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1.
Frontiers of Medicine ; (4): 1204-1218, 2023.
Artículo en Inglés | WPRIM | ID: wpr-1010813

RESUMEN

Brain development requires a delicate balance between self-renewal and differentiation in neural stem cells (NSC), which rely on the precise regulation of gene expression. Ten-eleven translocation 2 (TET2) modulates gene expression by the hydroxymethylation of 5-methylcytosine in DNA as an important epigenetic factor and participates in the neuronal differentiation. Yet, the regulation of TET2 in the process of neuronal differentiation remains unknown. Here, the protein level of TET2 was reduced by the ubiquitin-proteasome pathway during NSC differentiation, in contrast to mRNA level. We identified that TET2 physically interacts with the core subunits of the glucose-induced degradation-deficient (GID) ubiquitin ligase complex, an evolutionarily conserved ubiquitin ligase complex and is ubiquitinated by itself. The protein levels of GID complex subunits increased reciprocally with TET2 level upon NSC differentiation. The silencing of the core subunits of the GID complex, including WDR26 and ARMC8, attenuated the ubiquitination and degradation of TET2, increased the global 5-hydroxymethylcytosine levels, and promoted the differentiation of the NSC. TET2 level increased in the brain of the Wdr26+/- mice. Our results illustrated that the GID complex negatively regulates TET2 protein stability, further modulates NSC differentiation, and represents a novel regulatory mechanism involved in brain development.


Asunto(s)
Animales , Ratones , Proteínas de Unión al ADN/genética , Diferenciación Celular , Células-Madre Neurales , Translocación Genética , Ubiquitinas/genética , Ligasas/genética
2.
Chinese Journal of Cellular and Molecular Immunology ; (12): 846-851, 2023.
Artículo en Chino | WPRIM | ID: wpr-1009439

RESUMEN

Objective To prepare rabbit polyclonal antibody against mouse IQ and ubiquitin-like domain-containing protein (IQUB) and detect its expression in the mouse testis. Methods Full-length coding sequence of IQUB was inserted into the pET-30a(+) vector to construct pET-30a-IQUB recombinant prokaryotic plasmid. Transformation of pET-30a-IQUB plasmid into E. coli BL21 was performed, and protein expression was induced with isopropyl-beta-D-thiogalactoside (IPTG). The protein was purified through histidine-tagged fusion protein purification column, then denatured by treatment of urea with gradient concentration. New Zealand rabbits were immunized with the denatured protein to produce IQUB polyclonal antibody. Antibody titer was detected by ELISA, and Western blot analysis and immunofluorescence assay were employed to validate the effectiveness and specificity of IQUB antibody. Results pET-30a-IQUB recombinant plasmid was constructed, and protein expression of IQUB was induced successfully with IPTG. The titer of IQUB polyclonal antibody reached 1:1 000 000. The antibody specifically recognized the endogenous IQUB protein of testis in the wild-type adult mouse. IQUB was expressed in spermatogenic cells of different stages. It was localized in the acrosome and flagellum of mature sperms. Conclusion The highly specific rabbit anti-mouse IQUB polyclonal antibody is successfully prepared, which can be used for Western blot and immunofluorescence histochemistry.


Asunto(s)
Masculino , Conejos , Animales , Ratones , Ubiquitinas , Escherichia coli/genética , Isopropil Tiogalactósido , Anticuerpos , Ensayo de Inmunoadsorción Enzimática
3.
Biol. Res ; 56: 4-4, 2023. ilus, graf
Artículo en Inglés | LILACS | ID: biblio-1420302

RESUMEN

BACKGROUND: Spermatogonial stem cells (SSCs) are critical for sustaining spermatogenesis. Even though several regulators of SSC have been identified in rodents, the regulatory mechanism of SSC in humans has yet to be discovered. METHODS: To explore the regulatory mechanisms of human SSCs, we analyzed publicly available human testicular single-cell sequencing data and found that Ankyrin repeat and SOCS box protein 9 (ASB9) is highly expressed in SSCs. We examined the expression localization of ASB9 using immunohistochemistry and overexpressed ASB9 in human SSC lines to explore its role in SSC proliferation and apoptosis. Meanwhile, we used immunoprecipitation to find the target protein of ASB9 and verified its functions. In addition, we examined the changes in the distribution of ASB9 in non-obstructive azoospermia (NOA) patients using Western blot and immunofluorescence. RESULTS: The results of uniform manifold approximation and projection (UMAP) clustering and pseudotime analysis showed that ASB9 was highly expressed in SSCs, and its expression gradually increased during development. The immunohistochemical and dual-color immunofluorescence results displayed that ASB9 was mainly expressed in nonproliferating SSCs. Overexpression of ASB9 in the SSC line revealed significant inhibition of cell proliferation and increased apoptosis. We predicted the target proteins of ASB9 and verified that hypoxia-inducible factor 1-alpha inhibitor (HIF1AN), but not creatine kinase B-type (CKB), has a direct interaction with ASB9 in human SSC line using protein immunoprecipitation experiments. Subsequently, we re-expressed HIF1AN in ASB9 overexpressing cells and found that HIF1AN reversed the proliferative and apoptotic changes induced by ASB9 overexpression. In addition, we found that ABS9 was significantly downregulated in some NOA patients, implying a correlation between ASB9 dysregulation and impaired spermatogenesis. CONCLUSION: ASB9 is predominantly expressed in human SSCs, it affects the proliferation and apoptotic process of the SSC line through HIF1AN, and its abnormal expression may be associated with NOA.


Asunto(s)
Humanos , Masculino , Testículo/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Represoras/metabolismo , Espermatogénesis/fisiología , Ubiquitinas/metabolismo , Línea Celular , Apoptosis , Proliferación Celular , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Oxigenasas de Función Mixta/metabolismo
4.
Biol. Res ; 56: 21-21, 2023. ilus, graf
Artículo en Inglés | LILACS | ID: biblio-1513734

RESUMEN

BACKGROUND: Satellite cells are tissue-specific stem cells primarily responsible for the regenerative capacity of skeletal muscle. Satellite cell function and maintenance are regulated by extrinsic and intrinsic mechanisms, including the ubiquitin-proteasome system, which is key for maintaining protein homeostasis. In this context, it has been shown that ubiquitin-ligase NEDD4-1 targets the transcription factor PAX7 for proteasome-dependent degradation, promoting muscle differentiation in vitro. Nonetheless, whether NEDD4-1 is required for satellite cell function in regenerating muscle remains to be determined. RESULTS: Using conditional gene ablation, we show that NEDD4-1 loss, specifically in the satellite cell population, impairs muscle regeneration resulting in a significant reduction of whole-muscle size. At the cellular level, NEDD4-1-null muscle progenitors exhibit a significant decrease in the ability to proliferate and differentiate, contributing to the formation of myofibers with reduced diameter. CONCLUSIONS: These results indicate that NEDD4-1 expression is critical for proper muscle regeneration in vivo and suggest that it may control satellite cell function at multiple levels.


Asunto(s)
Músculo Esquelético/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Células Madre , Ubiquitinas/metabolismo , Diferenciación Celular , Desarrollo de Músculos/fisiología , Proliferación Celular/fisiología , Factor de Transcripción PAX7/genética , Factor de Transcripción PAX7/metabolismo
5.
Mem. Inst. Oswaldo Cruz ; 115: e190242, 2020. tab, graf
Artículo en Inglés | LILACS | ID: biblio-1091241

RESUMEN

BACKGROUND Ubiquitin (Ub) and Ub-like proteins (Ub-L) are critical regulators of complex cellular processes such as the cell cycle, DNA repair, transcription, chromatin remodeling, signal translation, and protein degradation. Giardia intestinalis possesses an experimentally proven Ub-conjugation system; however, a limited number of enzymes involved in this process were identified using basic local alignment search tool (BLAST). This is due to the limitations of BLAST's ability to identify homologous functional regions when similarity between the sequences dips to < 30%. In addition Ub-Ls and their conjugating enzymes have not been fully elucidated in Giardia. OBJETIVE To identify the enzymes involved in the Ub and Ub-Ls conjugation processes using intelligent systems based on the hidden Markov models (HMMs). METHODS We performed an HMM search of functional Pfam domains found in the key enzymes of these pathways in Giardia's proteome. Each open reading frame identified was analysed by sequence homology, domain architecture, and transcription levels. FINDINGS We identified 118 genes, 106 of which corresponded to the ubiquitination process (Ub, E1, E2, E3, and DUB enzymes). The E3 ligase group was the largest group with 82 members; 71 of which harbored a characteristic RING domain. Four Ub-Ls were identified and the conjugation enzymes for NEDD8 and URM1 were described for first time. The 3D model for Ub-Ls displayed the β-grasp fold typical. Furthermore, our sequence analysis for the corresponding activating enzymes detected the essential motifs required for conjugation. MAIN CONCLUSIONS Our findings highlight the complexity of Giardia's Ub-conjugation system, which is drastically different from that previously reported, and provides evidence for the presence of NEDDylation and URMylation enzymes in the genome and transcriptome of G. intestinalis.


Asunto(s)
Ubiquitinas/genética , Giardia lamblia/metabolismo , Ubiquitina/genética , Ubiquitinación , Ubiquitinas/metabolismo , Transducción de Señal , Modelos Moleculares , Giardia lamblia/genética , Ubiquitina/metabolismo
6.
The Korean Journal of Thoracic and Cardiovascular Surgery ; : 144-152, 2017.
Artículo en Inglés | WPRIM | ID: wpr-111254

RESUMEN

BACKGROUND: The ubiquitin-proteasome system (UPS) is an important pathway of proteolysis in pathologic hypertrophic cardiomyocytes. We hypothesize that MG132, a proteasome inhibitor, might prevent hypertrophic cardiomyopathy (CMP) by blocking the UPS. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and androgen receptor (AR) have been reported to be mediators of CMP and heart failure. This study drew upon pathophysiologic studies and the analysis of NF-κB and AR to assess the cardioprotective effects of MG132 in a left ventricular hypertrophy (LVH) rat model. METHODS: We constructed a transverse aortic constriction (TAC)-induced LVH rat model with 3 groups: sham (TAC-sham, n=10), control (TAC-cont, n=10), and MG132 administration (TAC-MG132, n=10). MG-132 (0.1 mg/kg) was injected for 4 weeks in the TAC-MG132 group. Pathophysiologic evaluations were performed and the expression of AR and NF-κB was measured in the left ventricle. RESULTS: Fibrosis was prevalent in the pathologic examination of the TAC-cont model, and it was reduced in the TAC-MG132 group, although not significantly. Less expression of AR, but not NF-κB, was found in the TAC-MG132 group than in the TAC-cont group (p<0.05). CONCLUSION: MG-132 was found to suppress AR in the TAC-CMP model by blocking the UPS, which reduced fibrosis. However, NF-κB expression levels were not related to UPS function.


Asunto(s)
Animales , Animales , Ratas , Linfocitos B , Cardiomiopatía Hipertrófica , Constricción , Fibrosis , Insuficiencia Cardíaca , Ventrículos Cardíacos , Hipertrofia , Hipertrofia Ventricular Izquierda , Modelos Animales , Miocitos Cardíacos , FN-kappa B , Complejo de la Endopetidasa Proteasomal , Inhibidores de Proteasoma , Proteolisis , Receptores Androgénicos , Ubiquitinas
7.
Protein & Cell ; (12): 63-77, 2016.
Artículo en Inglés | WPRIM | ID: wpr-757168

RESUMEN

SUMOylation is recently found to function as a targeting signal for the degradation of substrates through the ubiquitin-proteasome system. RNF4 is the most studied human SUMO-targeted ubiquitin E3 ligase. However, the relationship between SUMO proteases, SENPs, and RNF4 remains obscure. There are limited examples of the SENP regulation of SUMO2/3-targeted proteolysis mediated by RNF4. The present study investigated the role of SENP3 in the global protein turnover related to SUMO2/3-targeted ubiquitination and focused in particular on the SENP3 regulation of the stability of Sp1. Our data demonstrated that SENP3 impaired the global ubiquitination profile and promoted the accumulation of many proteins. Sp1, a cancer-associated transcription factor, was among these proteins. SENP3 increased the level of Sp1 protein via antagonizing the SUMO2/3-targeted ubiquitination and the consequent proteasome-dependent degradation that was mediated by RNF4. De-conjugation of SUMO2/3 by SENP3 attenuated the interaction of Sp1 with RNF4. In gastric cancer cell lines and specimens derived from patients and nude mice, the level of Sp1 was generally increased in parallel to the level of SENP3. These results provided a new explanation for the enrichment of the Sp1 protein in various cancers, and revealed a regulation of SUMO2/3 conjugated proteins whose levels may be tightly controlled by SENP3 and RNF4.


Asunto(s)
Animales , Humanos , Ratones , Cisteína Endopeptidasas , Genética , Metabolismo , Regulación Neoplásica de la Expresión Génica , Técnicas para Inmunoenzimas , Inmunoprecipitación , Ratones Endogámicos BALB C , Ratones Desnudos , Pronóstico , Procesamiento Proteico-Postraduccional , Proteolisis , ARN Mensajero , Genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina , Genética , Metabolismo , Factor de Transcripción Sp1 , Genética , Metabolismo , Neoplasias Gástricas , Genética , Metabolismo , Patología , Sumoilación , Células Tumorales Cultivadas , Ubiquitinación , Ubiquitinas , Genética , Metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
8.
Chinese Journal of Integrated Traditional and Western Medicine ; (12): 327-332, 2015.
Artículo en Chino | WPRIM | ID: wpr-297431

RESUMEN

<p><b>OBJECTIVE</b>To explore the scavenging action of tenuigenin (TEN) on intracerebral amyloid β protein (Aβ) aggregation and the abnormal phosphorylated tau protein and its mechanism in Alzheimer's disease (AD) rats' brain.</p><p><b>METHODS</b>Aβ1-40 was injected into the right CA1 region hippocampus to establish the AD model. Successfully modeled rats were divided into the model group, the low, middle, high TEN group. Rats were administered with TEN (18.5, 37.0, 74.0 mg/kg) by gastrogavage. Besides, a sham-operation group was set up. Expression levels of Aβ1-40 and Tau p-Ser262 were detected by immunohistochemistry. Expression levels of ubiquitin (Ub) and Ub-protein ligase E3 were measured by Western blotting.The content of 26S proteasome was detected by ELISA.</p><p><b>RESULTS</b>Immunohistochemical results showed that the number of Aβ and Tau p-Ser262 positively reacted neurons significantly increased in model group, when compared with the sham-operation group (P < 0.01). Results of Western blot showed expression levels of ubiquitinated protein were up-regulated and those of Ub-protein ligase E3 were down-regulated in the model group (P < 0.01). ELISA results showed that the content of 26S proteasome significantly decreased in AD rats' brain (P < 0.01). Compared with the model group, expression levels of Aβ1-40, Tau p-Ser262, and Ub significantly decreased; expression levels of Ub-protein ligase E3 apparently increased; the content of 26S proteasome significantly increased in each TEN treatment group (P < 0.05, P < 0.01). Best effect was shown in 37.0 mg/kg and 74.0 mg/kg TEN groups.</p><p><b>CONCLUSIONS</b>Ub proteasome pathway (UPP) participated in the occurrence of AD. TEN could obviously reduce intracere- bral Aβ1-40 accumulation and abnormal tau phosphorylation.</p>


Asunto(s)
Animales , Ratas , Enfermedad de Alzheimer , Metabolismo , Péptidos beta-Amiloides , Medicamentos Herbarios Chinos , Farmacología , Hipocampo , Metabolismo , Neuronas , Metabolismo , Fosforilación , Complejo de la Endopetidasa Proteasomal , Metabolismo , Ubiquitina-Proteína Ligasas , Metabolismo , Ubiquitinas
9.
Rev. latinoam. enferm ; 22(6): 966-972, 16/12/2014. tab
Artículo en Inglés | LILACS, BDENF | ID: lil-732947

RESUMEN

OBJECTIVES: to identify adaptation problems under Roy's Model in patients undergoing hemodialysis and to correlate them with the socioeconomic and clinical aspects. METHOD: a transversal study, undertaken using a questionnaire. The sample was made up of 178 individuals. The Chi-squared and Mann-Whitney U tests were undertaken. RESULTS: the adaptation problems and the socioeconomic and clinical aspects which presented statistical associations were: Hyperkalemia and age; Edema and income; Impairment of a primary sense: touch and income; Role failure and age; Sexual dysfunction and marital status and sex; Impairment of a primary sense: vision and years of education; Intolerance to activity and years of education; Chronic pain and sex and years of education; Impaired skin integrity and age: Hypocalcemia and access; Potential for injury and age and years of education; Nutrition below the organism's requirements and age; Impairment of a primary sense: hearing and sex and kinetic evaluation of urea; Mobility in gait and/or coordination restricted, and months of hemodialysis; and, Loss of ability for self-care, and months of hemodialysis and months of illness. CONCLUSION: adaptation problems in the clientele undergoing hemodialysis can be influenced by socioeconomic/clinical data. These findings contribute to the development of the profession, fostering the nurse's reflection regarding the care. .


OBJETIVOS: identificar os problemas adaptativos de Roy em pacientes submetidos a hemodiálise e correlacioná-los aos aspectos socioeconômicos e clínicos. MÉTODO: estudo transversal, realizado através de um formulário. A amostra foi de 178 indivíduos. Efetuaram-se os testes qui-quadrado e U de Mann-Whitney. RESULTADOS: os problemas adaptativos e os aspectos socioeconômicos e clínicos que apresentaram associações estatísticas foram: hipercalemia e idade; edema e renda; deficiência de um sentido primário: tátil e renda; falha no papel e idade; disfunção sexual e estado civil e sexo; deficiência de um sentido primário: visão e anos de estudo; intolerância à atividade e anos de estudo; dor crônica e sexo e anos de estudo; integridade da pele prejudicada e idade; hipocalcemia e acesso; potencial para lesão e idade e anos de estudo; nutrição menor que as necessidades do organismo e idade; deficiência de um sentido primário: audição e sexo e avaliação cinética da ureia; mobilidade andar e/ou coordenação restritas e meses de hemodiálise e perda de habilidade de autocuidado e meses de hemodiálise e meses de doença. CONCLUSÃO: problemas adaptativos da clientela hemodialítica podem sofrer influências de dados socioeconômicos/clínicos. Tais achados contribuem para o desenvolvimento da profissão, proporcionando reflexão por parte do enfermeiro acerca do cuidado. .


OBJETIVOS: identificar los problemas adaptativos de Roy en pacientes sometidos a hemodiálisis y correlacionarlos a los aspectos socioeconómicos y clínicos. MÉTODO: estudio transversal, realizado a través de un formulario. La muestra fue de 178 individuos. Se efectuaron las pruebas Chi-cuadrado y U de Mann-Whitney. RESULTADOS: los problemas adaptativos y los aspectos socioeconómicos y clínicos que presentaron asociaciones estadísticas fueron: Hiperkalemia y edad; Edema y renta; Deficiencia de un sentido primario: táctil y renta; Fracaso en el papel y edad; Disfunción sexual y estado civil y sexo; Deficiencia de un sentido primario: visión y años de estudio; Intolerancia a la actividad y años de estudio; Dolor crónico y sexo y años de estudio; Integridad de la piel perjudicada y edad; Hipocalcemia y acceso; Potencial para lesión y edad y años de estudio; Nutrición menor que las necesidades del organismo y edad; Deficiencia de un sentido primario: audición y sexo y evaluación cinética de la urea; Movilidad andar y/o coordinación restringidas y meses de hemodiálisis; y, Pérdida de habilidad de autocuidado y meses de hemodiálisis y meses de enfermedad. CONCLUSIÓN: los problemas adaptativos de la clientela hemodialítica pueden sufrir influencias de datos socioeconómicos/clínicos. Esos hallazgos contribuyen para el desarrollo de la profesión, permitiendo la reflexión del enfermero acerca del cuidado. .


Asunto(s)
Humanos , Lisosomas/metabolismo , Proteínas/metabolismo , Ubiquitinas/fisiología , Compartimento Celular , Inhibidores de Cisteína Proteinasa/farmacología , Filamentos Intermedios/fisiología , Leucina/análogos & derivados , Leucina/farmacología , Errores Innatos del Metabolismo/metabolismo , Orgánulos/ultraestructura
10.
Rev. bras. enferm ; 67(6): 891-897, Nov-Dec/2014. tab
Artículo en Portugués | LILACS, BDENF | ID: lil-732818

RESUMEN

A tomada de decisão é uma área de investigação na saúde que tem vindo a ganhar importância quer pelos modelos de parceria de cuidados que dão protagonismo ao paciente e família, quer pela preocupação crescente com a qualidade e satisfação do cliente com os cuidados disponibilizados. Assim, propusemo-nos efetuar a adaptação transcultural e avaliar as propriedades psicométricas da versão portuguesa da "The Satisfaction with Decision Scale" de Holmes-Rovner (1996), que visa avaliar a satisfação com as decisões tomadas em saúde. A amostra foi constituída por 521 estudantes da Escola Superior de Enfermagem do Porto. Os resultados obtidos nos testes de fiabilidade revelam uma boa consistência interna para o total dos itens (Alpha Cronbach = 0,88). O estudo psicométrico permite-nos afirmar que a versão em Português da "The Satisfaction with Decision Scale", que denominamos "Escala da Satisfação com a Decisão em Saúde", é um instrumento fidedigno e válido.


Decision making is an area of health research that has gained importance both for the partnership models of care that give prominence to the patient and family, either by growing concern about quality and customer satisfaction with the care provided. So we decided to make the cultural adaptation and to evaluate the psychometric properties of the Portuguese version "The Satisfaction with Decision Scale" de Holmes-Rovner (1996), which aims to assess satisfaction with the decisions taken in health. The sample consisted of 521 nursing students the School of Nursing of Porto. The results of reliability tests show good internal consistency for the total items (Alpha Cronbach = 0.88). The psychometric study allows us to state that the Portuguese version of "The Satisfaction with Decision Scale", we call "Escala da Satisfação com a Decisão em Saúde", is an instrument comparable with the original in terms of validity and reliability.


La toma de decisiones es un área de investigación en salud que ha ganado importancia tanto por los modelos de atención dirigida al paciente y su familia, como por la creciente preocupación por la calidad y satisfacción del cliente con la atención recibida. Por esta razón decidimos hacer la adaptación transcultural y evaluar las propiedades psicométricas de la versión portuguesa "The Satisfaction with Decision Scale" de Holmes-Rovner (1996), que tiene como objetivo evaluar la satisfacción con las decisiones adoptadas en materia de salud. La muestra consta de 521 estudiantes de la Escuela de Enfermería del Porto. Los resultados de las pruebas de fiabilidad muestran una buena consistencia interna para la escala total (Alpha Cronbach = 0,88). El estudio psicométrico nos permite afirmar que la versión en portugués de "The Satisfaction with Decision Scale", que nosotros llamamos "Escala da Satisfação com a Decisão em Saúde", es válida.


Asunto(s)
Fibroblastos/ultraestructura , Lisosomas/metabolismo , Proteínas/metabolismo , Ubiquitinas/metabolismo , Línea Celular , Inhibidores de Cisteína Proteinasa , Inmunohistoquímica , L-Lactato Deshidrogenasa/metabolismo , Leucina/análogos & derivados , Leucina/farmacología , Lisosomas/efectos de los fármacos , Microscopía Electrónica , ARN Mensajero/metabolismo , Ubiquitinas/genética
11.
Acta Academiae Medicinae Sinicae ; (6): 1-4, 2014.
Artículo en Chino | WPRIM | ID: wpr-285935

RESUMEN

<p><b>OBJECTIVE</b>To identify the specific protein interactions involved in Bat3-mediated apoptosis.</p><p><b>METHODS</b>Tandem affinity purification (TAP) was utilized to investigate Bat3-protein interactions, during which full-length human Bat3 fused with Strep2 and FLAG tag as a bait was used to screen the specific protein-protein interactions. The isolated proteins were identified with mass spectrometry.</p><p><b>RESULTS</b>TAP studies showed that Ubl4A was identified as a Bat3-binding partner. Further investigation using co-immunoprecipitation confirmed that Bat3 was associated with Ubl4A.</p><p><b>CONCLUSION</b>TAP was successfully established and is suitable for isolating the binding partners of Bat3.</p>


Asunto(s)
Humanos , Línea Celular , Chaperonas Moleculares , Unión Proteica , Ubiquitinas
12.
Campinas; s.n; abr. 2013. 67 p. ilus.
Tesis en Portugués | LILACS | ID: lil-691886

RESUMEN

A proteína PGC1α é um co-ativador de transcrição gênica que desempenha papel importante na regulação de uma série de fenômenos metabólicos que compreendem desde o controle da termogênese e mitocondriogênese até a regulação da secreção de insulina e a produção hepática de glicose. Como vários dos fenômenos biológicos controlados direta ou indiretamente pela PGC1α tem importância vital, a regulação dos níveis de PGC1α nos tecidos deve ser finamente ajustada. Nos últimos anos, inúmeros estudos exploraram os mecanismos envolvidos com o controle da expressão gênica e tradução da PGC1α. Entretanto, apenas alguns poucos estudos avaliaram a degradação da mesma. Um dos mais importantes mecanismos envolvidos com a regulação funcional e da meia-vida de proteínas é a ubiquitinação, que pode direcionar proteínas alvo ao proteassoma para degradação ou a outras modificações pós-traducionais. O objetivo do presente estudo foi avaliar a participação de uma proteína com atividade deubiquitinase e ubiquitina ligase, a A20, na manutenção da homeostase do tecido adiposo de animais submetidos à dieta rica em gordura e voluntários humanos magros e obesos antes e após cirurgia de redução de peso. Foram utilizados o tecido adiposo branco visceral e subcutâneo e o tecido adiposo marrom de camundongos Swiss machos submetidos a 16 semanas de dieta hiperlipídica e o tecido adiposo subcutâneo de voluntários magros e obesos antes e após a cirurgia bariátrica. Esses tecidos foram avaliados quanto ao conteúdo protéico e expressão gênica da proteína A20, e sua associação com a PGC1α por imunoprecipitação e imunofluorescência, bem como a ubiquitinação desta última. Os resultados obtidos a partir do tecido adiposo de humanos mostram uma diminuição na expressão da proteína A20 nos pacientes antes e após a cirurgia bariátrica com relação aos voluntários magros.


Peroxisome proliferator-activated receptor γ coactivator 1 alpha (PGC-1α) plays an important role in whole body metabolism and, particularly in glucose homeostasis. Its expression is tightly regulated and, small variations in tissue levels can have a major impact in a number of physiological and pathological conditions. Recent studies have shown that the ubiquitin/proteasome system plays a role in the control of PGC-1α degradation. Here we evaluated the interaction of PGC-1α with the protein A20, which plays a dual-role in the control of the ubiquitin/proteasome system acting as a deubiquitinase and as an E3 ligase. We employed immunoprecipitation, quantitative real-time PCR and immunofluorescence staining to evaluate PGC-1α, A20, PPARγ and ubiquitin in the adipose tissue of humans and mice. Our results show that, in distinct sites of the adipose tissue A20 binds to PGC-1α. At least in the subcutaneous fat of humans and mice the levels of PGC-1α decrease during obesity, while its physical association with A20 increases. The inhibition of A20 leads to a reduction of PGC-1α and PPARγ expression, suggesting that A20 acts as a protective factor against PGC-1α disposal. Thus, we provide evidence that mechanisms regulating PGC-1α ubiquitination are potentially involved in the control of the function of this transcriptional co-activator.


Asunto(s)
Humanos , Animales , Masculino , Femenino , Ratones , Grasa Abdominal , Obesidad , Tejido Adiposo , Inflamación , Ubiquitinas
13.
Chinese Journal of Virology ; (6): 480-487, 2013.
Artículo en Chino | WPRIM | ID: wpr-356678

RESUMEN

To investigate whether HIV-1 infection affects expression of interferon-stimulated gene 15 (ISG15) and determine the antiviral effect of ISG15 in vitro, ISG15 expression at transcription and protein level and supernatant p24 of HIV-1 was detected in various HIV-1 infected or transfected cell lines, respec tively. HIV-1 molecular clone pNL4-3 was used to transfect 293T, TZM-bl and HeLa cells while HIV-1 pseudo-typed virus was used to infect Jurkat, MT-1 and THP-1 cells. After twenty-four hours post infection or transfection, cells were harvested for extraction of total RNAs and subsequently used in real time PCR for quantification of ISG15 transcriptional expression. After forty-eight hours post infection or transfection, cells were harvested for extraction of total proteins to detect ISG15 protein expression. A significant up-regulation of ISG15 at transcription level was observed in HIV-1 infected or transfected cell lines, particulaly in THP-1 and TZM-bl cells. Up-regulation of ISG15 protein was observed only in TZM-bl cell. Cotransfection of ISG15 and HIV-1 indicated that ISG15 inhibited production of HIV-1 progeny virus in a dose and time depend manner in 293T cell but not TZM-bl cell. These results revealed upregulating ISG15 expression in transcriptional level and potential antagonistic mechanism against ISG15 by HIV-1 infection, simultanelusly.


Asunto(s)
Humanos , Secuencia de Bases , Línea Celular , Citocinas , Genética , Metabolismo , Infecciones por VIH , Genética , Metabolismo , Virología , VIH-1 , Fisiología , Interferones , Metabolismo , Datos de Secuencia Molecular , Ubiquitinas , Genética , Metabolismo , Regulación hacia Arriba
14.
Univ. sci ; 17(3): 273-281, Sep.-Dec. 2012. ilus
Artículo en Inglés | LILACS | ID: lil-669342

RESUMEN

Estudiamosel desdoblamiento forzado de una molécula de ubiquitina, usando elprotocolo de dinámica molecular “pull and wait” (PNW) a 300 K.Se implementó PNW en el programa CHARMM usando un tiempode integración de 1 fs y una constante dieléctrica de 1. La estructurasolvatada inicialmente, se colocó bajo tensión mecánica, ejerciéndosefuerzas en diferentes direcciones. El rompimiento de cinco enlacesde Hidrogeno entre los pliegues β1 y β5 que tiene lugar durante losprimeros 13 a 15 Å de extensión, marcan una barrera mecánica lacual define la máxima fuerza necesaria para el desdoblamiento. Lassimulaciones realizadas muestran que dado un tiempo adecuado, laaplicación de una fuerza pequeña puede desestabilizar los mencionadosenlaces de hidrógeno relativo a los enlaces que se pueden formar conmoléculas de agua; permitiendo la formación de enlaces de hidrógenoestables entre aguas y donadores o aceptores de la cadena principal.De esta forma, las simulaciones con PNW muestran que la tensiónmecánica no es responsable de separar los puentes de hidrógeno, estasólo los desestabiliza haciéndolos menos estables con respecto a losenlaces que se pueden formar con moléculas de agua. Simulacionesadicionales muestran que la fuerza necesaria para desestabilizarlos enlaces de hidrogeno, permitiendo su remplazo por enlaces conmoléculas de agua, depende fuertemente de la dirección de estiramiento.El protocolo de simulación que permite equilibrar a cada paso deextensión, nos evidenció eventos conducentes al desdoblamiento de lamolécula ubiquitina por fuerzas mecánicas...


Using the “pull and wait” (PNW) simulationprotocol at 300 K, we studied the unfolding of aubiquitin molecule by force. PNW was implemented inthe CHARMM program using an integration time step of1 fs and a uniform dielectric constant of 1. The ubiquitinmolecule, initially solvated, was put under mechanicalstress, exerting forces from different directions. Therupture of five hydrogen bonds between parallel strandsβ1 and β5 takes place during the extension from 13 to15 Å, defines a mechanical barrier for unfolding anddominates the point of maximum unfolding force. Thesimulations described here show that given adequatetime, a small applied force can destabilize those fiveH-bonds relative to the bonds that can be created towater molecules; allowing the formation of stableH-bonds between a single water molecule and the donorand acceptor groups of the interstrand H-bonds. Thus,simulations run with PNW show that the force is notresponsible for “ripping apart” the backbone H-bonds;it merely destabilizes them making them less stablethan the H-bonds they can make with water. Additionalsimulations show that the force necessary to destabilizethe H-bonds and allow them to be replaced by H-bondsto water molecules depends strongly on the pullingdirection. By using a simulation protocol that allowsequilibration at each extension we have been able toobserve the details of the events leading to the unfoldingof ubiquitin by mechanical force...


Estudamos o desdobramento forçado de uma molécula deubiquitina usando o protocolo de dinâmica molecular “pull and wait”(PNW) em 300 K. PNW foi implementado no programa CHARMMusando um tempo de integração de 1 fs e uma constante dielétrica de1. A estrutura solvatada, foi colocada sob estresse mecânico, exercendoseforças de diferentes direções. Simulações mostraram que a rupturade cinco ligações de Hidrogênio entre as dobras β1 e β5, que acontecedurante os primeiros 13 a 15 Å de extensão, define uma barreira, aqual determina a força máxima para o desdobramento. As simulaçõesmostram que, dado o tempo adequado, uma pequena força aplicadapode desestabilizar os mesmos cinco Hidrogênios relativos às ligaçõesH- que os grupos da cadeia principal podem fazer com a molécula deágua; permitindo a formação de ligações estáveis de H- entre moléculasde água e os grupos doadores e receptores da intercadeia. Desta forma,simulações que utilizam este protocolo mostram que a força nãoé utilizada para ®romper¼ as ligações H- da cadeia principal, apenasdesestabilizando-as e tornando-as menos estáveis do que as ligações queas mesmas podem fazer com a água. Simulações adicionais mostramque a força necessária para desestabilizar as ligações H- e permitir que asmesmas sejam substituídas por ligações do Hidrogênio com moléculasde água depende fortemente da direção da aplicação da força. Atravésda utilização de um protocolo de simulação que permite equilibrar emcada extensã, fomos capazes de observar os detalhes do mecanismo dedesdobramento da ubiquitina por força mecânica...


Asunto(s)
Ubiquitina/análisis , Ubiquitina/provisión & distribución , Ubiquitinas
15.
West China Journal of Stomatology ; (6): 97-102, 2012.
Artículo en Chino | WPRIM | ID: wpr-241853

RESUMEN

<p><b>OBJECTIVE</b>To investigate the association between small ubiquitin-related modifier-1 (SUMO-1) gene rs6709162, rs7599810, rs7580433 polymorphism and non-syndromic oral clefting (NSOC).</p><p><b>METHODS</b>Our study consisted of 208 Ningxia NSOC patients, their parents (189 fathers and 176 mothers), 172 nuclear families (patients and their parents), and 284 normal controls. DNA was extracted and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) was used to identify rs6709162, rs7599810, rs7580433 genotypes of the samples. The data was analyzed by case-control analysis, family based associated test (FBAT), and transmission disequilibrium test (TDT).</p><p><b>RESULTS</b>Case-control study found that TT genotype's frequency was significantly different in cleft lip and cleft palate group compared with the control group at rs7599810 of SUMO-1 (P=0.01, P=0.01). TDT test showed that rs7599810's T allele had over-transmitted (P=0.00) in cleft lip and palate group. FBAT analysis revealed that distribution of rs7599810's TT genotype and T allele was significantly different (P=0.00, P=0.00). TDT test showed that rs6709162's C allele in cleft palate and cleft lip and palate patients had over-transmitted (P=0.00, P=0.01). rs7580433's G allele in cleft lip group had over-transmitted (P=0.05).</p><p><b>CONCLUSION</b>SUMO-1 gene polymorphism is associated with NSOC.</p>


Asunto(s)
Femenino , Humanos , Masculino , Estudios de Casos y Controles , Ataxia Cerebelosa , Labio Leporino , Fisura del Paladar , Genotipo , Discapacidad Intelectual , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Proteína SUMO-1 , Genética , Ubiquitinas
16.
Acta Physiologica Sinica ; (6): 577-583, 2012.
Artículo en Inglés | WPRIM | ID: wpr-333165

RESUMEN

Activation of interferon (IFN) signaling in the central nervous system (CNS) is usually associated with inflammation. However, a robust activation of type I IFN-stimulated genes (ISGs) at pre-symptomatic stages occurs in the spinal cord of SOD1(G93A) mice, an amyotrophic lateral sclerosis (ALS) animal model, without obvious signs of inflammation. To determine if the same signaling pathway is elevated in other types of neuronal injuries, we examined the protein expression levels of an IFN-stimulated gene, ISG15, in mouse models of acute and chronic neuronal injuries. We found that ISG15 protein was dramatically increased in the brains of mice subjected to global ischemia and traumatic brain injury, and in transgenic mice overexpressing HIV gp120 protein. These results suggest that activation of ISGs is a shared feature of neuronal injuries and that ISG15 may be a suitable biomarker for detecting neuronal injuries in the CNS.


Asunto(s)
Animales , Ratones , Esclerosis Amiotrófica Lateral , Lesiones Encefálicas , Isquemia Encefálica , Sistema Nervioso Central , Citocinas , Metabolismo , Modelos Animales de Enfermedad , Ratones Transgénicos , Ubiquitinas , Metabolismo
17.
Chinese Journal of Virology ; (6): 78-83, 2012.
Artículo en Chino | WPRIM | ID: wpr-354767

RESUMEN

ISG15 is a 15kD ubiquitin-like protein (UBL) induced by interferon (IFN). ISG15 can be covalently attached to proteins, which is called ISGylation process. ISGylation system contains ISG15, UBE1L, UBCH8 and HERC5 proteins, which are all essential for ISGylation. ISG15 and ISGylation system have been found to have anti-viral effects. A better understanding of how ISG15 mediates the anti-viral activity will provide insights for new anti-viral drugs development and new therapeutic strategies. The mechanisms underlying the ISG15 mediated anti-viral response have been explored extensively in recent years. This minireview summarized the research advances of how ISG15 mediated the anti-viral effects against different kinds of viruses.


Asunto(s)
Animales , Humanos , Citocinas , Fisiología , Infecciones por VIH , Alergia e Inmunología , Gripe Humana , Alergia e Inmunología , Ubiquitinas , Fisiología , Virosis , Alergia e Inmunología
18.
Chinese Journal of Stomatology ; (12): 604-607, 2011.
Artículo en Chino | WPRIM | ID: wpr-306378

RESUMEN

<p><b>OBJECTIVE</b>To study the effect of diubiquitin (FAT10) down-regulation by small interfering RNA-mediated RNA interference (RNAi) on the biological features of tongue carcinoma cell line Tca8113.</p><p><b>METHODS</b>Tca8113 cells were transfected with synthetic small interfering RNA (siRNA) targeting FAT10. Expression of FAT10 mRNA and protein were respectively measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, transfection efficiencies were monitored. The distribution of cell cycle phases was determined using flow cytometry. The proliferative and invasive ability of Tca8113 cells in vitro was evaluated by the colony-forming unit assay and Transwell migration assay respectively.</p><p><b>RESULTS</b>Both FAT10 mRNA and protein expression were significantly decreased in the experimental group (pU-FAT10-siRNA: mRAN 0.36 ± 0.03, Protein 0.39 ± 0.04) compared with controls (</p><p><b>CONTROL</b>mRNA 0.95 ± 0.05, Protein 0.69 ± 0.05; pU-siRNA: mRNA 0.92 ± 0.07, Protein 0.64 ± 0.05) (P < 0.05). The cell cycle was arrested in the G(1) phase [pU-FAT10-siRNA: (72.45 ± 5.81)%,</p><p><b>CONTROL</b>(45.95 ± 3.80)%, pU-siRNA: (45.95 ± 3.80)%]. The proliferation and invasiveness of treated Tca8113 cells were inhibited in vitro (pU-FAT10-siRNA: 41.83 ± 8.19, CONTROL: 317.21 ± 69.48, pU-siRNA: 339.36 ± 73.84).</p><p><b>CONCLUSIONS</b>Delivery of siRNA targeting FAT10 seems efficient in down-regulating FAT10 expression and diminishing the growth, proliferation and invasiveness of Tca8113 cells, suggesting that siRNA-based strategy targeting FAT10 may lay a foundation for the clinical management of tongue carcinoma.</p>


Asunto(s)
Humanos , Carcinoma de Células Escamosas , Genética , Metabolismo , Patología , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación hacia Abajo , Interferencia de ARN , ARN Mensajero , Metabolismo , ARN Interferente Pequeño , Genética , Neoplasias de la Lengua , Genética , Metabolismo , Patología , Transfección , Ubiquitinas , Genética , Metabolismo
19.
Journal of Southern Medical University ; (12): 1493-1497, 2011.
Artículo en Chino | WPRIM | ID: wpr-333879

RESUMEN

<p><b>OBJECTIVE</b>To investigate whether progesterone receptor B (PRB) can be sumoylated by SUMO-2/3 and the effect of sumoylation on PRB transcriptional activity.</p><p><b>METHODS</b>SUMO-2/3 cDNA was amplified from MCF-7 cDNA and cloned into the eukaryotic expression vector pcDNA3-FLAG. The plasmid pXJ40-myc-PRB was cotransfected with pcDNA3FLAG-SUMO2, pcDNA3FLAG-SUMO3 or the mock control into 293T cells, and PRB sumoylation was detected by immunoprecipitation and Western blotting. The effect of PRB sumoylation on its transcriptional activity was determined using reporter luciferase assay.</p><p><b>RESULTS</b>pcDNA3FLAG-SUMO2 and pcDNA3FLAG-SUMO3 vectors were successfully constructed. SUMO-2/3 could bind covalently to PRB and increase its transcriptional dependent on the presence of progesterone.</p><p><b>CONCLUSION</b>PRB can be sumoylated by SUMO-2/3 and its function is regulated by this modification.</p>


Asunto(s)
Animales , Humanos , Línea Celular , Plásmidos , Genética , Receptores de Progesterona , Genética , Metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina , Genética , Metabolismo , Transcripción Genética , Transfección , Ubiquitinación , Ubiquitinas , Genética , Metabolismo
20.
Protein & Cell ; (12): 612-619, 2011.
Artículo en Inglés | WPRIM | ID: wpr-757060

RESUMEN

The ubiquitin-related modifier Urm1 can be covalently conjugated to lysine residues of other proteins, such as yeast Ahp1 and human MOCS3, through a mechanism involving the E1-like protein Uba4 (MOCS3 in humans). Similar to ubiquitination, urmylation requires a thioester intermediate and forms isopeptide bonds between Urm1 and its substrates. In addition, the urmylation process can be significantly enhanced by oxidative stress. Recent findings have demonstrated that Urm1 also acts as a sulfur carrier in the thiolation of eukaryotic tRNA via a mechanism that requires the formation of a thiocarboxylated Urm1. This role is very similar to that of prokaryotic sulfur carriers such as MoaD and ThiS. Evidence strongly supports the hypothesis that Urm1 is the molecular fossil in the evolutionary link between prokaryotic sulfur carriers and eukaryotic ubiquitin-like proteins. In the present review, we discuss the dual role of Urm1 in protein and tRNA modification.


Asunto(s)
Animales , Humanos , Modelos Biológicos , ARN de Transferencia , Metabolismo , Azufre , Metabolismo , Ubiquitina , Metabolismo , Ubiquitinas , Metabolismo
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