RESUMEN
Kidney injury molecule-1 (KIM-1) is a phosphatidylserine receptor that is specifically upregulated on proximal tubular epithelial cells (PTECs) during acute kidney injury and mitigates tissue damage by mediating efferocytosis (the phagocytic clearance of apoptotic cells). The signaling molecules that regulate efferocytosis in TECs are not well understood. Using a yeast two-hybrid screen, we identified the dynein light chain protein, Tctex-1, as a novel KIM-1-interacting protein. Immunoprecipitation and confocal imaging studies suggested that Tctex-1 associates with KIM-1 in cells at baseline, but, dissociates from KIM-1 within 90 min of initiation of efferocytosis. Interfering with actin or microtubule polymerization interestingly prevented the dissociation of KIM-1 from Tctex-1. Moreover, the subcellular localization of Tctex-1 changed from being microtubule-associated to mainly cytosolic upon expression of KIM-1. Short hairpin RNA-mediated silencing of endogenous Tctex-1 in cells significantly inhibited efferocytosis to levels comparable to that of knock down of KIM-1 in the same cells. Importantly, Tctex-1 was not involved in the delivery of KIM-1 to the cell-surface. On the other hand, KIM-1 expression significantly inhibited the phosphorylation of Tctex-1 at threonine 94 (T94), a post-translational modification which is known to disrupt the binding of Tctex-1 to dynein on microtubules. In keeping with this, we found that KIM-1 bound less efficiently to the phosphomimic (T94E) mutant of Tctex-1 compared to wild type Tctex-1. Surprisingly, expression of Tctex-1 T94E did not influence KIM-1-mediated efferocytosis. Our studies uncover a previously unknown role for Tctex-1 in KIM-1-dependent efferocytosis in epithelial cells.
Asunto(s)
Lesión Renal Aguda/metabolismo , Dineínas/metabolismo , Receptor Celular 1 del Virus de la Hepatitis A/metabolismo , Fagocitosis/fisiología , Actinas/metabolismo , Células Epiteliales/metabolismo , Humanos , Riñón/metabolismo , Microtúbulos/metabolismo , Fosforilación , Transducción de Señal/fisiologíaRESUMEN
Omi/HtrA2 is a nuclear encoded mitochondrial serine protease with dual and opposite functions that depend entirely on its subcellular localization. During apoptosis, Omi/HtrA2 is released into the cytoplasm where it participates in cell death. While confined in the inter-membrane space of the mitochondria, Omi/HtrA2 has a pro-survival function that may involve the regulation of protein quality control (PQC) and mitochondrial homeostasis. Loss of Omi/HtrA2's protease activity causes the neuromuscular disorder of the mnd2 (motor neuron degeneration 2) mutant mice. These mice develop multiple defects including neurodegeneration with parkinsonian features. Loss of Omi/HtrA2 in non-neuronal tissues has also been shown to cause premature aging. The normal function of Omi/HtrA2 in the mitochondria and how its deregulation causes neurodegeneration or premature aging are unknown. Here we report that the mitochondrial Mulan E3 ubiquitin ligase is a specific substrate of Omi/HtrA2. During exposure to H(2)O(2), Omi/HtrA2 degrades Mulan, and this regulation is lost in cells that carry the inactive protease. Furthermore, we show accumulation of Mulan protein in various tissues of mnd2 mice as well as in Omi/HtrA2(-/-) mouse embryonic fibroblasts (MEFs). This causes a significant decrease of mitofusin 2 (Mfn2) protein, and increased mitophagy. Our work describes a new stress-signaling pathway that is initiated in the mitochondria and involves the regulation of Mulan by Omi/HtrA2 protease. Deregulation of this pathway, as it occurs in mnd2 mutant mice, causes mitochondrial dysfunction and mitophagy, and could be responsible for the motor neuron disease and the premature aging phenotype observed in these animals.
Asunto(s)
Fibroblastos/metabolismo , GTP Fosfohidrolasas/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Mitofagia/genética , Serina Endopeptidasas/genética , Ubiquitina-Proteína Ligasas/genética , Envejecimiento Prematuro/genética , Envejecimiento Prematuro/metabolismo , Animales , Apoptosis , Línea Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Fibroblastos/patología , GTP Fosfohidrolasas/deficiencia , Regulación de la Expresión Génica , Células HEK293 , Serina Peptidasa A2 que Requiere Temperaturas Altas , Humanos , Ratones , Ratones Noqueados , Mitocondrias/patología , Proteínas Mitocondriales/deficiencia , Enfermedad de la Neurona Motora/genética , Enfermedad de la Neurona Motora/metabolismo , Enfermedad de la Neurona Motora/patología , Estrés Oxidativo , Transporte de Proteínas , Serina Endopeptidasas/deficiencia , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Obesity is a growing epidemic affecting millions of people worldwide and a major risk factor for a multitude of chronic diseases and premature mortality. Accumulating evidence suggests that mitochondria have a profound role in diet-induced obesity and the associated metabolic changes, but the molecular mechanisms linking mitochondria to obesity remain poorly understood. Our studies have identified a new function for mitochondrial MUL1 E3 ubiquitin ligase, a protein known to regulate mitochondrial dynamics and mitophagy, in the control of energy metabolism and lipogenesis. Genetic deletion of Mul1 in mice impedes mitophagy and presents a metabolic phenotype that is resistant to high-fat diet (HFD)-induced obesity and metabolic syndrome. Several metabolic and lipidomic pathways are perturbed in the liver and white adipose tissue (WAT) of Mul1(-/-) animals on HFD, including the one driven by Stearoyl-CoA Desaturase 1 (SCD1), a pivotal regulator of lipid metabolism and obesity. In addition, key enzymes crucial for lipogenesis and fatty acid oxidation such as ACC1, FASN, AMPK, and CPT1 are also modulated in the absence of MUL1. The concerted action of these enzymes, in the absence of MUL1, results in diminished fat storage and heightened fatty acid oxidation. Our findings underscore the significance of MUL1-mediated mitophagy in regulating lipogenesis and adiposity, particularly in the context of HFD. Consequently, our data advocate the potential of MUL1 as a therapeutic target for drug development in the treatment of obesity, insulin resistance, NAFLD, and cardiometabolic diseases.
RESUMEN
Abro1 (Abraxas brother 1), also known as KIAA0157, is a scaffold protein that recruits various polypeptides to assemble the BRISC (BRCC36 isopeptide) deubiquitinating enzyme (DUB) complex. The BRISC enzyme has a Lys63-linked deubiquitinating activity and is comprised of four known subunits: MERIT40 (mediator of Rap80 interactions and targeting 40kDa), BRE (brain and reproductive organ-expressed), BRCC36 (BRCA1/BRCA2-containing complex, subunit 3) and Abro1. We have previously shown that Abro1 has a cytoprotective role that involves the BRISC DUB complex acting on specific Lys63-linked polyubiquitinated substrates. In this report we identify three members of the AP-1 (activating protein-1) family, the ATF4, ATF5 (activating transcription factor) and JunD proteins, as specific interactors of Abro1. The function of ATF4-Abro1 interaction was investigated under normal conditions as well as under cellular stress. Abro1 is predominantly cytoplasmic, but during cellular stress it enters the nucleus and co-localizes with ATF4. Furthermore, this interaction with ATF4 is necessary and essential for the cytoprotective function of Abro1 following oxidative stress. The ability of Abro1 to specifically interact with a number of transcription factors suggests a new mechanism of regulation of the BRISC DUB complex. This regulation involves the participation of at least three known members of the AP-1 family of transcription factors.
Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Factores de Transcripción Activadores/metabolismo , Núcleo Celular/metabolismo , Citoprotección/fisiología , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Secuencia de Aminoácidos , Antivirales/farmacología , Núcleo Celular/efectos de los fármacos , Células Cultivadas , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Citoprotección/efectos de los fármacos , Humanos , Riñón/citología , Riñón/metabolismo , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Fracciones Subcelulares , Tunicamicina/farmacología , Técnicas del Sistema de Dos Híbridos , Proteasas Ubiquitina-Específicas , UbiquitinaciónRESUMEN
MUL1 is a multifunctional E3 ubiquitin ligase that is involved in various pathophysiological processes including apoptosis, mitophagy, mitochondrial dynamics, and innate immune response. We uncovered a new function for MUL1 in the regulation of mitochondrial metabolism. We characterized the metabolic phenotype of MUL1(-/-) cells using metabolomic, lipidomic, gene expression profiling, metabolic flux, and mitochondrial respiration analyses. In addition, the mechanism by which MUL1 regulates metabolism was investigated, and the transcription factor HIF-1α, as well as the serine/threonine kinase Akt2, were identified as the mediators of the MUL1 function. MUL1 ligase, through K48-specific polyubiquitination, regulates both Akt2 and HIF-1α protein level, and the absence of MUL1 leads to the accumulation and activation of both substrates. We used specific chemical inhibitors and activators of HIF-1α and Akt2 proteins, as well as Akt2(-/-) cells, to investigate the individual contribution of HIF-1α and Akt2 proteins to the MUL1-specific phenotype. This study describes a new function of MUL1 in the regulation of mitochondrial metabolism and reveals how its downregulation/inactivation can affect mitochondrial respiration and cause a shift to a new metabolic and lipidomic state.
RESUMEN
Abro1 (also known as KIAA0157) is a scaffold protein that recruits polypeptides to assemble the BRISC (BRCC36-containing isopeptidase complex) deubiquitinating (DUB) enzyme. The four subunits of BRISC enzyme include Abro1, NBA1, BRE, and BRCC36 proteins. The DUB activity of the BRISC enzyme is exclusively directed against Lys63-linked polyubiquitin that does not have a proteolytic role but regulates protein function. In this report, we identified Abro1 as a specific interactor of THAP5, a zinc finger transcription factor that is involved in G2/M control and apoptosis. Abro1 was predominantly expressed in the heart and its protein level was regulated following experimentally induced myocardial ischemia/reperfusion (MI/R) injury. Furthermore, in patients with coronary artery disease (CAD), there was a dramatic increase in Abro1 protein level in the myocardial infarction (MI) area. Increase in Abro1 leads to a significant reduction in Lys63-linked ubiquitination of specific protein targets. Reducing the Abro1 protein level exacerbated cellular damage and cell death of cardiomyocytes due to MI/R injury. Additionally, overexpression of Abro1 in a heterologous system provided significant protection against oxidative stress-induced apoptosis. In conclusion, our results demonstrate that Abro1 protein level substantially increases in myocardial injury and coronary artery disease and this up-regulation is part of a novel cardioprotective mechanism. In addition, our data suggest a potential new link between Lys63-specific ubiquitination, its modulation by the BRISC DUB enzyme, and the development and progression of heart disease.
Asunto(s)
Infarto del Miocardio/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Northern Blotting , Western Blotting , Línea Celular , Células Cultivadas , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Peróxido de Hidrógeno/farmacología , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/genética , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/metabolismo , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Ratas , Ratas Sprague-Dawley , Técnicas del Sistema de Dos HíbridosRESUMEN
THAP5 was originally isolated as a specific interactor and substrate of the mitochondrial pro-apoptotic Omi/HtrA2 protease. It is a human zinc finger protein characterized by a restricted pattern of expression and the lack of orthologs in mouse and rat. The biological function of THAP5 is unknown but our previous studies suggest it could regulate G2/M transition in kidney cells and could be involved in human cardiomyocyte cell death associated with coronary artery disease (CAD). In this report, we expanded our studies on the properties and function of THAP5 in human melanoma cells. THAP5 was expressed in primary human melanocytes as well as in all melanoma cell lines that were tested. THAP5 protein level was significantly induced by UV irradiation or cisplatin treatment, conditions known to cause DNA damage. The induction of THAP5 correlated with a significant increase in apoptotic cell death. In addition, we show that THAP5 is a nuclear protein that could recognize and bind a specific DNA motif. THAP5 could also repress the transcription of a reporter gene in a heterologous system. Our work suggests that THAP5 is a DNA-binding protein and a transcriptional repressor. Furthermore, THAP5 has a pro-apoptotic function and it was induced in melanoma cells under conditions that promoted cell death.
Asunto(s)
Apoptosis , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Melanoma/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Neoplasias Cutáneas/metabolismo , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Cisplatino/farmacología , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Humanos , Melanoma/genética , Ratones , Ratas , Neoplasias Cutáneas/genética , Transcripción GenéticaRESUMEN
UBXN7 is a cofactor protein that provides a scaffold for both CRL3KEAP1 and CRL2VHL ubiquitin ligase complexes involved in the regulation of the NRF2 and HIF-1α protein levels respectively. NRF2 and HIF-1α are surveillance transcription factors that orchestrate the cellular response to oxidative stress (NRF2) or to hypoxia (HIF-1α). Since mitochondria are the main oxygen sensors as well as the principal producers of ROS, it can be presumed that they may be able to modulate the activity of CRL3KEAP1 and CRL2VHL complexes in response to stress. We have uncovered a new mechanism of such regulation that involves the UBXN7 cofactor protein and its regulation by mitochondrial MUL1 E3 ubiquitin ligase. High level of UBXN7 leads to HIF-1α accumulation, whereas low level of UBXN7 correlates with an increase in NRF2 protein. The reciprocal regulation of HIF-1α and NRF2 by UBXN7 is coordinated under conditions of oxidative stress or hypoxia. In addition, this molecular mechanism leads to different metabolic states; high level of UBXN7 and accumulation of HIF-1α support glycolysis, whereas inactivation of UBXN7 and activation of NRF2 confer increased OXPHOS. We describe a new mechanism by which MUL1 E3 ubiquitin ligase modulates the UBXN7 cofactor protein level and provides a reciprocal regulation of CRL3KEAP1 and CRL2VHL ubiquitin ligase complexes. Furthermore, we delineate how this regulation is reflected in NRF2 and HIF-1α accumulation and determines the metabolic state as well as the adaptive response to mitochondrial stress.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Hipoxia de la Célula , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glucólisis , Células HEK293 , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Fosforilación Oxidativa , Estrés Oxidativo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Kidney fibrosis, a typical characteristic of chronic renal disease, is associated with tubular epithelial cell apoptosis. The results of our recent studies have shown that Omi/HtrA2 (Omi), a proapoptotic mitochondrial serine protease, performs a crucial function in renal tubular epithelial apoptotic cell death in animal models of acute kidney injury, including cisplatin toxicity and ischemia-reperfusion insult. However, the role of Omi in tubulointerstitial disease-associated fibrosis in the kidney remains to be clearly defined. We evaluated the potential function and molecular mechanism of Omi in ureteral obstruction-induced kidney epithelial cell apoptosis and fibrosis. The mice were subjected to unilateral ureteral obstruction (UUO) via the ligation of the left ureter near the renal pelvis. UUO increased the protein level of Omi in the cytosolic fraction of the kidney, with a concomitant reduction in the mitochondrial fraction. UUO reduced the X-linked inhibitor of apoptosis protein (XIAP), a substrate of Omi, and pro-caspase-3, whereas it increased cleaved poly(ADP-ribose) polymerase (cleaved PARP) and the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells. When mice were treated with ucf-101, an inhibitor of the proteolytic activity of Omi (6.19 microg/day ip), on a daily basis beginning 2 days before UUO and continuing until the end of the experiment, the Omi inhibitor protected XIAP cleavage after UUO and reduced the increment of PARP cleavage and the numbers of TUNEL-positive cells. Furthermore, the Omi inhibitor significantly attenuated UUO-induced increases in fibrotic characteristics in the kidney, including the atrophy and dilation of tubules, expansion of the interstitium, and increases in the expression of collagens, alpha-smooth muscle actin, and fibronectin. In conclusion, Omi/HtrA2 is associated with apoptotic signaling pathways in tubular epithelial cells activated by unilateral ureteral obstruction, thereby resulting in kidney fibrosis.
Asunto(s)
Apoptosis , Enfermedades Renales/enzimología , Túbulos Renales/enzimología , Proteínas Mitocondriales/metabolismo , Serina Endopeptidasas/metabolismo , Obstrucción Ureteral/enzimología , Actinas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/enzimología , Fibrosis , Serina Peptidasa A2 que Requiere Temperaturas Altas , Etiquetado Corte-Fin in Situ , Enfermedades Renales/etiología , Enfermedades Renales/patología , Enfermedades Renales/prevención & control , Túbulos Renales/efectos de los fármacos , Túbulos Renales/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Mitocondrias/enzimología , Proteínas Mitocondriales/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Proteasas/farmacología , Pirimidinonas/farmacología , Transducción de Señal , Tionas/farmacología , Factores de Tiempo , Obstrucción Ureteral/complicaciones , Obstrucción Ureteral/tratamiento farmacológico , Obstrucción Ureteral/patología , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
MUL1 is a multifunctional E3 ubiquitin ligase anchored in the outer mitochondrial membrane with its RING finger domain facing the cytoplasm. MUL1 participates in various biological pathways involved in apoptosis, mitochondrial dynamics, and innate immune response. The unique topology of MUL1 enables it to "sense" mitochondrial stress in the intermembrane mitochondrial space and convey these signals through the ubiquitination of specific cytoplasmic substrates. We have identified UBXN7, the cofactor protein of the CRL2VHL ligase complex, as a specific substrate of MUL1 ligase. CRL2VHL ligase complex regulates HIF-1α protein levels under aerobic (normoxia) or anaerobic (hypoxia) conditions. Inactivation of MUL1 ligase leads to accumulation of UBXN7, with concomitant increase in HIF-1α protein levels, reduction in oxidative phosphorylation, and increased glycolysis. We describe a novel pathway that originates in the mitochondria and operates upstream of the CRL2VHL ligase complex. Furthermore, we delineate the mechanism by which the mitochondria, through MUL1 ligase, can inhibit the CRL2VHL complex leading to high HIF-1α protein levels and a metabolic shift to glycolysis under normoxic conditions.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Mitocondrias/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular , Línea Celular Tumoral , Glucólisis/fisiología , Células HEK293 , Células HeLa , Humanos , Dinámicas Mitocondriales/fisiología , Membranas Mitocondriales/metabolismo , Ubiquitinación/fisiologíaRESUMEN
Omi/HtrA2 is a mitochondrial serine protease that has a dual function: while confined in the mitochondria, it promotes cell survival, but when released into the cytoplasm, it participates in caspase-dependent as well as caspase-independent cell death. To investigate the mechanism of Omi/HtrA2's function, we set out to isolate and characterize novel substrates for this protease. We have identified Thanatos-associated protein 5 (THAP5) as a specific interactor and substrate of Omi/HtrA2 in cells undergoing apoptosis. This protein is an uncharacterized member of the THAP family of proteins. THAP5 has a unique pattern of expression and is found predominantly in the human heart, although a very low expression is also seen in the human brain and muscle. THAP5 protein is localized in the nucleus and, when ectopically expressed, induces cell cycle arrest. During apoptosis, THAP5 protein is degraded, and this process can be blocked using a specific Omi/HtrA2 inhibitor, leading to reduced cell death. In patients with coronary artery disease, THAP5 protein levels substantially decrease in the myocardial infarction area, suggesting a potential role of this protein in human heart disease. This work identifies human THAP5 as a cardiac-specific nuclear protein that controls cell cycle progression. Furthermore, during apoptosis, THAP5 is cleaved and removed by the proapoptotic Omi/HtrA2 protease. Taken together, we provide evidence to support that THAP5 and its regulation by Omi/HtrA2 provide a new link between cell cycle control and apoptosis in cardiomyocytes.
Asunto(s)
Apoptosis/fisiología , Enfermedad de la Arteria Coronaria/fisiopatología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Mitocondriales/metabolismo , Miocardio/enzimología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/fisiología , Núcleo Celular/enzimología , Cisplatino/farmacología , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/patología , Regulación Enzimológica de la Expresión Génica/fisiología , Células HeLa , Serina Peptidasa A2 que Requiere Temperaturas Altas , Homeostasis/fisiología , Humanos , Peróxido de Hidrógeno/farmacología , Riñón/citología , Mitocondrias Cardíacas/enzimología , Proteínas Mitocondriales/antagonistas & inhibidores , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Oxidantes/farmacología , Pirimidinonas/farmacología , ARN Mensajero/metabolismo , Especificidad por Sustrato/fisiología , Tionas/farmacología , Transfección , Técnicas del Sistema de Dos Híbridos , LevadurasRESUMEN
Resveratrol is a naturally occurring phytoalexin with antioxidant and antiinflammatory properties. Recent studies suggest that resveratrol possesses anticancer effects, although its mechanism of action is not well understood. We now show that resveratrol inhibits Src tyrosine kinase activity and thereby blocks constitutive signal transducer and activator of transcription 3 (Stat3) protein activation in malignant cells. Analyses of resveratrol-treated malignant cells harboring constitutively-active Stat3 reveal irreversible cell cycle arrest of v-Src-transformed mouse fibroblasts (NIH3T3/v-Src), human breast (MDA-MB-231), pancreatic (Panc-1), and prostate carcinoma (DU145) cell lines at the G0-G1 phase or at the S phase of human breast cancer (MDA-MB-468) and pancreatic cancer (Colo-357) cells, and loss of viability due to apoptosis. By contrast, cells treated with resveratrol, but lacking aberrant Stat3 activity, show reversible growth arrest and minimal loss of viability. Moreover, in malignant cells harboring constitutively-active Stat3, including human prostate cancer DU145 cells and v-Src-transformed mouse fibroblasts (NIH3T3/v-Src), resveratrol treatment represses Stat3-regulated cyclin D1 as well as Bcl-xL and Mcl-1 genes, suggesting that the antitumor cell activity of resveratrol is in part due to the blockade of Stat3-mediated dysregulation of growth and survival pathways. Our study is among the first to identify Src-Stat3 signaling as a target of resveratrol, further defining the mechanism of antitumor cell activity of resveratrol and raising its potential application in tumors with an activated Stat3 profile.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis , Neoplasias/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Estilbenos/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Animales , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclina D1/genética , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Resveratrol , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína bcl-X/genéticaRESUMEN
BACKGROUND: Omi/HtrA2 is a proapoptotic mitochondrial serine protease involved in caspase-dependent as well as caspase-independent cell death. However, the role of Omi/HtrA2 in the apoptotic cell death that occurs in vivo under pathological conditions remains unknown. The present study was designed to investigate whether Omi/HtrA2 plays an important role in postischemic myocardial apoptosis. METHODS AND RESULTS: Male adult mice were subjected to 30 minutes of myocardial ischemia followed by reperfusion and treated with vehicle or ucf-101, a novel and specific Omi/HtrA2 inhibitor, 10 minutes before reperfusion. Myocardial ischemia/reperfusion significantly increased cytosolic Omi/HtrA2 content and markedly increased apoptosis. Treatment with ucf-101 exerted significant cardioprotective effects, as evidenced by less terminal dUTP nick end-labeling staining, a lower incidence of DNA ladder fragmentation, and smaller infarct size. Furthermore, treatment with ucf-101 before reperfusion attenuated X-linked inhibitor of apoptosis protein degradation and inhibited caspase-9 and caspase-3 activities. CONCLUSIONS: Taken together, these results demonstrate for the first time that ischemia/reperfusion results in Omi/HtrA2 translocation from the mitochondria to the cytosol, where it promotes cardiomyocyte apoptosis via a protease activity-dependent, caspase-mediated pathway.
Asunto(s)
Apoptosis/fisiología , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/patología , Serina Endopeptidasas/fisiología , Animales , Apoptosis/efectos de los fármacos , Cardiotónicos/farmacología , Caspasa 3 , Caspasa 9 , Inhibidores de Caspasas , Caspasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Citosol/enzimología , Serina Peptidasa A2 que Requiere Temperaturas Altas , Masculino , Ratones , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/enzimología , Proteínas Mitocondriales , Infarto del Miocardio/enzimología , Daño por Reperfusión Miocárdica/enzimología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Transporte de Proteínas/efectos de los fármacos , Proteínas/antagonistas & inhibidores , Pirimidinonas/farmacología , Tionas/farmacología , Proteína Inhibidora de la Apoptosis Ligada a XRESUMEN
Omi is a mammalian serine protease that is localized in the mitochondria and released to the cytoplasm in response to apoptotic stimuli. Omi induces cell death in a caspase-dependent manner by interacting with the X-chromosome linked inhibitor of apoptosis protein, as well as in a caspase-independent way that relies on its proteolytic activity. Omi is synthesized as a precursor polypeptide and is processed to an active serine protease with a unique PDZ domain. PDZ domains recognize the extreme carboxyl terminus of target proteins. Internal peptides that are able to fold into a beta-finger are also reported to bind some PDZ domains. Using a modified yeast two-hybrid system, PDZ(Omi) mutants were isolated by their ability to bind the carboxyl terminus of human Myc oncoprotein in yeast as well as in mammalian cells. One such PDZ(m) domain (PDZ-M1), when transfected into mammalian cells, was able to bind to endogenous Myc protein and induce cell death. PDZ-M1-induced apoptosis was entirely dependent on the presence of Myc protein and was not observed when c-myc null fibroblasts were used. Our studies indicate that the PDZ domain of Omi can provide a prototype that could easily be exploited to target specifically and inactivate oncogenes by binding to their unique carboxyl terminus.
Asunto(s)
Serina Endopeptidasas/genética , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Cartilla de ADN , Células HeLa , Serina Peptidasa A2 que Requiere Temperaturas Altas , Humanos , Cinética , Proteínas Mitocondriales , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Pliegue de Proteína , Estructura Secundaria de Proteína , Proto-Oncogenes Mas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismoRESUMEN
Ischemic stroke, or a brain attack, is the third leading cause of death in developed countries. A critical feature of the disease is a highly selective pattern of neuronal loss; certain identifiable subsets of neurons--particularly CA1 pyramidal neurons in the hippocampus are severely damaged, whereas others remain intact. A key step in this selective neuronal injury is Ca2+/Zn2+ entry into vulnerable neurons through alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor channels, a principle subtype of glutamate receptors. AMPA receptor channels are assembled from glutamate receptor (GluR)1, -2, -3, and -4 subunits. Circumstance data have indicated that the GluR2 subunits dictate Ca2+/Zn2+ permeability of AMPA receptor channels and gate injurious Ca2+/Zn2+ signals in vulnerable neurons. Therefore, targeting to the AMPA receptor subunit GluR2 can be considered a practical strategy for stroke therapy.
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Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Receptores AMPA/metabolismo , Glutamatos/toxicidad , Humanos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Receptores AMPA/genética , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Mulan is an E3 ubiquitin ligase embedded in the outer mitochondrial membrane (OMM) with its RING finger facing the cytoplasm and a large domain located in the intermembrane space (IMS). Mulan is known to have an important role in cell growth, cell death, and more recently in mitophagy. The mechanism of its function is poorly understood; but as an E3 ligase it is expected to interact with specific E2 ubiquitin conjugating enzymes and these complexes will bind and ubiquitinate specific substrates. The unique topology of Mulan can provide a direct link of communicating mitochondrial signals to the cytoplasm. Our studies identified four different E2 conjugating enzymes (Ube2E2, Ube2E3, Ube2G2 and Ube2L3) as specific interactors of Mulan. Each of these E2 conjugating enzymes was fused to the RING finger domain of Mulan and used in a modified yeast two-hybrid screen. Several unique interactors for each Mulan-E2 complex were isolated. One such specific interactor of Mulan-Ube2E3 was the GABARAP (GABAA receptor-associated protein). GABARAP is a member of the Atg8 family of proteins that plays a major role in autophagy/mitophagy. The interaction of GABARAP with Mulan-Ube2E3 required an LC3-interacting region (LIR) located in the RING finger domain of Mulan as well as the presence of Ube2E3. The isolation of four different E2 conjugating enzymes, as specific partners of Mulan E3 ligase, suggests that Mulan is involved in multiple biological pathways. In addition, the interaction of GABARAP with Mulan-Ube2E3 supports the role of Mulan as an important regulator of mitophagy and provides a plausible mechanism for its function in this process.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitofagia , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Células HEK293 , Células HeLa , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Unión Proteica , Dominios RING Finger , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato , Técnicas del Sistema de Dos Híbridos , Ubiquitina-Proteína Ligasas/químicaRESUMEN
Acute renal failure (ARF) is characterized by a very high mortality essentially unchanged over the past 40 years. Simple vertebrate models are needed to improve our understanding of ARF and facilitate the development of novel therapies for this clinical syndrome. Here, we demonstrate that gentamicin, a commonly used nephrotoxic antibiotic, causes larval zebrafish to develop ARF characterized by histological and functional changes that mirror aminoglycoside toxicity in higher vertebrates and inability of zebrafish to maintain fluid homeostasis. We developed a novel method to quantitate renal function in larval zebrafish and demonstrate a decline in glomerular filtration rate after gentamicin exposure. The antineoplastic drug cisplatin, whose use in humans is limited by kidney toxicity, also causes typical histological changes and a decline in renal function in larval zebrafish. A specific inhibitor of Omi/HtrA2, a serine protease implicated in cisplatin-induced apoptosis, prevented renal failure and increased survival. This protective effect was confirmed in a mouse model of cisplatin-induced nephrotoxicity. Therefore, zebrafish provides a unique model system, amenable to genetic manipulation and drug screening, to explore the pathophysiology of ARF and establish novel therapies with potential use in mammals.
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Lesión Renal Aguda/inducido químicamente , Antibacterianos/toxicidad , Modelos Animales de Enfermedad , Gentamicinas/toxicidad , Pez Cebra , Lesión Renal Aguda/fisiopatología , Animales , Antineoplásicos/toxicidad , Cisplatino/toxicidad , Tasa de Filtración Glomerular , Serina Peptidasa A2 que Requiere Temperaturas Altas , Riñón/crecimiento & desarrollo , Riñón/patología , Riñón/fisiología , Lisosomas/metabolismo , Lisosomas/patología , Ratones , Ratones Endogámicos BALB C , Proteínas Mitocondriales , Fosfolípidos/metabolismo , Serina Endopeptidasas/metabolismoRESUMEN
Omi/HtrA2 is a mitochondrial proapoptotic serine protease that is able to induce both caspase-dependent and caspase-independent cell death. After apoptotic stimuli, Omi is released to the cytoplasm where it binds and cleaves inhibitor of apoptosis proteins. In this report, we investigated the role of Omi in renal cell death following cisplatin treatment. Using primary mouse proximal tubule cells, as well as established renal cell lines, we show that the level of Omi protein is upregulated after treatment with cisplatin. This upregulation is followed by the release of Omi from mitochondria to the cytoplasm and degradation of XIAP. Reducing the endogenous level of Omi protein using RNA interference renders renal cells resistant to cisplatin-induced cell death. Furthermore, we show that the proteolytic activity of Omi is necessary and essential for cisplatin-induced cell death in this system. When renal cells are treated with Omi's specific inhibitor, ucf-101, they become significantly resistant to cisplatin-induced cell death. Ucf-101 was also able to minimize cisplatin-induced nephrotoxic injury in animals. Our results demonstrate that Omi is a major mediator of cisplatin-induced cell death in renal cells and suggest a way to limit renal injury by specifically inhibiting its proteolytic activity.
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Antineoplásicos/toxicidad , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Cisplatino/toxicidad , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/patología , Serina Endopeptidasas/farmacología , Animales , Técnicas de Cultivo de Célula , Serina Peptidasa A2 que Requiere Temperaturas Altas , Humanos , Túbulos Renales Proximales/citología , Ratones , Ratones Endogámicos C57BL , Proteínas Mitocondriales , Proteínas/metabolismoRESUMEN
Omi/HtrA2 is a nuclear-encoded mitochondrial serine protease that has a pro-apoptotic function in mammalian cells. Upon induction of apoptosis, Omi translocates to the cytoplasm and participates in caspase-dependent apoptosis by binding and degrading inhibitor of apoptosis proteins. Omi can also initiate caspase-independent apoptosis in a process that relies entirely on its ability to function as an active protease. To investigate the mechanism of Omi-induced apoptosis, we set out to isolate novel substrates that are cleaved by this protease. We identified HS1-associated protein X-1 (HAX-1), a mitochondrial anti-apoptotic protein, as a specific Omi interactor that is cleaved by Omi both in vitro and in vivo. HAX-1 degradation follows Omi activation in cells treated with various apoptotic stimuli. Using a specific inhibitor of Omi, HAX-1 degradation is prevented and cell death is reduced. Cleavage of HAX-1 was not observed in a cell line derived from motor neuron degeneration 2 mice that carry a mutated form of Omi that affects its proteolytic activity. Degradation of HAX-1 is an early event in the apoptotic process and occurs while Omi is still confined in the mitochondria. Our results suggest that Omi has a unique pro-apoptotic function in mitochondria that involves removal of the HAX-1 anti-apoptotic protein. This function is distinct from its ability to activate caspase-dependent apoptosis in the cytoplasm by degrading inhibitor of apoptosis proteins.
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Apoptosis/fisiología , Proteínas/metabolismo , Serina Endopeptidasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Serina Peptidasa A2 que Requiere Temperaturas Altas , Humanos , Péptidos y Proteínas de Señalización Intracelular , Potenciales de la Membrana/fisiología , Ratones , Mitocondrias/metabolismo , Proteínas Mitocondriales , Técnicas del Sistema de Dos HíbridosRESUMEN
Omi/HtrA2 is a mammalian serine protease with high homology to bacterial HtrA chaperones. Omi/HtrA2 is localized in mitochondria and is released to the cytoplasm in response to apoptotic stimuli. Omi/HtrA2 induces cell death in a caspase-dependent manner by interacting with the inhibitor of apoptosis protein as well as in a caspase-independent manner that relies on its protease activity. We describe the identification and characterization of a novel compound as a specific inhibitor of the proteolytic activity of Omi/HtrA2. This compound (ucf-101) was isolated in a high throughput screening of a combinatorial library using bacterially made Omi-(134-458) protease and fluorescein-casein as a generic substrate. ucf-101 showed specific activity against Omi/HtrA2 and very little activity against various other serine proteases. This compound has a natural fluorescence that was used to monitor its ability to enter mammalian cells. ucf-101, when tested in caspase-9 (-/-) null fibroblasts, was found to inhibit Omi/HtrA2-induced cell death.