjuvant CCRT progress to recurrence or distant metastases. Therefore, identifying a predictive biomarker is necessary for treating CCRT. MATERIALS AND METHODS: We investigated the relationship between the levels of histone ubiquitination enzyme and clinicopathological outcomes in patients with rectal cancer who were administered CCRT and confirm the role of histone ubiquitination enzyme in regulating the cell response to ionizing radiation (IR). RESULTS: Clinical data indicated that UBE2B expression was significantly correlated with tumor regression grade. Inhibition of UBE2B elevated the genotoxicity of IR to radioresistant cell lines. In contrast, UBE2B over-expression reduced cell sensitivity to IR. Importantly, the recruitment of 53BP1 and Rad51 was remarkably prolonged in cells after pre-treatment with UBE2B inhibitor, TZ9, suggesting a defective DNA repair pathway in UBE2B-deficient cells. CONCLUSION: These results indicate that over-expression of UBE2B correlates with poor response and low survival rate in patients who are administered preoperative CCRT.


Assuntos
Quimiorradioterapia , Regulação Neoplásica da Expressão Gênica , Neoplasias Retais/genética , Neoplasias Retais/terapia , Enzimas de Conjugação de Ubiquitina/genética , Idoso , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Dano ao DNA , Reparo do DNA , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Tolerância a Radiação/efeitos da radiação , Radiação Ionizante , Neoplasias Retais/patologia , Enzimas de Conjugação de Ubiquitina/metabolismo
5.
Virology ; 548: 174-181, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838940

RESUMO

The incidence of head and neck squamous cell carcinomas (HNSCCs) is rising in developed countries. This is driven by an increase in HNSCCs caused by high-risk human papillomavirus (HPV) infections or HPV + HNSCCs. Compared to HNSCCs not caused by HPV (HPV- HNSCCs), HPV + HNSCCs are more responsive to therapy and associated with better oncologic outcomes. As a result, the HPV status of an HNSCC is an important determinant in medical management. One method to determine the HPV status of an HNSCC is increased expression of p16 caused by the HPV E7 oncogene. We identified novel expression changes in HPV + HNSCCs. A comparison of gene expression among HPV+ and HPV- HNSCCs in The Cancer Genome Atlas demonstrated increased DNA repair gene expression in HPV + HNSCCs. Further, DNA repair gene expression correlated with HNSCC survival. Immunohistochemical analysis of a novel HNSCC microarray confirmed that DNA repair protein abundance is elevated in HPV + HNSCCs.


Assuntos
Alphapapillomavirus/metabolismo , Infecções por Papillomavirus/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adulto , Idoso , Alphapapillomavirus/genética , Alphapapillomavirus/isolamento & purificação , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Reparo do DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
6.
Nat Commun ; 11(1): 2751, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488130

RESUMO

The bacterial effector MavC catalyzes non-canonical ubiquitination of host E2 enzyme UBE2N without engaging any of the conventional ubiquitination machinery, thereby abolishing UBE2N's function in forming K63-linked ubiquitin (Ub) chains and dampening NF-кB signaling. We now report the structures of MavC in complex with conjugated UBE2N~Ub and an inhibitor protein Lpg2149, as well as the structure of its ortholog, MvcA, bound to Lpg2149. Recognition of UBE2N and Ub depends on several unique features of MavC, which explains the inability of MvcA to catalyze ubiquitination. Unexpectedly, MavC and MvcA also possess deubiquitinase activity against MavC-mediated ubiquitination, highlighting MavC as a unique enzyme possessing deamidation, ubiquitination, and deubiquitination activities. Further, Lpg2149 directly binds and inhibits both MavC and MvcA by disrupting the interactions between enzymes and Ub. These results provide detailed insights into catalysis and regulation of MavC-type enzymes and the molecular mechanisms of this non-canonical ubiquitination machinery.


Assuntos
Bactérias/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Catálise , Cristalografia por Raios X , Humanos , Modelos Moleculares , NF-kappa B/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas
7.
Nat Commun ; 11(1): 2846, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503993

RESUMO

Based on extensive structural analysis it was proposed that RING E3 ligases prime the E2~ubiquitin conjugate (E2~Ub) for catalysis by locking it into a closed conformation, where ubiquitin is folded back onto the E2 exposing the restrained thioester bond to attack by substrate nucleophile. However the proposal that the RING dependent closed conformation of E2~Ub represents the active form that mediates ubiquitin transfer has yet to be experimentally tested. To test this hypothesis we use single molecule Förster Resonance Energy Transfer (smFRET) to measure the conformation of a FRET labelled E2~Ub conjugate, which distinguishes between closed and alternative conformations. We describe a real-time FRET assay with a thioester linked E2~Ub conjugate to monitor single ubiquitination events and demonstrate that ubiquitin is transferred to substrate from the closed conformation. These findings are likely to be relevant to all RING E3 catalysed reactions ligating ubiquitin and other ubiquitin-like proteins (Ubls) to substrates.


Assuntos
Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Estrutura Terciária de Proteína , Domínios RING Finger , Imagem Individual de Molécula , Ubiquitinação
8.
Proc Natl Acad Sci U S A ; 117(20): 10778-10788, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32366662

RESUMO

The Arg/N-degron pathway targets proteins for degradation by recognizing their N-terminal (Nt) residues. If a substrate bears, for example, Nt-Asn, its targeting involves deamidation of Nt-Asn, arginylation of resulting Nt-Asp, binding of resulting (conjugated) Nt-Arg to the UBR1-RAD6 E3-E2 ubiquitin ligase, ligase-mediated synthesis of a substrate-linked polyubiquitin chain, its capture by the proteasome, and substrate's degradation. We discovered that the human Nt-Asn-specific Nt-amidase NTAN1, Nt-Gln-specific Nt-amidase NTAQ1, arginyltransferase ATE1, and the ubiquitin ligase UBR1-UBE2A/B (or UBR2-UBE2A/B) form a complex in which NTAN1 Nt-amidase binds to NTAQ1, ATE1, and UBR1/UBR2. In addition, NTAQ1 Nt-amidase and ATE1 arginyltransferase also bind to UBR1/UBR2. In the yeast Saccharomyces cerevisiae, the Nt-amidase, arginyltransferase, and the double-E3 ubiquitin ligase UBR1-RAD6/UFD4-UBC4/5 are shown to form an analogous targeting complex. These complexes may enable substrate channeling, in which a substrate bearing, for example, Nt-Asn, would be captured by a complex-bound Nt-amidase, followed by sequential Nt modifications of the substrate and its polyubiquitylation at an internal Lys residue without substrate's dissociation into the bulk solution. At least in yeast, the UBR1/UFD4 ubiquitin ligase interacts with the 26S proteasome, suggesting an even larger Arg/N-degron-targeting complex that contains the proteasome as well. In addition, specific features of protein-sized Arg/N-degron substrates, including their partly sequential and partly nonsequential enzymatic modifications, led us to a verifiable concept termed "superchanneling." In superchanneling, the synthesis of a substrate-linked poly-Ub chain can occur not only after a substrate's sequential Nt modifications, but also before them, through a skipping of either some or all of these modifications within a targeting complex.


Assuntos
Proteólise , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitinação , Amidoidrolases/metabolismo , Aminoaciltransferases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
9.
Nat Struct Mol Biol ; 27(6): 550-560, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393902

RESUMO

The interplay between E2 and E3 enzymes regulates the polyubiquitination of substrates in eukaryotes. Among the several RING-domain E3 ligases in humans, many utilize two distinct E2s for polyubiquitination. For example, the cell cycle regulatory E3, human anaphase-promoting complex/cyclosome (APC/C), relies on UBE2C to prime substrates with ubiquitin (Ub) and on UBE2S to extend polyubiquitin chains. However, the potential coordination between these steps in ubiquitin chain formation remains undefined. While numerous studies have unveiled how RING E3s stimulate individual E2s for Ub transfer, here we change perspective to describe a case where the chain-elongating E2 UBE2S feeds back and directly stimulates the E3 APC/C to promote substrate priming and subsequent multiubiquitination by UBE2C. Our work reveals an unexpected model for the mechanisms of RING E3-dependent ubiquitination and for the diverse and complex interrelationship between components of the ubiquitination cascade.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/química , Ciclossomo-Complexo Promotor de Anáfase/genética , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/genética , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Citidina Trifosfato/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Células HeLa , Humanos , Poliubiquitina/metabolismo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/química , Ubiquitinação
10.
Nat Commun ; 11(1): 2365, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398758

RESUMO

The bacterial effector MavC modulates the host immune response by blocking Ube2N activity employing an E1-independent ubiquitin ligation, catalyzing formation of a γ-glutamyl-ε-Lys (Gln40Ub-Lys92Ube2N) isopeptide crosslink using a transglutaminase mechanism. Here we provide biochemical evidence in support of MavC targeting the activated, thioester-linked Ube2N~ubiquitin conjugate, catalyzing an intramolecular transglutamination reaction, covalently crosslinking the Ube2N and Ub subunits effectively inactivating the E2~Ub conjugate. Ubiquitin exhibits weak binding to MavC alone, but shows an increase in affinity when tethered to Ube2N in a disulfide-linked substrate that mimics the charged E2~Ub conjugate. Crystal structures of MavC in complex with the substrate mimic and crosslinked product provide insights into the reaction mechanism and underlying protein dynamics that favor transamidation over deamidation, while revealing a crucial role for the structurally unique insertion domain in substrate recognition. This work provides a structural basis of ubiquitination by transglutamination and identifies this enzyme's true physiological substrate.


Assuntos
Proteínas de Bactérias/metabolismo , Legionella pneumophila/enzimologia , Transglutaminases/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Domínio Catalítico/genética , Clonagem Molecular , Cristalografia por Raios X , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Especificidade por Substrato , Transglutaminases/genética , Transglutaminases/isolamento & purificação , Transglutaminases/ultraestrutura , Ubiquitina/isolamento & purificação , Ubiquitina/ultraestrutura , Enzimas de Conjugação de Ubiquitina/isolamento & purificação , Enzimas de Conjugação de Ubiquitina/ultraestrutura , Ubiquitinação
11.
Mol Cell ; 78(4): 641-652.e9, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32330457

RESUMO

Ubiquitination is essential for numerous eukaryotic cellular processes. Here, we show that the type III effector CteC from Chromobacterium violaceum functions as an adenosine diphosphate (ADP)-ribosyltransferase that specifically modifies ubiquitin via threonine ADP-ribosylation on residue T66. The covalent modification prevents the transfer of ubiquitin from ubiquitin-activating enzyme E1 to ubiquitin-conjugating enzyme E2, which inhibits subsequent ubiquitin activation by E2 and E3 enzymes in the ubiquitination cascade and leads to the shutdown of polyubiquitin synthesis in host cells. This unique modification also causes dysfunction of polyubiquitin chains in cells, thereby blocking host ubiquitin signaling. The disruption of host ubiquitination by CteC plays a crucial role in C. violaceum colonization in mice during infection. CteC represents a family of effector proteins in pathogens of hosts from different kingdoms. All the members of this family specifically ADP-ribosylate ubiquitin. The action of CteC reveals a new mechanism for interfering with host ubiquitination by pathogens.


Assuntos
ADP-Ribosilação , Proteínas de Bactérias/metabolismo , Chromobacterium/metabolismo , Poliubiquitina/metabolismo , Treonina/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Proteínas de Bactérias/genética , Chromobacterium/genética , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Processamento de Proteína Pós-Traducional , Treonina/genética , Enzimas Ativadoras de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitinação
12.
Nature ; 579(7800): 592-597, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32214243

RESUMO

The conserved yeast E3 ubiquitin ligase Bre1 and its partner, the E2 ubiquitin-conjugating enzyme Rad6, monoubiquitinate histone H2B across gene bodies during the transcription cycle1. Although processive ubiquitination might-in principle-arise from Bre1 and Rad6 travelling with RNA polymerase II2, the mechanism of H2B ubiquitination across genic nucleosomes remains unclear. Here we implicate liquid-liquid phase separation3 as the underlying mechanism. Biochemical reconstitution shows that Bre1 binds the scaffold protein Lge1, which possesses an intrinsically disordered region that phase-separates via multivalent interactions. The resulting condensates comprise a core of Lge1 encapsulated by an outer catalytic shell of Bre1. This layered liquid recruits Rad6 and the nucleosomal substrate, which accelerates the ubiquitination of H2B. In vivo, the condensate-forming region of Lge1 is required to ubiquitinate H2B in gene bodies beyond the +1 nucleosome. Our data suggest that layered condensates of histone-modifying enzymes generate chromatin-associated 'reaction chambers', with augmented catalytic activity along gene bodies. Equivalent processes may occur in human cells, and cause neurological disease when impaired.


Assuntos
Nucleossomos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Ubiquitinação , Biocatálise , Histonas/química , Histonas/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Viabilidade Microbiana , Transição de Fase , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo
13.
Proc Natl Acad Sci U S A ; 117(11): 6223-6230, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123097

RESUMO

All multicellular organisms keep a balance between sink and source activities by controlling nutrient transport at strategic positions. In most plants, photosynthetically produced sucrose is the predominant carbon and energy source, whose transport from leaves to carbon sink organs depends on sucrose transporters. In the model plant Arabidopsis thaliana, transport of sucrose into the phloem vascular tissue by SUCROSE TRANSPORTER 2 (SUC2) sets the rate of carbon export from source leaves, just like the SUC2 homologs of most crop plants. Despite their importance, little is known about the proteins that regulate these sucrose transporters. Here, identification and characterization of SUC2-interaction partners revealed that SUC2 activity is regulated via its protein turnover rate and phosphorylation state. UBIQUITIN-CONJUGATING ENZYME 34 (UBC34) was found to trigger turnover of SUC2 in a light-dependent manner. The E2 enzyme UBC34 could ubiquitinate SUC2 in vitro, a function generally associated with E3 ubiquitin ligases. ubc34 mutants showed increased phloem loading, as well as increased biomass and yield. In contrast, mutants of another SUC2-interaction partner, WALL-ASSOCIATED KINASE LIKE 8 (WAKL8), showed decreased phloem loading and growth. An in vivo assay based on a fluorescent sucrose analog confirmed that SUC2 phosphorylation by WAKL8 can increase transport activity. Both proteins are required for the up-regulation of phloem loading in response to increased light intensity. The molecular mechanism of SUC2 regulation elucidated here provides promising targets for the biotechnological enhancement of source strength.


Assuntos
Arabidopsis/fisiologia , Sequestro de Carbono , Carbono/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Mutação , Floema/metabolismo , Fosforilação/fisiologia , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação/fisiologia
14.
Proc Natl Acad Sci U S A ; 117(14): 7792-7798, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32209662

RESUMO

A significant fraction of the glycerophospholipids in the human body is composed of plasmalogens, particularly in the brain, cardiac, and immune cell membranes. A decline in these lipids has been observed in such diseases as Alzheimer's and chronic obstructive pulmonary disease. Plasmalogens contain a characteristic 1-O-alk-1'-enyl ether (vinyl ether) double bond that confers special biophysical, biochemical, and chemical properties to these lipids. However, the genetics of their biosynthesis is not fully understood, since no gene has been identified that encodes plasmanylethanolamine desaturase (E.C. 1.14.99.19), the enzyme introducing the crucial alk-1'-enyl ether double bond. The present work identifies this gene as transmembrane protein 189 (TMEM189). Inactivation of the TMEM189 gene in human HAP1 cells led to a total loss of plasmanylethanolamine desaturase activity, strongly decreased plasmalogen levels, and accumulation of plasmanylethanolamine substrates and resulted in an inability of these cells to form labeled plasmalogens from labeled alkylglycerols. Transient expression of TMEM189 protein, but not of other selected desaturases, recovered this deficit. TMEM189 proteins contain a conserved protein motif (pfam10520) with eight conserved histidines that is shared by an alternative type of plant desaturase but not by other mammalian proteins. Each of these histidines is essential for plasmanylethanolamine desaturase activity. Mice homozygous for an inactivated Tmem189 gene lacked plasmanylethanolamine desaturase activity and had dramatically lowered plasmalogen levels in their tissues. These results assign the TMEM189 gene to plasmanylethanolamine desaturase and suggest that the previously characterized phenotype of Tmem189-deficient mice may be caused by a lack of plasmalogens.


Assuntos
Lipídeos/genética , Oxirredutases/genética , Plasmalogênios/genética , Enzimas de Conjugação de Ubiquitina/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Linhagem Celular , Humanos , Camundongos , Oxirredução , Oxirredutases/metabolismo , Fenótipo , Plasmalogênios/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Compostos de Vinila/metabolismo
15.
Biochem Biophys Res Commun ; 525(3): 668-674, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32139119

RESUMO

Moyamoya disease (MMD) is a cerebrovascular disease characterized by progressive occlusion of the internal carotid arteries. Genetic studies originally identified RNF213 as an MMD susceptibility gene that encodes a large 591 kDa protein with a functional RING domain and dual AAA+ ATPase domains. As the functions of RNF213 and its relationship to MMD onset are unknown, we set out to characterize the ubiquitin ligase activity of RNF213, and the effects of MMD patient mutations on these activities and on other cellular processes. In vitro ubiquitination assays, using the RNF213 RING domain, identified Ubc13/Uev1A as a key ubiquitin conjugating enzyme that together generate K63-linked polyubiquitin chains. However, nearly all MMD patient mutations in the RING domain greatly reduced this activity. When full-length proteins were overexpressed in HEK293T cells, patient mutations that abolished the ubiquitin ligase activities conversely enhanced nuclear factor κB (NFκB) activation and induced apoptosis accompanied with Caspase-3 activation. These induced activities were dependent on the RNF213 AAA+ domain. Our results suggest that the NFκB- and apoptosis-inducing functions of RNF213 may be negatively regulated by its ubiquitin ligase activity and that disruption of this regulation could contribute towards MMD onset.


Assuntos
Domínio AAA , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Apoptose , Doença de Moyamoya/genética , Mutação/genética , NF-kappa B/metabolismo , Domínios RING Finger , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Sequência de Aminoácidos , Células HEK293 , Humanos , Lisina/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Poliubiquitina/metabolismo , Fatores de Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo
16.
Biochemistry ; 59(8): 921-932, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32049508

RESUMO

OTUB1 is a highly expressed cysteine protease that specifically cleaves K48-linked polyubiquitin chains. This unique deubiquitinating enzyme (DUB) can bind to a subset of E2 ubiquitin conjugating enzymes, forming complexes in which the two enzymes can regulate one another's activity. OTUB1 can noncatalytically suppress the ubiquitin conjugating activity of its E2 partners by sequestering the charged E2∼Ub thioester and preventing ubiquitin transfer. The same E2 enzymes, when uncharged, can stimulate the DUB activity of OTUB1 in vitro, although the importance of OTUB1 stimulation in vivo remains unclear. To assess the potential balance between these activities that might occur in cells, we characterized the kinetics and thermodynamics governing the formation and activity of OTUB1:E2 complexes. We show that both stimulation of OTUB1 by E2 enzymes and noncatalytic inhibition of E2 enzymes by OTUB1 occur at physiologically relevant concentrations of both partners. Whereas E2 partners differ in their ability to stimulate OTUB1 activity, we find that this variability is not correlated with the affinity of each E2 for OTUB1. In addition to UBE2N and the UBE2D isoforms, we find that OTUB1 inhibits the polyubiquitination activity of all three UBE2E enzymes, UBE2E1, UBE2E2, and UBE2E3. Interestingly, although OTUB1 also inhibits the auto-monoubiquitination and autopolyubiquitination activity of UBE2E1 and UBE2E2, it is unable to suppress autoubiquitination by UBE2E3. Our quantitative analysis provides a basis for further exploring the biological roles of OTUB1:E2 complexes in cells.


Assuntos
Cisteína Endopeptidases/metabolismo , Enzimas Desubiquitinantes/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Humanos , Cinética , Poliubiquitina/metabolismo , Ligação Proteica , Multimerização Proteica , Saccharomyces cerevisiae/enzimologia , Termodinâmica , Enzimas de Conjugação de Ubiquitina/química , Ubiquitinação/efeitos dos fármacos
17.
Biochem Biophys Res Commun ; 524(4): 910-915, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32051088

RESUMO

S-Nitrosylation of protein cysteine thiol is a post-translational modification mediated by nitric oxide (NO). The overproduction of NO causes nitrosative stress, which is known to induce endoplasmic reticulum (ER) stress. We previously reported that S-nitrosylation of protein disulfide isomerase (PDI) and the ER stress sensor inositol-requiring enzyme 1 (IRE1) decreases their enzymatic activities. However, it remains unclear whether nitrosative stress affects ER-associated degradation (ERAD), a separate ER stress regulatory system responsible for the degradation of substrates via the ubiquitin-proteasomal pathway. In the present study, we found that the ubiquitination of a known ERAD substrate, serine/threonine-protein kinase 1 (SGK1), is attenuated by nitrosative stress. C-terminus of Hsc70-interacting protein (CHIP) together with ubiquitin-conjugating enzyme E2 D1 (UBE2D1) are involved in this modification. We detected that UBE2D1 is S-nitrosylated at its active site, Cys85 by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, in vitro and cell-based experiments revealed that S-nitrosylated UBE2D1 has decreased ubiquitin-conjugating activity. Our results suggested that nitrosative stress interferes with ERAD, leading to prolongation of ER stress by co-disruption of various pathways, including the molecular chaperone and ER stress sensor pathways. Given that nitrosative stress and ER stress are upregulated in the brains of patient with Parkinson's disease (PD) and of those with Alzheimer's disease (AD), our findings may provide further insights into the pathogenesis of these neurodegenerative disorders.


Assuntos
Proteínas Imediatamente Precoces/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/genética , Domínio Catalítico , Cromonas/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Degradação Associada com o Retículo Endoplasmático/efeitos dos fármacos , Degradação Associada com o Retículo Endoplasmático/genética , Células HEK293 , Humanos , Proteínas Imediatamente Precoces/genética , Leupeptinas/farmacologia , Morfolinas/farmacologia , Estresse Nitrosativo , Compostos Nitrosos/metabolismo , Oxirredução/efeitos dos fármacos , Fosforilação , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/genética , Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
18.
Int J Med Sci ; 17(2): 274-279, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32038111

RESUMO

Ubiquitin conjugating enzyme E2S (Ube2S) plays important roles in cancer development in some malignant tumors. However, the functions and related molecular network of Ube2S in non-small cell lung cancer are not fully understood. In the current study, we examined the expression of Ube2S in non-small cell lung cancer and its clinicopathological significance. We also investigated the molecules and pathways regulated by Ube2S. An immunostaining study showed that the positive rate of Ube2s expression in lung cancer tissues was higher than that in normal lung tissues (p < 0.05). Upregulated Ube2S expression in cancer tissues significantly correlated with clinical progression (TNM III versus I + II), lymph node metastasis, and shorter survival time of the patients (p < 0.05). When Ube2S was overexpressed in A549 cells, the abilities of these cells to proliferate and migrate were increased (p < 0.05). Moreover, Ube2S significantly upregulated the expression of ß-catenin, cyclin D1, and MMP7 (novel molecules of the Wnt/ß-catenin pathway), and the activity of this pathway (p < 0.05). In addition, a Wnt/ß-catenin signaling inhibitor effectively abolished the function of Ube2S. These results indicate that Ube2S may be a novel marker contributing to lung cancer development, possibly through regulating canonical Wnt signaling.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Enzimas de Conjugação de Ubiquitina/metabolismo , beta Catenina/metabolismo , Células A549 , Western Blotting , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Enzimas de Conjugação de Ubiquitina/genética , Via de Sinalização Wnt/fisiologia , beta Catenina/genética
19.
Proc Natl Acad Sci U S A ; 117(8): 4117-4124, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32047038

RESUMO

The Cullin-RING ligases (CRLs) are the largest family of ubiquitin E3s activated by neddylation and regulated by the deneddylase COP9 signalosome (CSN). The inositol polyphosphate metabolites promote the formation of CRL-CSN complexes, but with unclear mechanism of action. Here, we provide structural and genetic evidence supporting inositol hexakisphosphate (IP6) as a general CSN cofactor recruiting CRLs. We determined the crystal structure of IP6 in complex with CSN subunit 2 (CSN2), based on which we identified the IP6-corresponding electron density in the cryoelectron microscopy map of a CRL4A-CSN complex. IP6 binds to a cognate pocket formed by conserved lysine residues from CSN2 and Rbx1/Roc1, thereby strengthening CRL-CSN interactions to dislodge the E2 CDC34/UBE2R from CRL and to promote CRL deneddylation. IP6 binding-deficient Csn2 K70E/K70E knockin mice are embryonic lethal. The same mutation disabled Schizosaccharomyces pombe Csn2 from rescuing UV-hypersensitivity of csn2-null yeast. These data suggest that CRL transition from the E2-bound active state to the CSN-bound sequestered state is critically assisted by an interfacial IP6 small molecule, whose metabolism may be coupled to CRL-CSN complex dynamics.


Assuntos
Complexo do Signalossomo COP9/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sítios de Ligação , Calorimetria/métodos , Deleção de Genes , Técnicas de Introdução de Genes , Genes Transgênicos Suicidas , Genótipo , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae , Organismos Livres de Patógenos Específicos , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
20.
Nature ; 578(7795): 461-466, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32051583

RESUMO

Eukaryotic cell biology depends on cullin-RING E3 ligase (CRL)-catalysed protein ubiquitylation1, which is tightly controlled by the modification of cullin with the ubiquitin-like protein NEDD82-6. However, how CRLs catalyse ubiquitylation, and the basis of NEDD8 activation, remain unknown. Here we report the cryo-electron microscopy structure of a chemically trapped complex that represents the ubiquitylation intermediate, in which the neddylated CRL1ß-TRCP promotes the transfer of ubiquitin from the E2 ubiquitin-conjugating enzyme UBE2D to its recruited substrate, phosphorylated IκBα. NEDD8 acts as a nexus that binds disparate cullin elements and the RING-activated ubiquitin-linked UBE2D. Local structural remodelling of NEDD8 and large-scale movements of CRL domains converge to juxtapose the substrate and the ubiquitylation active site. These findings explain how a distinctive ubiquitin-like protein alters the functions of its targets, and show how numerous NEDD8-dependent interprotein interactions and conformational changes synergistically configure a catalytic CRL architecture that is both robust, to enable rapid ubiquitylation of the substrate, and fragile, to enable the subsequent functions of cullin-RING proteins.


Assuntos
Microscopia Crioeletrônica , Proteína NEDD8/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Biocatálise , Humanos , Modelos Moleculares , Proteína NEDD8/química , Proteína NEDD8/ultraestrutura , Inibidor de NF-kappaB alfa/química , Inibidor de NF-kappaB alfa/metabolismo , Inibidor de NF-kappaB alfa/ultraestrutura , Fosforilação , Conformação Proteica , Especificidade por Substrato , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/ultraestrutura , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/ultraestrutura , Ubiquitinação
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