RESUMO
BACKGROUND: Cardiomyopathy is a common and lethal complication in patients with limb-girdle muscular dystrophy (LGMD), one of the most prevalent forms of muscular dystrophy. The pathogenesis underlying LGMD-related cardiomyopathy remains unclear. NRIP (gene name DCAF6), a Ca2+-dependent calmodulin binding protein, was reduced in dystrophic muscles from LGMD patients. Mice lacking NRIP exhibit a myopathic phenotype resembling that in LGMD patients, making NRIP deficiency a potential culprit leading to cardiomyopathy. This study aimed to determine if NRIP deficiency leads to cardiomyopathy and to explore the underlying molecular mechanisms. METHODS AND RESULTS: NRIP expression was reduced in both human and mouse failing hearts. Muscle-specific NRIP knockout (MCK-Cre::Dcaf6flox/flox) mouse heart and isolated cardiomyocytes exhibited markedly reduced contractility. Transmission electron microscopy revealed abnormal sarcomere structures and mitochondrial morphology in MCK-Cre::Dcaf6flox/flox hearts. Protein co-immunoprecipitation and confocal imaging revealed that NRIP interacts with α-actinin 2 (ACTN2) at the Z-disc. We found that NRIP facilitated ACTN2-mediated F-actin bundling, and that NRIP deficiency resulted in reduced binding between Z-disc proteins ACTN2 and Cap-Z. In addition, NRIP-deficiency led to increased mitochondrial ROS and impaired mitochondrial respiration/ATP production owing to elevated cellular NADH/NAD+ ratios. Treatment with mitochondria-directed antioxidant mitoTEMPO or NAD+ precursor nicotinic acid restored mitochondrial function and cardiac contractility in MCK-Cre::Dcaf6flox/flox mice. CONCLUSIONS: NRIP is essential to maintain sarcomere structure and mitochondrial/contractile function in cardiomyocytes. Our results revealed a novel role for NRIP deficiency in the pathogenesis of LGMD and heart failure. Targeting NRIP, therefore, could be a powerful new approach to treat myocardial dysfunction in LGMD and heart failure patients.
Assuntos
Cardiomiopatias/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteína 1 de Interação com Receptor Nuclear/metabolismo , Sarcômeros/metabolismo , Actinina/metabolismo , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Cardiomiopatias/fisiopatologia , Respiração Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Insuficiência Cardíaca/genética , Homeostase/efeitos dos fármacos , Humanos , Masculino , Camundongos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/ultraestrutura , Modelos Biológicos , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , NAD/metabolismo , Niacina/farmacologia , Proteína 1 de Interação com Receptor Nuclear/química , Fenótipo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Espécies Reativas de Oxigênio/metabolismo , Sarcômeros/efeitos dos fármacos , Sarcômeros/ultraestruturaRESUMO
Nuclear receptor interaction protein (NRIP, also known as DCAF6 and IQWD1) is a Ca(2+)-dependent calmodulin-binding protein. In this study, we newly identify NRIP as a Z-disc protein in skeletal muscle. NRIP-knockout mice were generated and found to have reduced muscle strength, susceptibility to fatigue and impaired adaptive exercise performance. The mechanisms of NRIP-regulated muscle contraction depend on NRIP being downstream of Ca(2+) signaling, where it stimulates activation of both 'calcineurin-nuclear factor of activated T-cells, cytoplasmic 1' (CaN-NFATc1; also known as NFATC1) and calmodulin-dependent protein kinase II (CaMKII) through interaction with calmodulin (CaM), resulting in the induction of mitochondrial activity and the expression of genes encoding the slow class of myosin, and in the regulation of Ca(2+) homeostasis through the internal Ca(2+) stores of the sarcoplasmic reticulum. Moreover, NRIP-knockout mice have a delayed regenerative capacity. The amount of NRIP can be enhanced after muscle injury and is responsible for muscle regeneration, which is associated with the increased expression of myogenin, desmin and embryonic myosin heavy chain during myogenesis, as well as for myotube formation. In conclusion, NRIP is a novel Z-disc protein that is important for skeletal muscle strength and regenerative capacity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Calmodulina/metabolismo , Músculo Esquelético/fisiologia , Proteínas Nucleares/metabolismo , Regeneração/fisiologia , Animais , Camundongos , Camundongos Knockout , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Transdução de SinaisRESUMO
Previously, we found a gene named nuclear receptor interaction protein (NRIP) (or DCAF6 or IQWD1). We demonstrate that NRIP is a novel binding protein for human papillomavirus 16 (HPV-16) E2 protein. HPV-16 E2 and NRIP can directly associate into a complex in vivo and in vitro, and the N-terminal domain of NRIP interacts with the transactivation domain of HPV-16 E2. Only full-length NRIP can stabilize E2 protein and induce HPV gene expression, and NRIP silenced by two designed small interfering RNAs (siRNAs) decreases E2 protein levels and E2-driven gene expression. We found that NRIP can directly bind with calmodulin in the presence of calcium through its IQ domain, resulting in decreased E2 ubiquitination and increased E2 protein stability. Complex formation between NRIP and calcium/calmodulin activates the phosphatase calcineurin to dephosphorylate E2 and increase E2 protein stability. We present evidences for E2 phosphorylation in vivo and show that NRIP acts as a scaffold to recruit E2 and calcium/calmodulin to prevent polyubiquitination and degradation of E2, enhancing E2 stability and E2-driven gene expression.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Calcineurina/metabolismo , Proteínas de Ligação a Calmodulina/genética , Linhagem Celular , Células HEK293 , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , FosforilaçãoRESUMO
BACKGROUND: Nuclear receptor interaction protein (NRIP) is a calcium/calmodulin (CaM) binding protein. Nuclear receptor interaction protein interacts with CaM to activate calcineurin and CaMKII signalling. The conventional NRIP knockout mice (global knockout) showed muscular abnormality with reduction of muscle oxidative functions and motor function defects. METHODS: To investigate the role of NRIP on neuromuscular system, we generated muscle-restricted NRIP knockout mice [conditional knockout (cKO)]. The muscle functions (including oxidative muscle markers and muscle strength) and lumbar motor neuron functions [motor neuron number, axon denervation, neuromuscular junction (NMJ)] were tested. The laser-captured microdissection at NMJ of skeletal muscles and adenovirus gene therapy for rescued effects were performed. RESULTS: The cKO mice showed muscular abnormality with reduction of muscle oxidative functions and impaired motor performances as global knockout mice. To our surprise, cKO mice also displayed motor neuron degeneration with abnormal architecture of NMJ. Specifically, the cKO mice revealed reduced motor neuron number with small neuronal size in lumbar spinal cord as well as denervating change, small motor endplates, and decreased myonuclei number at NMJ in skeletal muscles. To explore the mechanisms, we screened various muscle-derived factors and found that myogenin is a potential candidate that myogenin expression was lower in skeletal muscles of cKO mice than wild-type mice. Because NRIP and myogenin were colocalized around acetylcholine receptors at NMJ, we extracted RNA from synaptic and extrasynaptic regions of muscles using laser capture microdissection and showed that myogenin expression was especially lower at synaptic region in cKO than wild-type mice. Notably, overexpression of myogenin using intramuscular adenovirus encoding myogenin treatment rescued abnormal NMJ architecture and preserved motor neuron death in cKO mice. CONCLUSIONS: In summary, we demonstrated that deprivation of NRIP decreases myogenin expression at NMJ, possibly leading to abnormal NMJ formation, denervation of acetylcholine receptor, and subsequent loss of spinal motor neuron. Overexpression of myogenin in cKO mice can partially rescue abnormal NMJ architecture and motor neuron death. Therefore, muscular NRIP is a novel trophic factor supporting spinal motor neuron via stabilization of NMJ by myogenin expression.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neurônios Motores/metabolismo , Miogenina/genética , Junção Neuromuscular/metabolismo , Proteínas Nucleares/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Biomarcadores , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Miogenina/metabolismo , Degeneração Neural , Proteínas Nucleares/metabolismo , Fenótipo , Transdução GenéticaRESUMO
Both nuclear receptor interaction protein (NRIP) and DNA damage binding protein 2 (DDB2) belong to the Cullin 4 (CUL4)-DDB1 binding protein family and are androgen receptor (AR)-interacting proteins. Here, we investigated the expression patterns of the NRIP, DDB2 and AR proteins in human prostate cancer tissues and found that the expression levels of NRIP and AR were higher, but the DDB2 level was lower, in prostate cancer tissues than in non-neoplastic controls, suggesting NRIP as a candidate tumor promoter and DDB2 as a tumor suppressor in prostate cancer. Furthermore, both NRIP and DDB2 shared the same AR binding domain; they were competitors for the AR, but not for DDB1 binding, in the AR-DDB2-DDB1-CUL4A complex. Conclusively, NRIP stabilizes the AR protein by displacing DDB2 from the AR-DDB2 complex. Consistent with our hypothesis, a specific expression pattern with high levels of NRIP and AR, together with a low level of DDB2, was found more frequently in the human prostate cancer tissues with a cribriform pattern than in non-cribriform tumors, suggesting that disruption of the balance between NRIP and DDB2 may change AR protein homeostasis and contribute to pathogenesis in certain aggressive types of prostate cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Western Blotting , Proteínas Culina/metabolismo , Humanos , Imuno-Histoquímica , Imunoprecipitação , Masculino , Estabilidade Proteica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The papillomavirus E2 protein is involved in the maintenance of persistent infection and known to bind either to cellular factors or directly to mitotic chromosomes in order to partition the viral genome into the daughter cells. However, how the HPV-16 E2 protein acts to facilitate partitioning of the viral genome remains unclear. In this study, we found that serine 243 of HPV-16 E2, located in the hinge region, is crucial for chromosome binding during mitosis. Bromodomain protein 4 (Brd4) has been identified as a cellular binding target through which the E2 protein of bovine papillomavirus type 1 (BPV-1) tethers the viral genome to mitotic chromosomes. Mutation analysis showed that, when the residue serine 243 was substituted by glutamic acid or aspartic acid, whose negative charges mimic the effect of constitutive phosphorylation, the protein still can interact with Brd4 and colocalize with Brd4 in condensed metaphase and anaphase chromosomes. However, substitution by the polar uncharged residues asparagine or glutamine abrogated Brd4 and mitotic chromosome binding. Moreover, following treatment with the inhibitor JQ1 to release Brd4 from the chromosomes, Brd4 and E2 formed punctate foci separate from the chromosomes, further supporting the hypothesis that the association of the HPV-16 E2 protein with the chromosomes is Brd4-dependent. In addition, the S243A E2 protein has a shorter half-life than the wild type, indicating that phosphorylation of the HPV-16 E2 protein at serine 243 also increases its half-life. Thus, phosphorylation of serine 243 in the hinge region of HPV-16 E2 is essential for interaction with Brd4 and required for host chromosome binding.
Assuntos
Proteínas de Ligação a DNA/química , Papillomavirus Humano 16/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/química , Serina/química , Fatores de Transcrição/metabolismo , Anáfase , Animais , Ácido Aspártico/química , Células COS , Proteínas de Ciclo Celular , Chlorocebus aethiops , Genoma Viral , Ácido Glutâmico/química , Glutamina/química , Células HEK293 , Humanos , Metáfase , Mutação , Fenótipo , Fosforilação , Ligação ProteicaRESUMO
We previously identified a gene, nuclear receptor-interaction protein (NRIP), which functions as a transcription cofactor in glucocorticoid receptor (GR) and human papillomavirus E2 (HPV E2)-driven gene expression. Here, we comprehensively evaluated the role of NRIP in HPV-16 gene expression. NRIP acts as a transcription cofactor to enhance GR-regulated HPV-16 gene expression in the presence of hormone. NRIP also can form complex with E2 that caused NRIP-induced HPV gene expression via E2-binding sites in a hormone-independent manner. Furthermore, NRIP can associate with GR and E2 to form tri-protein complex to activate HPV gene expression via GRE, not the E2-binding site, in a hormone-dependent manner. These results indicate that NRIP and GR are viral E2-binding proteins and that NRIP regulates HPV gene expression via GRE and/or E2 binding site in the HPV promoter in a hormone-dependent or independent manner, respectively.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Papillomavirus Humano 16/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular , Proteínas de Ligação a DNA/genética , Papillomavirus Humano 16/genética , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Ligação Proteica , Receptores de Glucocorticoides/genética , Transcrição Gênica , Regulação para CimaRESUMO
Damaged DNA-binding protein 2 (DDB2), a protein that binds damaged DNA, is a DDB1 and CUL4-associated factor. This study is the first to demonstrate that DDB2 is a novel androgen receptor (AR)-interacting protein; and mediating contact with AR and CUL4A-DDB1 complex for AR ubiquitination/degradation. DNA damage induces both p53 and DDB2 gene expression those two can inhibit AR expression. The former reduces AR via transcription regulation but the latter via proteosome degradation. Thereby DDB2 can inhibit cell growth rate in AR-expressing cells (LNCaP) but not in AR-null cells (PC3). Hence DDB2 may be a potential regimen for prostate cancer treatment, especially in androgen-refractory patients harboring high amount of AR who cannot be cured by androgen ablation.