RESUMO
HECT ubiquitin ligases play essential roles in metazoan development and physiology. The HECT ligase HUWE1 is central to the cellular stress response by mediating degradation of key death or survival factors, including Mcl1, p53, DDIT4, and Myc. Although mutations in HUWE1 and related HECT ligases are widely implicated in human disease, our molecular understanding remains limited. Here we present a comprehensive investigation of full-length HUWE1, deepening our understanding of this class of enzymes. The N-terminal â¼3,900 amino acids of HUWE1 are indispensable for proper ligase function, and our cryo-EM structures of HUWE1 offer a complete molecular picture of this large HECT ubiquitin ligase. HUWE1 forms an alpha solenoid-shaped assembly with a central pore decorated with protein interaction modules. Structures of HUWE1 variants linked to neurodevelopmental disorders as well as of HUWE1 bound to a model substrate link the functions of this essential enzyme to its three-dimensional organization.
Assuntos
Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Microscopia Crioeletrônica/métodos , Células HEK293 , Humanos , Estresse Fisiológico/fisiologia , Relação Estrutura-Atividade , Proteínas Supressoras de Tumor/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The MYC oncoprotein globally affects the function of RNA polymerase II (RNAPII). The ability of MYC to promote transcription elongation depends on its ubiquitylation. Here, we show that MYC and PAF1c (polymerase II-associated factor 1 complex) interact directly and mutually enhance each other's association with active promoters. PAF1c is rapidly transferred from MYC onto RNAPII. This transfer is driven by the HUWE1 ubiquitin ligase and is required for MYC-dependent transcription elongation. MYC and HUWE1 promote histone H2B ubiquitylation, which alters chromatin structure both for transcription elongation and double-strand break repair. Consistently, MYC suppresses double-strand break accumulation in active genes in a strictly PAF1c-dependent manner. Depletion of PAF1c causes transcription-dependent accumulation of double-strand breaks, despite widespread repair-associated DNA synthesis. Our data show that the transfer of PAF1c from MYC onto RNAPII efficiently couples transcription elongation with double-strand break repair to maintain the genomic integrity of MYC-driven tumor cells.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Elongação da Transcrição Genética , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Linhagem Celular Tumoral , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-myc/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Alternative polyadenylation (APA) contributes to transcriptome complexity by generating mRNA isoforms with varying 3' UTR lengths. APA leading to 3' UTR shortening (3' US) is a common feature of most cancer cells; however, the molecular mechanisms are not understood. Here, we describe a widespread mechanism promoting 3' US in cancer through ubiquitination of the mRNA 3' end processing complex protein, PCF11, by the cancer-specific MAGE-A11-HUWE1 ubiquitin ligase. MAGE-A11 is normally expressed only in the male germline but is frequently re-activated in cancers. MAGE-A11 is necessary for cancer cell viability and is sufficient to drive tumorigenesis. Screening for targets of MAGE-A11 revealed that it ubiquitinates PCF11, resulting in loss of CFIm25 from the mRNA 3' end processing complex. This leads to APA of many transcripts affecting core oncogenic and tumor suppressors, including cyclin D2 and PTEN. These findings provide insights into the molecular mechanisms driving APA in cancer and suggest therapeutic strategies.
Assuntos
Regiões 3' não Traduzidas/genética , Antígenos de Neoplasias/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/patologia , RNA Mensageiro/metabolismo , Ubiquitina/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Animais , Antígenos de Neoplasias/genética , Apoptose , Biomarcadores Tumorais , Carcinogênese , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células , Fator de Especificidade de Clivagem e Poliadenilação/genética , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Poliadenilação , Splicing de RNA , RNA Mensageiro/genética , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
mTORC1, the major homeostatic sensor and responder, regulates cell catabolism mainly by targeting autophagy. Here, we show that mTORC1 directly controls autophagosome formation via phosphorylation of WIPI2, a critical protein in isolation membrane growth and elongation. mTORC1 phosphorylates Ser395 of WIPI2, directing WIPI2 to interact specifically with the E3 ubiquitin ligase HUWE1 for ubiquitination and proteasomal degradation. Physiological or pharmacological inhibition of mTORC1 in cells promotes WIPI2 stabilization, autophagosome formation, and autophagic degradation. In mouse liver, fasting significantly increases the WIPI2 protein level, while silencing HUWE1 enhances autophagy, and introducing WIPI2 improves lipid clearance. Thus, regulation of the intracellular WIPI2 protein level by mTORC1 and HUWE1 is a key determinant of autophagy flux and may coordinate the initiation, progression, and completion of autophagy.
Assuntos
Autofagia/fisiologia , Proteínas de Transporte/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a Fosfato , Ubiquitinação/fisiologiaRESUMO
Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) open reading frame 9b (ORF9b) antagonizes the antiviral type I and III interferon (IFN) responses and is ubiquitinated and degraded via the ubiquitin-proteasome pathway. However, E3 ubiquitin ligases that mediate the polyubiquitination and degradation of ORF9b remain unknown. In this study, we identified 14 E3 ligases that specifically bind to SARS-CoV-2 ORF9b. Specifically, three E3 ligases, HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 (HUWE1), ubiquitin protein ligase E3 component n-recognin 4 (UBR4), and UBR5, induced K48-linked polyubiquitination and degradation of ORF9b, thereby attenuating ORF9b-mediated inhibition of the IFN response and SARS-CoV-2 replication. Moreover, each E3 ligase performed this function independent of the other two E3 ligases. Therefore, the three E3 ligases identified in this study as anti-SARS-CoV-2 host factors provide novel molecular insight into the virus-host interaction.IMPORTANCEUbiquitination is an important post-translational modification that regulates multiple biological processes, including viral replication. Identification of E3 ubiquitin ligases that target viral proteins for degradation can provide novel targets for antagonizing viral infections. Here, we identified multiple E3 ligases, including HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 (HUWE1), ubiquitin protein ligase E3 component n-recognin 4 (UBR4), and UBR5, that ubiquitinated and induced the degradation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frame 9b (ORF9b), an interferon (IFN) antagonist, thereby enhancing IFN production and attenuating SARS-CoV-2 replication. Our study provides new possibilities for drug development targeting the interaction between E3 ligases and ORF9b.
Assuntos
COVID-19 , Interações entre Hospedeiro e Microrganismos , Proteólise , SARS-CoV-2 , Ubiquitina-Proteína Ligases , Ubiquitinação , Proteínas Virais , Humanos , Células CACO-2 , COVID-19/enzimologia , COVID-19/imunologia , COVID-19/metabolismo , COVID-19/virologia , Células HEK293 , Interferons/imunologia , Fases de Leitura Aberta , Ligação Proteica , SARS-CoV-2/imunologia , SARS-CoV-2/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
Inflammation plays an important role in the initiation and progression of colorectal cancer (CRC) and leads to ß-catenin accumulation in colitis-related CRC. However, the mechanism remains largely unknown. Here, pancreatic progenitor cell differentiation and proliferation factor (PPDPF) is found to be upregulated in CRC and significantly correlated with tumor-node-metastasis (TNM) stages and survival time. Knockout of PPDPF in the intestinal epithelium shortens crypts, decreases the number of stem cells, and inhibits the growth of organoids and the occurrence of azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CRC. Mechanistically, PPDPF is found to interact with Casein kinase 1α (CK1α), thereby disrupting its binding to Axin, disassociating the ß-catenin destruction complex, decreasing the phosphorylation of ß-catenin, and activating the Wnt/ß-catenin pathway. Furthermore, interleukin 6 (IL6)/Janus kinase 2 (JAK2)-mediated inflammatory signals lead to phosphorylation of PPDPF at Tyr16 and Tyr17, stabilizing the protein. In summary, this study demonstrates that PPDPF is a key molecule in CRC carcinogenesis and progression that connects inflammatory signals to the Wnt/ß-catenin signaling pathway, providing a potential novel therapeutic target.
Assuntos
Neoplasias Colorretais , Interleucina-6 , Humanos , Interleucina-6/efeitos adversos , Interleucina-6/metabolismo , Fosforilação , beta Catenina/metabolismo , Via de Sinalização Wnt , Janus Quinase 2/metabolismo , Neoplasias Colorretais/genética , Proliferação de Células , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão GênicaRESUMO
Several ubiquitin chain types have remained unstudied, mainly because tools and techniques to detect these posttranslational modifications are scarce. Linkage-specific antibodies have shaped our understanding of the roles and dynamics of polyubiquitin signals but are available for only five out of eight linkage types. We here characterize K6- and K33-linkage-specific "affimer" reagents as high-affinity ubiquitin interactors. Crystal structures of affimers bound to their cognate chain types reveal mechanisms of specificity and a K11 cross-reactivity in the K33 affimer. Structure-guided improvements yield superior affinity reagents suitable for western blotting, confocal fluorescence microscopy and pull-down applications. This allowed us to identify RNF144A and RNF144B as E3 ligases that assemble K6-, K11-, and K48-linked polyubiquitin in vitro. A protocol to enrich K6-ubiquitinated proteins from cells identifies HUWE1 as a main E3 ligase for this chain type, and we show that mitofusin-2 is modified with K6-linked polyubiquitin in a HUWE1-dependent manner.
Assuntos
GTP Fosfo-Hidrolases/química , Proteínas Mitocondriais/química , Sondas Moleculares/química , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Ubiquitina-Proteína Ligases/química , Ubiquitina/química , Motivos de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Cinética , Lisina/química , Lisina/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Proteínas Supressoras de Tumor , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Two sphingosine kinase isoforms, sphingosine kinase 1 (SPHK1) and sphingosine kinase 2 (SPHK2), synthesize the lipid sphingosine-1-phosphate (S1P) by phosphorylating sphingosine. SPHK1 is a cytoplasmic kinase, and SPHK2 is localized to the nucleus and other organelles. In the cytoplasm, the SPHK1/S1P pathway modulates autophagy and protein ubiquitination, among other processes. In the nucleus, the SPHK2/S1P pathway regulates transcription. Here, we hypothesized that the SPHK2/S1P pathway governs protein ubiquitination in neurons. We found that ectopic expression of SPHK2 increases ubiquitinated substrate levels in cultured neurons and pharmacologically inhibiting SPHK2 decreases protein ubiquitination. With mass spectrometry, we discovered that inhibiting SPHK2 affects lipid and synaptic protein networks as well as a ubiquitin-dependent protein network. Several ubiquitin-conjugating and hydrolyzing proteins, such as the E3 ubiquitin-protein ligases HUWE1 and TRIP12, the E2 ubiquitin-conjugating enzyme UBE2Z, and the ubiquitin-specific proteases USP15 and USP30, were downregulated by SPHK2 inhibition. Using RNA sequencing, we found that inhibiting SPHK2 altered lipid and neuron-specific gene networks, among others. Genes that encode the corresponding proteins from the ubiquitin-dependent protein network that we discovered with mass spectrometry were not affected by inhibiting SPHK2, indicating that the SPHK2/S1P pathway regulates ubiquitination at the protein level. We also show that both SPHK2 and HUWE1 were upregulated in the striatum of a mouse model of Huntington's disease, the BACHD mice, indicating that our findings are relevant to neurodegenerative diseases. Our results identify SPHK2/S1P as a novel regulator of protein ubiquitination networks in neurons and provide a new target for developing therapies for neurodegenerative diseases.
RESUMO
Polyubiquitin chains of different topologies regulate diverse cellular processes. K48- and K63-linked chains, the two most abundant chain types, regulate proteolytic and signaling pathways, respectively. Although recent studies reported important roles for heterogeneous chains, the functions of branched ubiquitin chains remain unclear. Here, we show that the ubiquitin chain branched at K48 and K63 regulates nuclear factor κB (NF-κB) signaling. A mass-spectrometry-based quantification strategy revealed that K48-K63 branched ubiquitin linkages are abundant in cells. In response to interleukin-1ß, the E3 ubiquitin ligase HUWE1 generates K48 branches on K63 chains formed by TRAF6, yielding K48-K63 branched chains. The K48-K63 branched linkage permits recognition by TAB2 but protects K63 linkages from CYLD-mediated deubiquitylation, thereby amplifying NF-κB signals. These results reveal a previously unappreciated cooperation between K48 and K63 linkages that generates a unique coding signal: ubiquitin chain branching differentially controls readout of the ubiquitin code by specific reader and eraser proteins to activate NF-κB signaling.
Assuntos
Lisina/química , NF-kappa B/química , Poliubiquitina/química , Fator 6 Associado a Receptor de TNF/química , Ubiquitina-Proteína Ligases/química , Ubiquitina/química , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Enzima Desubiquitinante CYLD , Expressão Gênica , Humanos , Interleucina-1beta/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Lisina/metabolismo , Modelos Moleculares , NF-kappa B/genética , NF-kappa B/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Poliubiquitina/genética , Poliubiquitina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Especificidade por Substrato , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
With the outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), coronaviruses have begun to attract great attention across the world. Of the known human coronaviruses, however, Middle East respiratory syndrome coronavirus (MERS-CoV) is the most lethal. Coronavirus proteins can be divided into three groups: nonstructural proteins, structural proteins, and accessory proteins. While the number of each of these proteins varies greatly among different coronaviruses, accessory proteins are most closely related to the pathogenicity of the virus. We found for the first time that the ORF3 accessory protein of MERS-CoV, which closely resembles the ORF3a proteins of severe acute respiratory syndrome coronavirus and SARS-CoV-2, has the ability to induce apoptosis in cells in a dose-dependent manner. Through bioinformatics analysis and validation, we revealed that ORF3 is an unstable protein and has a shorter half-life in cells compared to that of severe acute respiratory syndrome coronavirus and SARS-CoV-2 ORF3a proteins. After screening, we identified a host E3 ligase, HUWE1, that specifically induces MERS-CoV ORF3 protein ubiquitination and degradation through the ubiquitin-proteasome system. This results in the diminished ability of ORF3 to induce apoptosis, which might partially explain the lower spread of MERS-CoV compared to other coronaviruses. In summary, this study reveals a pathological function of MERS-CoV ORF3 protein and identifies a potential host antiviral protein, HUWE1, with an ability to antagonize MERS-CoV pathogenesis by inducing ORF3 degradation, thus enriching our knowledge of the pathogenesis of MERS-CoV and suggesting new targets and strategies for clinical development of drugs for MERS-CoV treatment.
Assuntos
Apoptose , Infecções por Coronavirus/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas não Estruturais Virais/metabolismo , Células A549 , Linhagem Celular , Biologia Computacional , Infecções por Coronavirus/fisiopatologia , Infecções por Coronavirus/virologia , Células Epiteliais/fisiologia , Células Epiteliais/virologia , Células HEK293 , Interações Hospedeiro-Patógeno , HumanosRESUMO
Mesenchymal stem cells (MSCs) are adult stem cell populations and exhibit great potential in regenerative medicine and oncology. Platelet-derived growth factors (PDGFs) are well known to regulate MSC biology through their chemotactic and mitogenic properties. However, their direct roles in the regulation of MSC lineage commitment are unclear. Here, we show that PDGF D promotes the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) into osteoblasts and inhibits hBMSC differentiation into adipocytes. We demonstrate that PDGF D-induced ß-actin expression and polymerization are essential for mediating this differential regulation of osteoblastogenesis and adipogenesis. Interestingly, we found that PDGF D induces massive upward molecular weight shifts of its cognate receptor, PDGF receptor beta (ß-PDGFR) in hBMSCs, which was not observed in fibroblasts. Proteomic analysis indicated that the E3 ubiquitin ligase HECT, UBA, and WWE domain-containing protein 1 (HUWE1) associates with the PDGF D-activated ß-PDGFR signaling complex in hBMSCs, resulting in ß-PDGFR polyubiquitination. In contrast to the well-known role of ubiquitin in protein degradation, we provide evidence that HUWE1-mediated ß-PDGFR polyubiquitination delays ß-PDGFR internalization and degradation, thereby prolonging AKT signaling. Finally, we demonstrate that HUWE1-regulated ß-PDGFR signaling is essential for osteoblastic differentiation of hBMSCs, while being dispensable for PDGF D-induced hBMSC migration and proliferation as well as PDGF D-mediated inhibition of hBMSC differentiation into adipocytes. Taken together, our findings provide novel insights into the molecular mechanism by which PDGF D regulates the commitment of hBMSCs into the osteoblastic lineage.
Assuntos
Linfocinas/metabolismo , Células-Tronco Mesenquimais , Fator de Crescimento Derivado de Plaquetas/metabolismo , Ubiquitina-Proteína Ligases , Diferenciação Celular , Proliferação de Células , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Proteômica , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The ERK1/2 (also known as MAPK3 and MAPK1, respectively) signaling pathway is critical in organismal development and tissue morphogenesis. Deregulation of this pathway leads to congenital abnormalities with severe developmental dysmorphisms. The core ERK1/2 cascade relies on scaffold proteins, such as Shoc2 to guide and fine-tune its signals. Mutations in SHOC2 lead to the development of the pathology termed Noonan-like Syndrome with Loose Anagen Hair (NSLAH). However, the mechanisms underlying the functions of Shoc2 and its contributions to disease progression remain unclear. Here, we show that ERK1/2 pathway activation triggers the interaction of Shoc2 with the ubiquitin-specific protease USP7. We reveal that, in the Shoc2 module, USP7 functions as a molecular 'switch' that controls the E3 ligase HUWE1 and the HUWE1-induced regulatory feedback loop. We also demonstrate that disruption of Shoc2-USP7 binding leads to aberrant activation of the Shoc2-ERK1/2 axis. Importantly, our studies reveal a possible role for USP7 in the pathogenic mechanisms underlying NSLAH, thereby extending our understanding of how ubiquitin-specific proteases regulate intracellular signaling.
Assuntos
Síndrome dos Cabelos Anágenos Frouxos , Sistema de Sinalização das MAP Quinases , Síndrome de Noonan , Peptidase 7 Específica de Ubiquitina , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Sistema de Sinalização das MAP Quinases/genética , Síndrome de Noonan/genética , Transdução de Sinais , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Peptidase 7 Específica de Ubiquitina/genéticaRESUMO
BACKGROUND: Intellectual disability is a form of neurodevelopmental disorders that begin in childhood and is characterized by substantial intellectual difficulties as well as difficulties in conceptual, social, and practical areas of living. Several genetic and nongenetic factors contribute to its development; however, its most severe forms are generally attributed to single-gene defects. High-throughput technologies and data sharing contributed to the diagnosis of hundreds of single-gene intellectual disability subtypes. METHOD: We applied exome sequencing to identify potential variants causing syndromic intellectual disability in six Sudanese patients from four unrelated families. Data sharing through the Varsome portal corroborated the diagnosis of one of these patients and a Tunisian patient investigated through exome sequencing. Sanger sequencing validated the identified variants and their segregation with the phenotypes in the five studied families. RESULT: We identified three pathogenic/likely pathogenic variants in CCDC82, ADAT3, and HUWE1 and variants of uncertain significance in HERC2 and ATP2B3. The patients with the CCDC82 variants had microcephaly and spasticity, two signs absent in the two previously reported families with CCDC82-related intellectual disability. CONCLUSION: In conclusion, we report new patients with pathogenic mutations in the genes CCDC82, ADAT3, and HUWE1. We also highlight the possibility of extending the CCDC82-linked phenotype to include spastic paraplegia and microcephaly.
Assuntos
Adenosina Desaminase , Deficiência Intelectual , Proteínas de Ligação a RNA , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Adenosina Desaminase/genética , Exoma , Humanos , Deficiência Intelectual/diagnóstico , Microcefalia/genética , Mutação , Paraplegia/genética , Linhagem , Fenótipo , Proteínas de Ligação a RNA/genética , Sudão , Proteínas Supressoras de Tumor/genética , Tunísia , Ubiquitina-Proteína Ligases/genética , Sequenciamento do ExomaRESUMO
Tripartite motif containing 37 (TRIM37) was reported to function as a tumor promoter in the development of various cancers. However, the biological role of TRIM37 in ovarian cancer (OC) was still unclear. Expressions of TRIM37 and HUWE1 were detected by qRT-PCR and western blotting in OC cells. Cell proliferation was evaluated by CCK-8 assay and colony formation assay. Cell migration and invasion capabilities were examined by wound healing and transwell assay. Flow cytometry and western blotting were performed to measure cell apoptosis. Wnt/ß-catenin pathway and the expression of c-Myc were identified by qRT-PCR and western blotting. The binding of TRIM37 and HUWE1 was predicted by STRING database and verified by co-immunoprecipitation. In addition, a xenograft mouse model was established to evaluate the effects of TRIM37 and HUWE1 on tumor growth and c-MYC expression in vivo. The present study revealed that TRIM37 expression was upregulated in OC tissues and cells. TRIM37 silencing inhibited OC cell migration and invasion, promoted OC cell apoptosis, and blocked Wnt/ß-catenin signaling pathway, as well as suppressing c-MYC protein expression. Mechanistic studies suggested that TRIM37 binds to HUWE1. HUWE1 was upregulated in OC cells and TRIM37 promoted the c-MYC expression through targeting HUWE1. Animal experiments showed that TRIM37 silencing significantly repressed the tumor growth and c-MYC protein level, but HUWE1 overexpression reversed the effects of TRIM37 knockdown on mice with OC. Our findings revealed that TRIM37 accelerated the progression of OC and promoted c-MYC expression by binding to HUWE1, which provides therapeutic targets for OC treatment.
Assuntos
Neoplasias Ovarianas , Ubiquitina-Proteína Ligases , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Proteínas com Motivo Tripartido , Proteínas Supressoras de TumorRESUMO
Retinitis pigmentosa (RP) is the most common inherited retinal degenerative disease which is the major cause of vision loss. X-linked RP patients account for 5%-15% of all inherited RP cases and mutations in RP2 (Retinitis pigmentosa 2) were responsible for about 20% X-linked RP families. A majority of RP2 pathogenic mutations displayed a vulnerable protein stability and degraded rapidly through ubiquitin-proteasome system (UPS). Though the RP2 protein could be readily recovered by proteasome inhibitors, e.g., MG132, their applications for RP2-related RP therapy were limited by their nonspecific characterization. In the present study, we aimed to identify UPS-related factors, such as E3 ligases, which are specifically involved in degradation of RP2 pathogenic mutants. We identified several E3 ligases, such as HUWE1, and the co-chaperon BAG6 specifically interacting with RP2 pathogenic mutants. Knockdown of HUWE1 and BAG6 could partially rescue the reduced protein levels of RP2 mutants. BAG6 is required for recruitment of HUWE1 to ubiquitinate RP2 mutants at the K268 site. The HUWE1 inhibitor BI8622 could restore the levels of RP2 mutant and then the binding to its partner ARL3 in retina cell lines. This study revealed the details of UPS-related degradation of RP2 mutants and possibly provided a potential treatment for RP2-related RP.
Assuntos
Proteínas do Olho , Retinose Pigmentar , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligases/metabolismo , Proteínas de Membrana/genética , Chaperonas Moleculares/metabolismo , Retinose Pigmentar/patologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
HUWE1, as a well-known E3 ligase, has an oncogenic or tumor-suppressive role in various cancers by mediating ubiquitination and degradation of substrates. However, the role and action mechanism of HUWE1 in gastric cancer (GC) remain unclear. This study aimed to investigate whether HUWE1 plays a role in GC development in vitro by mediating transforming growth factor-ß receptor 2 (TGFBR2) ubiquitination and degradation. HUWE1 was overexpressed in SGCA-7901 GC cells and silenced in MGC-803 GC cells. Then, GC cell proliferation, migration, and invasion were evaluated by MTT assay and Transwell migration and invasion assay, respectively. The regulatory effect and mechanism of HUWE1 on TGFBR2 expression were assessed by qRT-PCR, western blot, and ubiquitination assay. HUWE1 mRNA and protein levels were higher in human GC tissues than in matched noncancerous gastric tissues. HUWE1 overexpression promoted, whereas HUWE1 silencing inhibited GC cell proliferation, migration, and invasion. HUWE1 promoted ubiquitination and degradation of TGFBR2. TGFBR2 overexpression impaired the tumor-promoting effect of HUWE1 overexpression in regulating GC cell behaviors. HUWE1 promoted GC cell proliferation, migration, and invasion, at least partially, by mediating TGFBR2 ubiquitination. Our data indicated a novel regulatory mechanism of HUWE1 in GC development, and provided a potential idea for the disease treatment.
Assuntos
Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Neoplasias Gástricas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Humanos , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Neoplasias Gástricas/patologia , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Injury of renal tubular epithelial cells is a key feature of the pathogenicity associated with tubulointerstitial fibrosis and other kidney diseases. HUWE1, an E3 ubiquitin ligase, acts by participating in ubiquitination and degradation of its target proteins. However, the detailed mechanisms by which HUWE1 might regulate fibrosis in renal tubular epithelial cells have not been established. Here, the possible regulation of renal tubulointerstitial fibrosis by HUWE1 was investigated by examining the expression of HUWE1 and EGFR in unilateral ureteral obstruction (UUO) mice. Markedly consistent reciprocal changes in HUWE1 and EGFR expression were observed at the protein and mRNA levels in the kidney after UUO injury. Expression of HUWE1 inhibited TGF-ß-induced injury to HK-2 cells, while HUWE1 overexpression decreased the expression of EGFR. Further analysis indicated that HUWE1 physically interacted with EGFR and promoted its ubiquitination and degradation. HUWE1 expression also showed clinical relevance in renal disease, as it notably decreased in multiple types of clinical nephropathy, while EGFR expression significantly increased when compared to the normal kidney. Therefore, this study demonstrated that HUWE1, which serves as an E3 ubiquitin ligase specific for EGFR, promotes EGFR ubiquitination and degradation, thereby regulating EGFR expression and providing protection against kidney injury.
Assuntos
Fibrose/metabolismo , Fibrose/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Rim/metabolismo , Rim/patologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Western Blotting , Linhagem Celular , Receptores ErbB/genética , Receptores ErbB/metabolismo , Imunofluorescência , Humanos , Imuno-Histoquímica , Nefropatias/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética , Ubiquitinação/fisiologia , Obstrução Ureteral/genética , Obstrução Ureteral/metabolismoRESUMO
Tumorigenesis results from dysregulation of oncogenes and tumor suppressors that influence cellular proliferation, differentiation, apoptosis, and/or senescence. Many gene products involved in these processes are substrates of the E3 ubiquitin ligase Mule/Huwe1/Arf-BP1 (Mule), but whether Mule acts as an oncogene or tumor suppressor in vivo remains controversial. We generated K14Cre;Mule(flox/flox(y)) (Mule kKO) mice and subjected them to DMBA/PMA-induced skin carcinogenesis, which depends on oncogenic Ras signaling. Mule deficiency resulted in increased penetrance, number, and severity of skin tumors, which could be reversed by concomitant genetic knockout of c-Myc but not by knockout of p53 or p19Arf. Notably, in the absence of Mule, c-Myc/Miz1 transcriptional complexes accumulated, and levels of p21CDKN1A (p21) and p15INK4B (p15) were down-regulated. In vitro, Mule-deficient primary keratinocytes exhibited increased proliferation that could be reversed by Miz1 knockdown. Transfer of Mule-deficient transformed cells to nude mice resulted in enhanced tumor growth that again could be abrogated by Miz1 knockdown. Our data demonstrate in vivo that Mule suppresses Ras-mediated tumorigenesis by preventing an accumulation of c-Myc/Miz1 complexes that mediates p21 and p15 down-regulation.
Assuntos
Transformação Celular Neoplásica , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo , Proteínas Nucleares/antagonistas & inibidores , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Inibidoras de STAT Ativados/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismo , 9,10-Dimetil-1,2-benzantraceno/farmacologia , Animais , Transformação Celular Neoplásica/genética , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p15/biossíntese , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/genética , Feminino , Genes ras , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/patologia , Masculino , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína Oncogênica p21(ras)/antagonistas & inibidores , Proteína Oncogênica p21(ras)/genética , Proteínas Inibidoras de STAT Ativados/deficiência , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Proto-Oncogênicas c-myc/deficiência , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Acetato de Tetradecanoilforbol/farmacologia , Proteína Supressora de Tumor p53 , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genéticaRESUMO
Autophagy is a critical cytoprotective mechanism against stress, which is initiated by the protein kinase Unc-51-like kinase 1 (ULK1) complex. Autophagy plays a role in both inhibiting the progression of diseases and facilitating pathogenesis, so it is critical to elucidate the mechanisms regulating individual components of the autophagy machinery under various conditions. Here, we examined whether ULK1 complex component autophagy-related protein 101 (ATG101) is downregulated via ubiquitination, and whether this in turn suppresses autophagy activity in cancer cells. Knockout of ATG101 in cancer cells using CRISPR resulted in severe growth retardation and lower survival under nutrient starvation. Transfection of mutant ATG101 revealed that the C-terminal region is a key domain of ubiquitination, while co-immunoprecipitation and knockdown experiments revealed that HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1(HUWE1) is a major E3 ubiquitin ligase targeting ATG101. Protein levels of ATG101 was more stable and the related-autophagy activity was higher in HUWE1-depleted cancer cells compared to wild type (WT) controls, indicating that HUWE1-mediated ubiquitination promotes ATG101 degradation. Moreover, enhanced autophagy in HUWE1-depleted cancer cells was reversed by siRNA-mediated ATG101 knockdown. Stable ATG101 level in HUWE1-depleted cells was a strong driver of autophagosome formation similar to upregulation of the known HUWE1 substrate WD repeat domain, phosphoinositide interacting 2 (WIPI2). Cellular survival rates were higher in HUWE1-knockdown cancer cells compared to controls, while concomitant siRNA-mediated ATG101 knockdown tends to increase apoptosis rate. Collectively, these results suggest that HUWE1 normally serves to suppress autophagy by ubiquitinating and triggering degradation of ATG101 and WIPI2, which in turn represses the survival of cancer cells. Accordingly, ATG101-mediated autophagy may play a critical role in overcoming metabolic stress, thereby contributing to the growth, survival, and treatment resistance of certain cancers.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Neoplasias/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas de Transporte Vesicular/metabolismo , Proteínas Relacionadas à Autofagia/genética , Sobrevivência Celular , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
Inhibitory GABAergic transmission is required for proper circuit function in the nervous system. However, our understanding of molecular mechanisms that preferentially influence GABAergic transmission, particularly presynaptic mechanisms, remains limited. We previously reported that the ubiquitin ligase EEL-1 preferentially regulates GABAergic presynaptic transmission. To further explore how EEL-1 functions, here we performed affinity purification proteomics using Caenorhabditis elegans and identified the O-GlcNAc transferase OGT-1 as an EEL-1 binding protein. This observation was intriguing, as we know little about how OGT-1 affects neuron function. Using C. elegans biochemistry, we confirmed that the OGT-1/EEL-1 complex forms in neurons in vivo and showed that the human orthologs, OGT and HUWE1, also bind in cell culture. We observed that, like EEL-1, OGT-1 is expressed in GABAergic motor neurons, localizes to GABAergic presynaptic terminals, and functions cell-autonomously to regulate GABA neuron function. Results with catalytically inactive point mutants indicated that OGT-1 glycosyltransferase activity is dispensable for GABA neuron function. Consistent with OGT-1 and EEL-1 forming a complex, genetic results using automated, behavioral pharmacology assays showed that ogt-1 and eel-1 act in parallel to regulate GABA neuron function. These findings demonstrate that OGT-1 and EEL-1 form a conserved signaling complex and function together to affect GABA neuron function.