RESUMEN

Biomolecular condensates (BCs) are formed by proteins with intrinsically disordered regions (IDRs) via liquid-liquid phase separation. Mieap/Spata18, a p53-inducible protein, participates in suppression of colorectal tumors by promoting mitochondrial quality control. However, the regulatory mechanism involved remains unclear. Here, we report that Mieap is an IDR-containing protein that drives formation of BCs involved in cardiolipin metabolism. Mieap BCs specifically phase separate the mitochondrial phospholipid, cardiolipin. Mieap directly binds to cardiolipin in vitro. Lipidomic analysis of cardiolipin suggests that Mieap promotes enzymatic reactions in cardiolipin biosynthesis and remodeling. Accordingly, four cardiolipin biosynthetic enzymes, TAMM41, PGS1, PTPMT1, and CRLS1 and two remodeling enzymes, PLA2G6 and TAZ, are phase-separated by Mieap BCs. Mieap-deficient cells exhibit altered crista structure, leading to decreased respiration activity and ATP production in mitochondria. These results suggest that Mieap may form membrane-less organelles to compartmentalize and facilitate cardiolipin metabolism, thus potentially contributing to mitochondrial quality control.

Asunto(s)

Mitocondrias , Enfermedades Mitocondriales , Humanos , Mitocondrias/genética , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/terapia , ADN Mitocondrial

RESUMEN

Cathepsin D (CTSD) is a major lysosomal protease harboring an N-terminal signal peptide (amino acids 1-20) to enable vesicular transport from endoplasmic reticulum to lysosomes. Here, we report the possibility of a mitochondrial targeting sequence and mitochondrial localization of CTSD in cells. Live-cell imaging analysis with C-terminal enhanced green fluorescent protein-tagged CTSD (EGFP-CTSD) indicated that CTSD localizes to mitochondria. CTSD amino acids 21-35 are responsible for its mitochondrial localization, which exhibit typical features of mitochondrial targeting sequences, and are evolutionarily conserved. A proteinase K protection assay and sucrose gradient analysis showed that a small population of endogenous CTSD molecules exists in mitochondria. These results suggest that CTSD is a dual-targeted protein that may localize in both lysosomes and mitochondria.

Asunto(s)

Catepsina D , Mitocondrias , Catepsina D/genética , Catepsina D/metabolismo , Mitocondrias/metabolismo , Péptido Hidrolasas/metabolismo

RESUMEN

Mitochondria-eating protein (MIEAP) is a molecule important for non-canonical mitophagy and thought to be a tumor suppressor. Our previous study found that MIEAP expression is defective in thyroid oncocytomas, irrespective of being benign or malignant, and also in non-oncocytic thyroid cancers. Thyroid oncocytomas are composed of large polygonal cells with eosinophilic cytoplasm that is rich in abnormal mitochondria. Thus, our data indicate that, together with increased mitochondrial biogenesis that compensates for the dysfunction of the mitochondria, MIEAP plays a critical role in the accumulation of mitochondria in thyroid oncocytic tumors, whereas a defective MIEAP expression alone is not sufficient for mitochondrial accumulation in non-oncocytic cancers with normal mitochondria. To clarify whether MIEAP is a tumor suppressor in the thyroids and whether MIEAP knockout (KO) alone is sufficient for the oncocytic phenotype and also to extend our effort toward canonical mitophagy (a selective autophagy), we here conducted mouse studies using genetically engineered mice. BrafCA/wt mice developed thyroid cancers 1 year after intrathyroidal injection of adenovirus expressing Cre, while cancer development was observed at 6 months in adenovirus-Cre-injected BrafCA/wt;MieapKO/KO and BrafCA/wt;Atg5flox/flox mice [where autophagy-related 5 (ATG5) is a component of autophagic machinery], although KO of either molecule alone was not sufficient for cancer development. These data demonstrate that MIEAP or ATG5 KO accelerated thyroid cancer development. However, cancers in adenovirus-Cre-injected BrafCA/wt ;MieapKO/KO and BrafCA/wt ;Atg5flox/flox mice were not oncocytic. In conclusion, we here show that MIEAP and ATG5 are both tumor suppressors in thyroid carcinogenesis, but as we have anticipated from our previous data, KO of either molecule does not confer the oncocytic phenotype to BRAFV600E-positive thyroid cancers. The combination of disruptive mitochondrial function and impaired mitochondrial quality control may be necessary to establish a mouse model of thyroid oncocytoma.

Asunto(s)

Adenoma Oxifílico , Proteínas Mitocondriales , Neoplasias de la Tiroides , Animales , Ratones , Adenoma Oxifílico/patología , Autofagia/genética , Proteína 5 Relacionada con la Autofagia/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Mitofagia/genética , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias de la Tiroides/patología

RESUMEN

Mitochondria-eating protein (MIEAP; also known as SPATA18), a p53-downstream gene, is involved in mitochondrial quality control (MQC). Enforced MIEAP expression induces caspase-dependent cell death in vitro, and impairment of the p53/MIEAP-regulated MQC pathway is frequently observed in breast cancer (BC), resulting in poor disease-free survival (DFS). To investigate the clinical significance of MIEAP in BC, we identified 2,980 patients from two global, large-scale primary BC cohorts: the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC; n=1,904) and the Cancer Genome Atlas (TCGA; n=1,076). We divided patients in each cohort into high and low groups based on median gene expression levels and analyzed the association between MIEAP expression and clinical outcomes. Compared with normal tumors, MIEAP expression was significantly downregulated in all patients with p53-mutant BC regardless of subtype. MIEAP expression was negatively correlated with KI67 expression. Gene set enrichment analysis demonstrated that cell cycle- and proliferation-associated gene sets were significantly enriched in MIEAP-low tumors compared to MIEAP-high tumors. Patients with MIEAP-high luminal subtype were associated with significantly longer DFS than those with MIEAP-low luminal tumors in both cohorts, whereas significantly longer overall survival was observed only in the METABRIC cohort, which has roughly double the number of samples. These results indicated that the mechanistic role of MIEAP is clinically relevant in the two independent cohorts. This is the first study to use large cohorts to demonstrate the association between MIEAP expression and survival in patients with luminal subtype BC.

RESUMEN

Mitochondria-eating protein (Mieap) plays a critical role in mitochondrial quality control (MQC) and functions as a p53-inducible tumor suppressor. This study aimed to examine its role in gastric cancer (GC) and esophageal cancer (EC). GC cells were infected with Mieap-overexpressing adenovirus (Ad-Mieap) and subjected to fluorescence-activated cell sorting (FACS), western blotting, and caspase assays. Thereafter, we evaluated the potential disruption of the p53/Mieap-regulated MQC pathway in vivo. Methylation-specific PCR (MSP) for Mieap, NIX, and BNIP3 promoters was performed and p53 mutations were detected using cryopreserved surgical specimens. Exogenous Mieap in GC cells induced the formation of vacuole-like structures (called MIVs, Mieap-induced vacuoles) and caspase-dependent cell death, with the activation of both caspase-3 and caspase-9. Of the 47 GC patients, promoter methylation in Mieap, BNIP3, and NIX was identified in two (4.3%), 29 (61.7%), and zero (0%) specimens, respectively. In total, 33 GC patients (70.2%) inactivated this MQC pathway. Amazingly, BNIP3 promoter in the normal epithelium was highly methylated in 18 of the 47 GC patients (38.3%). In EC patients, this MQC pathway was also inactivated in ten of 12 patients (83.3%). These results indicate that p53/Mieap-regulated MQC plays an important role in upper gastrointestinal (GI) tumor suppression, possibly, in part, through the mitochondrial apoptotic pathway.

Asunto(s)

Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Neoplasias Gástricas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Anciano , Apoptosis/genética , Caspasas/metabolismo , Línea Celular Tumoral , Metilación de ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Mitocondrias/genética , Proteínas Mitocondriales/genética , Mutación , Regiones Promotoras Genéticas/genética , Transducción de Señal/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética

RESUMEN

Kaposi's sarcoma-associated herpesvirus (KSHV) causes both AIDS-related Kaposi's sarcoma (KS) and classic KS, but their clinical presentations are different, and respective mechanisms remain to be elucidated. The KSHV K1 gene is reportedly involved in tumorigenesis through the immunoreceptor tyrosine-based activation motif (ITAM). Since we found the sequence variations in the K1 gene of KSHV isolated from AIDS-related KS and classic KS, we hypothesized that the transformation activity of the K1 gene contributes to the different clinical presentations. To evaluate our hypothesis, we compared the transformation activities of the K1 gene between AIDS-related KS and classic KS. We also analyzed ITAM activities and the downstream AKT and NF-κB. We found that the transformation activity of AIDS-related K1 was greater than that of classic K1, and that AIDS-related K1 induced higher ITAM activity than classic K1, causing more potent Akt and NF-κB activities. K1 downregulation by siRNA in AIDS-related K1 expressing cells induced a loss of transformation properties and decreased both Akt and NF-κB activities, suggesting a correlation between the transformation activity of K1 and ITAM signaling. Our study indicates that the increased transformation activity of AIDS-related K1 is associated with its clinical aggressiveness, whereas the weak transformation activity of classic type K1 is associated with a mild clinical presentation and spontaneous regression. The mechanism of spontaneous regression of classic KS may provide new therapeutic strategy to cancer.

Asunto(s)

Infecciones Oportunistas Relacionadas con el SIDA/genética , Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica , Herpesvirus Humano 8/genética , Interacciones Huésped-Patógeno/genética , Sarcoma de Kaposi/genética , Neoplasias Cutáneas/genética , Proteínas Virales/genética , Infecciones Oportunistas Relacionadas con el SIDA/diagnóstico , Infecciones Oportunistas Relacionadas con el SIDA/patología , Infecciones Oportunistas Relacionadas con el SIDA/virología , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Fibroblastos/metabolismo , Fibroblastos/virología , Células HeLa , Herpesvirus Humano 8/crecimiento & desarrollo , Herpesvirus Humano 8/patogenicidad , Humanos , Ratones , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Remisión Espontánea , Sarcoma de Kaposi/diagnóstico , Sarcoma de Kaposi/patología , Sarcoma de Kaposi/virología , Índice de Severidad de la Enfermedad , Transducción de Señal , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/virología , Transformación Genética , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/metabolismo

RESUMEN

Mitochondria-eating protein (Mieap), encoded by a p53-target gene, plays an important role in mitochondrial quality control (MQC). Mieap has been reported to have a critical role in tumor suppression in colorectal cancer. Here, we investigated its role as a tumor suppressor in breast cancer. The enforced expression of exogenous Mieap in breast cancer cells induced caspase-dependent apoptosis, with activation of both caspase-3/7 and caspase-9. Immunohistochemistry revealed endogenous Mieap in the cytoplasm in 24/75 (32%) invasive ductal carcinomas (IDC), 15/27 (55.6%) cases of ductal carcinoma in situ (DCIS) and 16/18 (88.9%) fibroadenomas (FA) (IDC vs DCIS; P = 0.0389, DCIS vs FA; P = 0.0234, IDC vs FA; P < 0.0001). In IDC, the Mieap promoter was methylated in 6/46 (13%) cases, whereas p53 was mutated in 6/46 (13%) cases. Therefore, the p53/Mieap-regulated MQC pathway was inactivated in 12/46 IDC (26.1%). Interestingly, all tumors derived from the 12 patients with Mieap promoter methylation or p53 mutations pathologically exhibited more aggressive and malignant breast cancer phenotypes. Impairment of p53/Mieap-regulated MQC pathway resulted in significantly shorter disease-free survival (DFS) (P = 0.021), although p53 status is more prognostic in DFS than Mieap promoter methylation. These results indicate that p53/Mieap-regulated MQC has a critical role in tumor suppression in breast cancer, possibly in part through mitochondrial apoptotic pathway.

Asunto(s)

Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis , Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Caspasas/metabolismo , Línea Celular Tumoral , Citoplasma/metabolismo , Metilación de ADN , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Persona de Mediana Edad , Mitocondrias/metabolismo , Mutación , Regiones Promotoras Genéticas

RESUMEN

Oncogenic mutations of RAS genes, found in about 30% of human cancers, are considered to play important roles in cancer development. However, oncogenic RAS can also induce senescence in mouse and human normal fibroblasts. In some cell lines, oncogenic RAS has been reported to induce non-apoptotic programed cell death (PCD). Here, we investigated effects of oncogenic RAS expression in several types of normal human epithelial cells. Oncogenic RAS but not wild-type RAS stimulated macropinocytosis with accumulation of large-phase lucent vacuoles in the cytoplasm, subsequently leading to cell death which was indistinguishable from a recently proposed new type of PCD, methuosis. A RAC1 inhibitor suppressed accumulation of macropinosomes and overexpression of MYC attenuated oncogenic RAS-induced such accumulation, cell cycle arrest and cell death. MYC suppression or rapamycin treatment in some cancer cell lines harbouring oncogenic mutations in RAS genes induced cell death with accumulation of macropinosomes. These results suggest that this type of non-apoptotic PCD is a tumour-suppressing mechanism acting against oncogenic RAS mutations in normal human epithelial cells, which can be overcome by MYC overexpression, raising the possibility that its induction might be a novel approach to treatment of RAS-mutated human cancers.

Asunto(s)

Muerte Celular/genética , Células Epiteliales/patología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas ras/genética , Línea Celular Tumoral , Células Cultivadas , Humanos , Neoplasias/genética , Neoplasias/patología , Proteína de Unión al GTP rac1/metabolismo

RESUMEN

Human angiosarcoma is a rare malignant vascular tumor associated with extremely poor clinical outcome and generally arising in skin of the head and neck region. However, little is known about the molecular pathogeneses and useful immunohistochemical markers of angiosarcoma. To investigate the mechanisms of angiosarcoma progression, we collected 85 cases of human angiosarcoma specimens with clinical records and analyzed ISO-HAS-B patient-derived angiosarcoma cells. As control subjects, 54 cases of hemangioma and 34 of pyogenic granuloma were collected. Remarkably, consistent with our recent observations regarding the involvement of survivin expression following Hippo pathway inactivation in the neoplastic proliferation of murine hemangioendothelioma cells and human infantile hemangioma, nuclear survivin expression was observed in all cases of angiosarcoma but not in hemangiomas and pyogenic granulomas, and the Hippo pathway was inactivated in 90.3% of yes-associated protein (YAP) -positive angiosarcoma cases. However, survivin expression modes and YAP localization (Hippo pathway activation modes) were not correlated with survival. In addition, we confirmed that survivin small interference RNA (siRNA) transfection and YM155, an anti-survivin drug, elicited decreased nuclear survivin expression and cell proliferation in ISO-HAS-B cells which expressed survivin consistently. Conclusively, these findings support the importance of survivin as a good marker and critical regulator of cellular proliferation for human angiosarcoma and YM155 as a potential therapeutic agent.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/genética , Biomarcadores de Tumor/genética , Hemangiosarcoma/genética , Proteínas Inhibidoras de la Apoptosis/genética , Fosfoproteínas/genética , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hemangiosarcoma/patología , Vía de Señalización Hippo , Humanos , Imidazoles , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Masculino , Persona de Mediana Edad , Naftoquinonas , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/genética , Survivin , Factores de Transcripción , Proteínas Señalizadoras YAP

RESUMEN

The tumor suppressor p53 gene is frequently mutated in human cancers, and the p53 protein suppresses cancer. However, the mechanism behind the p53-mediated tumor suppression is still unclear. Recently, the mitochondria-eating protein (Mieap) was identified as a p53-inducible protein. Mieap induces the accumulation of lysosomal proteins within mitochondria (Mieap-induced accumulation of lysosome-like organelles within mitochondria, or MALM) in response to mitochondrial damage, and eliminates the oxidized mitochondrial proteins to repair unhealthy mitochondria. Furthermore, Mieap also induces vacuole-like structures (Mieap-induced vacuole, or MIV) to eat and degrade unhealthy mitochondria. Therefore, Mieap controls mitochondrial quality by repairing or eliminating unhealthy mitochondria by MALM or MIV, respectively. This mechanism is not mediated by canonical autophagy. Mieap-deficient ApcMin/+ mice show strikingly high rates of intestinal tumor development as well as advanced-grade adenomas and adenocarcinomas. The p53/Mieap/BCL2 interacting protein 3 mitochondrial quality control pathway is frequently inactivated in human colorectal cancers. Defects in Mieap-regulated mitochondrial quality control lead to accumulation of unhealthy mitochondria in cancer cells. Cancer-specific unhealthy mitochondria could contribute to cancer development and aggressiveness through mitochondrial reactive oxygen species and altered metabolism. Mieap-regulated mitochondrial quality control is a newly discovered function of p53 that plays a critical role in tumor suppression.

Asunto(s)

Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Neoplasias/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Autofagia/fisiología , Humanos , Lisosomas/metabolismo , Oxidación-Reducción

RESUMEN

Cholangiocarcinoma is a relatively rare cancer, but its incidence is increasing worldwide. Although several risk factors have been suggested, the etiology and pathogenesis of the majority of cholangiocarcinomas remain unclear. Recently, a high incidence of early-onset cholangiocarcinoma was reported among the workers of a printing company in Osaka, Japan. These workers underwent high exposure to organic solvents, mainly haloalkanes such as 1,2-dichloropropane (1,2-DCP) and/or dichloromethane. We performed whole-exome analysis on four cases of cholangiocarcinoma among the printing workers. An average of 44.8 somatic mutations was detected per Mb in the genome of the printing workers' cholangiocarcinoma tissues, approximately 30-fold higher than that found in control common cholangiocarcinoma tissues. Furthermore, C:G-to-T:A transitions with substantial strand bias as well as unique trinucleotide mutational changes of GpCpY to GpTpY and NpCpY to NpTpY or NpApY were predominant in all of the printing workers' cholangiocarcinoma genomes. These results were consistent with the epidemiological observation that they had been exposed to high concentrations of chemical compounds. Whole-genome analysis of Salmonella typhimurium strain TA100 exposed to 1,2-DCP revealed a partial recapitulation of the mutational signature in the printing workers' cholangiocarcinoma. Although our results provide mutational signatures unique to occupational cholangiocarcinoma, the underlying mechanisms of the disease should be further investigated by using appropriate model systems and by comparison with genomic data from other cancers.

Asunto(s)

Colangiocarcinoma/epidemiología , Colangiocarcinoma/genética , Exoma/genética , Exposición Profesional , Adulto , Colangiocarcinoma/inducido químicamente , Colangiocarcinoma/patología , Exoma/efectos de los fármacos , Humanos , Japón/epidemiología , Masculino , Cloruro de Metileno/toxicidad , Mutación/efectos de los fármacos , Impresión , Propano/análogos & derivados , Propano/toxicidad , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética

RESUMEN

Mieap, a novel p53-inducible protein, plays a key role in maintaining healthy mitochondria in various pathophysiological states. Here, we show that Mieap deficiency in Apc(Min/+) mice is strikingly associated with the malignant progression of murine intestinal tumors. To understand the role that Mieap plays in in vivo tumorigenesis, we generated Mieap heterozygous (Apc(Min/+) Mieap(+/-)) and homozygous (Apc(Min/+) Mieap(-/-)) Apc(Min/+) mice. Interestingly, the Apc(Min/+) mice with the Mieap(+/-) and Mieap(-/-) genetic background revealed remarkable shortening of the lifetime compared to Apc(Min/+) mice because of severe anemia. A substantial increase in the number and size of intestinal polyps was associated with Mieap gene deficiency. Histopathologically, intestinal tumors in the Mieap-deficient Apc(Min/+) mice clearly demonstrated advanced grades of adenomas and adenocarcinomas. We demonstrated that the significant increase in morphologically unhealthy mitochondria and trace accumulations of reactive oxygen species may be mechanisms underlying the increased malignant progression of the intestinal tumors of Mieap-deficient Apc(Min/+) mice. These findings suggest that the Mieap-regulated mitochondrial quality control plays a critical role in preventing mouse intestinal tumorigenesis.

Asunto(s)

Neoplasias Intestinales/patología , Mitocondrias/patología , Proteínas Mitocondriales/fisiología , Animales , Humanos , Ratones

RESUMEN

Spontaneous regression of neuroblastoma (NB) resembles the developmentally regulated programmed cell death (PCD) of sympathetic neurons. Regressing tumor cells express high levels of the nerve growth factor (NGF) receptors TRKA and p75NTR and are dependent on NGF for survival; however, the underlying molecular mechanism remains elusive. Here, we show that UNC5D, a dependence receptor that is directly targeted by p53 family members, is highly expressed in favorable NBs. NGF withdrawal strongly upregulated UNC5D, E2F1, and p53 in human primary favorable NBs. The induced UNC5D was cleaved by caspases 2/3, and the released intracellular fragment translocated into the nucleus and interacted with E2F1 to selectively transactivate the proapoptotic target gene. The cleavage of UNC5D and its induction of apoptosis were strongly inhibited by addition of netrin-1. Unc5d(-/-) mice consistently exhibited a significant increase in dorsal root ganglia neurons and resistance to NGF depletion-induced apoptosis in sympathetic neurons compared with wild-type cells. Our data suggest that UNC5D forms a positive feedback loop with p53 and E2F1 to promote NGF dependence-mediated PCD during NB regression.

Asunto(s)

Regresión Neoplásica Espontánea , Factor de Crecimiento Nervioso/fisiología , Neuroblastoma/metabolismo , Receptores de Superficie Celular/metabolismo , Transporte Activo de Núcleo Celular , Animales , Apoptosis , Caspasa 2/metabolismo , Caspasa 3/metabolismo , Cisteína Endopeptidasas/metabolismo , Factor de Transcripción E2F1/metabolismo , Retroalimentación Fisiológica , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Crecimiento Nervioso/metabolismo , Netrina-1 , Neuroblastoma/mortalidad , Neuroblastoma/patología , Células PC12 , Pronóstico , Proteolisis , Ratas , Receptores de Superficie Celular/genética , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Regulación hacia Arriba

RESUMEN

The p53 tumor suppressor protein is a transcription factor controlling various outcomes, such as growth arrest and apoptosis, through the regulation of different sets of target genes. The nuclear mitotic apparatus protein (NuMA) plays important roles in spindle pole organization during mitosis and in chromatin regulation in the nucleus during interphase. Although NuMA has been shown to colocalize with several nuclear proteins, including high-mobility-group proteins I and Y and GAS41, the role of NuMA during interphase remains unclear. Here we report that NuMA binds to p53 to modulate p53-mediated transcription. Acute and partial ablation of NuMA attenuates the induction of the proarrested p21 gene following DNA damage, subsequently causing impaired cell cycle arrest. Interestingly, NuMA knockdown had little effect on the induction of the p53-dependent proapoptotic PUMA gene. Furthermore, NuMA is required for the recruitment of cyclin-dependent kinase 8 (Cdk8), a component of the Mediator complex and a promoter of p53-mediated p21 gene function. These data demonstrate that NuMA is critical for the target selectivity of p53-mediated transcription.

Asunto(s)

Antígenos Nucleares/metabolismo , Quinasa 8 Dependiente de Ciclina/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Antígenos Nucleares/genética , Neoplasias de la Mama/metabolismo , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN , Femenino , Fibrosarcoma/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas Asociadas a Matriz Nuclear/genética , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño , Transcripción Genética

RESUMEN

Mieap, a p53-inducible protein, controls mitochondrial integrity by inducing the accumulation of lysosomal proteins within mitochondria. This phenomenon is designated MALM, for Mieap-induced accumulation of lysosome-like organelles within mitochondria. To identify this novel Mieap-interacting protein(s), we performed a two-dimensional image-converted analysis of liquid chromatography and mass spectrometry (2DICAL) on the proteins immunoprecipitated by an anti-Mieap antibody. We indentified 14-3-3γ as one of the proteins that was included in the Mieap-binding protein complex when MALM was induced. The interaction between Mieap and 14-3-3γ was confirmed on the exogenous and endogenous proteins. Interestingly, 14-3-3γ was localized within mitochondria when MALM occurred. A 14-3-3γ deficiency did not affect the accumulation of Mieap and lysosomal proteins within mitochondria, but dramatically inhibited the elimination of oxidized mitochondrial proteins. These results suggest that 14-3-3γ plays a critical role in eliminating oxidized mitochondrial proteins during the MALM process by interacting with Mieap within mitochondria.

RESUMEN

Mieap, a p53-inducible protein, controls mitochondrial quality by repairing unhealthy mitochondria. During repair, Mieap induces the accumulation of intramitochondrial lysosomal proteins (designated MALM for Mieap-induced accumulation of lysosome-like organelles within mitochondria) by interacting with NIX, leading to the elimination of oxidized mitochondrial proteins. Here, we report that an additional mitochondrial outer membrane protein, BNIP3, is also involved in MALM. BNIP3 interacts with Mieap in a reactive oxygen species (ROS)-dependent manner via the BH3 domain of BNIP3 and the coiled-coil domains of Mieap. The knockdown of endogenous BNIP3 expression severely inhibited MALM. Although the overexpression of either BNIP3 or NIX did not cause a remarkable change in the mitochondrial membrane potential (MMP), the co-expression of all three exogenous proteins, Mieap, BNIP3 and NIX, caused a dramatic reduction in MMP, implying that the physical interaction of Mieap, BNIP3 and NIX at the mitochondrial outer membrane may regulate the opening of a pore in the mitochondrial double membrane. This effect was not related to cell death. These results suggest that two mitochondrial outer membrane proteins, BNIP3 and NIX, mediate MALM in order to maintain mitochondrial integrity. The physical interaction of Mieap, BNIP3 and NIX at the mitochondrial outer membrane may play a critical role in the translocation of lysosomal proteins from the cytoplasm to the mitochondrial matrix.

Asunto(s)

Proteínas de la Membrana/fisiología , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Proteínas Supresoras de Tumor/fisiología , Línea Celular Tumoral , Células HCT116 , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/fisiología , Modelos Biológicos , Orgánulos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica/fisiología , Dominios y Motivos de Interacción de Proteínas/genética , Dominios y Motivos de Interacción de Proteínas/fisiología , Transporte de Proteínas/genética , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/fisiología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

The accumulation of unhealthy mitochondria results in mitochondrial dysfunction, which has been implicated in aging, cancer, and a variety of degenerative diseases. However, the mechanism by which mitochondrial quality is regulated remains unclear. Here, we show that Mieap, a novel p53-inducible protein, induces intramitochondrial lysosome-like organella that plays a critical role in mitochondrial quality control. Mieap expression is directly regulated by p53 and is frequently lost in human cancer as result of DNA methylation. Mieap dramatically induces the accumulation of lysosomal proteins within mitochondria and mitochondrial acidic condition without destroying the mitochondrial structure (designated MALM, for Mieap-induced accumulation of lysosome-like organelles within mitochondria) in response to mitochondrial damage. MALM was not related to canonical autophagy. MALM is involved in the degradation of oxidized mitochondrial proteins, leading to increased ATP synthesis and decreased reactive oxygen species generation. These results suggest that Mieap induces intramitochondrial lysosome-like organella that plays a critical role in mitochondrial quality control by eliminating oxidized mitochondrial proteins. Cancer cells might accumulate unhealthy mitochondria due to p53 mutations and/or Mieap methylation, representing a potential cause of the Warburg effect.

Asunto(s)

Lisosomas , Mitocondrias/ultraestructura , Proteínas Mitocondriales/fisiología , Orgánulos/química , Proteína p53 Supresora de Tumor/fisiología , Humanos , Lisosomas/metabolismo , Mitocondrias/patología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Oxidación-Reducción , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Activación Transcripcional

RESUMEN

Maintenance of healthy mitochondria prevents aging, cancer, and a variety of degenerative diseases that are due to the result of defective mitochondrial quality control (MQC). Recently, we discovered a novel mechanism for MQC, in which Mieap induces intramitochondrial lysosome-like organella that plays a critical role in the elimination of oxidized mitochondrial proteins (designated MALM for Mieap-induced accumulation of lysosome-like organelles within mitochondria). However, a large part of the mechanisms for MQC remains unknown. Here, we report additional mechanisms for Mieap-regulated MQC. Reactive oxygen species (ROS) scavengers completely inhibited MALM. A mitochondrial outer membrane protein NIX interacted with Mieap in a ROS-dependent manner via the BH3 domain of NIX and the coiled-coil domain of Mieap. Deficiency of NIX also completely impaired MALM. When MALM was inhibited, Mieap induced vacuole-like structures (designated as MIV for Mieap-induced vacuole), which engulfed and degraded the unhealthy mitochondria by accumulating lysosomes. The inactivation of p53 severely impaired both MALM and MIV generation, leading to accumulation of unhealthy mitochondria. These results suggest that (1) mitochondrial ROS and NIX are essential factors for MALM, (2) MIV is a novel mechanism for lysosomal degradation of mitochondria, and (3) the p53-Mieap pathway plays a pivotal role in MQC by repairing or eliminating unhealthy mitochondria via MALM or MIV generation, respectively.

Asunto(s)

Lisosomas , Proteínas de la Membrana/fisiología , Mitocondrias/patología , Proteínas Mitocondriales/fisiología , Orgánulos/química , Proteínas Proto-Oncogénicas/fisiología , Proteína p53 Supresora de Tumor/fisiología , Proteínas Supresoras de Tumor/fisiología , Línea Celular , Humanos , Lisosomas/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/ultraestructura , Proteínas Mitocondriales/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Especies Reactivas de Oxígeno/farmacología , Activación Transcripcional , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

NEEP21, also designated D4S234E or NSG1, is an endosomal protein expressed in neuronal cells under normal conditions. Here, we report that NEEP21 is a direct transcriptional target gene of the tumor suppressor p53. NEEP21 expression is inducible in non-neuronal human cancer cell lines by exposure to adriamycin, hydrogen peroxide, UV and γ-ray in a p53-dependent manner. Chromatin immunoprecipitation assay indicated that a potential p53-binding site (p53BS) is located in intron 1 of the NEEP21 gene. A reporter assay confirmed that p53BS has p53-responsive activity. The heterologous luciferase gene containing p53BS is also transactivated by p73-ß and p63-γ. The introduction of the NEEP21 gene into various cancer cell lines suppressed cell growth. Infection with an adenovirus vector containing NEEP21 induced apoptotic cell death via caspase-3 activation in many cancer cell lines. The expression of NEEP21 mRNA was remarkably induced by γ-ray irradiation in the spleen of p53+/+ mice but not in that of p53-/- mice. These results suggest that NEEP21 may play a critical role in apoptosis as a mediator of p53.

Asunto(s)

Proteínas Portadoras/genética , Regulación Neoplásica de la Expresión Génica/genética , Proteínas del Tejido Nervioso/genética , Activación Transcripcional/genética , Proteína p53 Supresora de Tumor/genética , Animales , Línea Celular Tumoral , Separación Celular , Inmunoprecipitación de Cromatina , Citometría de Flujo , Expresión Génica , Humanos , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa