RESUMEN

TIMM50 is a core subunit of the TIM23 complex, the mitochondrial inner membrane translocase responsible for the import of pre-sequence-containing precursors into the mitochondrial matrix and inner membrane. Here we describe a mitochondrial disease patient who is homozygous for a novel variant in TIMM50 and establish the first proteomic map of mitochondrial disease associated with TIMM50 dysfunction. We demonstrate that TIMM50 pathogenic variants reduce the levels and activity of endogenous TIM23 complex, which significantly impacts the mitochondrial proteome, resulting in a combined oxidative phosphorylation (OXPHOS) defect and changes to mitochondrial ultrastructure. Using proteomic data sets from TIMM50 patient fibroblasts and a TIMM50 HEK293 cell model of disease, we reveal that laterally released substrates imported via the TIM23SORT complex pathway are most sensitive to loss of TIMM50. Proteins involved in OXPHOS and mitochondrial ultrastructure are enriched in the TIM23SORT substrate pool, providing a biochemical mechanism for the specific defects in TIMM50-associated mitochondrial disease patients. These results highlight the power of using proteomics to elucidate molecular mechanisms of disease and uncovering novel features of fundamental biology, with the implication that human TIMM50 may have a more pronounced role in lateral insertion than previously understood.

Asunto(s)

Mitocondrias , Enfermedades Mitocondriales , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Fosforilación Oxidativa , Transporte de Proteínas , Humanos , Fibroblastos/metabolismo , Células HEK293 , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Enfermedades Mitocondriales/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Membranas Mitocondriales/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Mutación/genética , Proteómica/métodos

RESUMEN

BACKGROUND: Metabolic reprogramming is one of hallmarks of cancer progression and is of great importance for the tumor microenvironment (TME). As an abundant metabolite, lactate has been found to play a critical role in cancer development and immunosuppression of TME. However, the potential role of lactate metabolism-related genes in endometrial cancer (EC) remains obscure. METHODS: RNA sequencing data and clinical information of EC were obtained from The Cancer Genome Atlas (TCGA) database. Lactate metabolism-related genes (LMRGs) WERE from Molecular Signature Database v7.4 and then compared the candidate genes from TCGA to obtain final genes. Univariate analysis and Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression were performed to screen prognostic genes. A lactate metabolism-related risk profile was constructed using multivariate Cox regression analysis. The signature was validated by time-dependent ROC curve analysis and Kaplan-Meier analysis. The relationship between the risk score and age, grade, stage, tumor microenvironmental characteristics, and drug sensitivity was as well explored by correlation analyses. Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway functional analysis between the high and low-risk groups were performed. CCK8, EdU, and clone formation assays were applied to detect the proliferation ability of EC cells, Transwell assay was performed to detect the migration ability of EC cells, and intracellular lactate and glucose content was used to asses lactate metabolism. RESULTS: We constructed a risk signature based on 18 LMRGs. Kaplan-Meier curves confirmed that the high-risk group had poorer prognosis compared to the low-risk group. A nomogram was then constructed to predict the probability of EC survival. We also performed GO enrichment analysis and KEGG pathway functional analysis between the high and low-risk groups, and the outcome revealed that the features were significantly associated with energy metabolism. There was a significant correspondence between LMRGs and tumor mutational load, checkpoints and immune cell infiltration. C1, C2, and C4 were the most infiltrated in the high-risk group. The high-risk group showed increased dendritic cell activation, while the low-risk group showed increased plasma cells and Treg cells. Drug sensitivity analysis showed LMRGs risk was more resistant to Scr kinase inhibitors. We further proved that one of the lactate metabolism related genes, TIMM50 could promote EC cell proliferation, migration and lactate metabolism. CONCLUSION: In conclusion, we have established an effective prognostic signature based on LMRG expression patterns, which may greatly facilitate the assessment of prognosis, molecular features and treatment modalities in EC patients and may be useful in the future translation to clinical applications. TIMM50 was identified as a novel molecule that mediates lactate metabolism in vitro and in vivo, maybe a promising target for EC prognosis.

Asunto(s)

Neoplasias Endometriales , Humanos , Femenino , Neoplasias Endometriales/genética , Metabolismo Energético , Factores de Riesgo , Pronóstico , Microambiente Tumoral/genética

RESUMEN

BACKGROUND: Intellectual disability (ID) is a heterogeneous group of neurodevelopmental disorders that is characterized by significant impairment in intellectual and adaptive functioning with onset during the developmental period. Whole-exome sequencing (WES)-based studies in the consanguineous families with individuals affected with ID have shown a high burden of relevant variants. So far, over 700 genes have been reported in syndromic and non-syndromic ID. However, genetic causes in more than 50% of ID patients still remain unclear. METHODS: Whole-exome sequencing was applied for investigation of various variants of ID, then Sanger sequencing and in silico analysis in ten patients from five Iranian consanguineous families diagnosed with autosomal recessive neurodevelopmental disorders, intellectual disability, performed for confirming the causative mutation within the probands. The most patients presented moderate-to-severe intellectual disability, developmental delay, seizure, speech problem, high level of lactate, and onset before 10 years. RESULTS: Filtering the data identified by WES, two novel homozygous missense variants in FBXO31 and TIMM50 genes and one previously reported mutation in the CEP290 gene in the probands were found. Sanger sequencing confirmed the homozygote variant's presence of TIMM50 and FBXO31 genes in six patients and two affected siblings in their respective families. Our computational results predicted that the variants are located in the conserved regions across different species and have the impacts on the protein stability. CONCLUSION: Hence, we provide evidence for the pathogenicity of two novel variants in the patients which will expand our knowledge about potential mutation involved in the heterogeneous disease.

Asunto(s)

Consanguinidad , Proteínas F-Box/genética , Discapacidad Intelectual/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/genética , Mutación Missense , Trastornos del Neurodesarrollo/genética , Proteínas Supresoras de Tumor/genética , Adolescente , Antígenos de Neoplasias/genética , Proteínas de Ciclo Celular/genética , Niño , Preescolar , Trastornos de los Cromosomas , Proteínas del Citoesqueleto/genética , Femenino , Genes Recesivos , Homocigoto , Humanos , Patrón de Herencia , Irán , Masculino

RESUMEN

Mitochondria are involved in a variety of developmental processes and neurodegenerative diseases. The translocase complexes of the outer and inner mitochondrial membranes (TOM and TIM) are protein complexes involved in transporting protein precursors across mitochondrial membranes. Although rabbits are important animal models for neurodegenerative diseases, the expression of TOM and TIM complexes has yet to be examined in the rabbit brain. In the present study, we quantitatively evaluated the protein expression of the translocase of outer mitochondrial membrane 40 (TOMM40) and inner mitochondrial membrane 50 (TIMM50) complexes, two of the TOM/TIM complexes, in the cerebral, cerebellar, and hippocampal cortices of the New Zealand white rabbit brain, using immunohistochemistry. Sections from brain specimens were initially stained for cytochrome c oxidase (COX), a well-known mitochondrial marker, which was found to be homogeneously expressed in the cerebrum, but localized to the Purkinje and pyramidal neurons of the cerebellum and hippocampus, respectively. TOMM40 and TIMM50 proteins consistently revealed a similar expression pattern, although at different ratios. In the cerebrum, TOMM40 and TIMM50 immunoreactions were homogeneously distributed within the cytoplasm of various neurons. Meanwhile, Purkinje cells in the cerebellum and pyramidal neurons in the hippocampus displayed higher intensities in their cytoplasm. The specific cellular localization of TOMM40 and TIMM50 proteins in various regions of the rabbit brain suggests a distinct function of each protein in these regions. Further analysis will be required to evaluate the molecular functions of these proteins.

Asunto(s)

Encéfalo , Mitocondrias , Membranas Mitocondriales , Animales , Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Células Piramidales , Conejos

RESUMEN

Mitochondria are essential in eukaryotes. Besides producing 80% of total cellular ATP, mitochondria are involved in various cellular functions such as apoptosis, inflammation, innate immunity, stress tolerance, and Ca2+ homeostasis. Mitochondria are also the site for many critical metabolic pathways and are integrated into the signaling network to maintain cellular homeostasis under stress. Mitochondria require hundreds of proteins to perform all these functions. Since the mitochondrial genome only encodes a handful of proteins, most mitochondrial proteins are imported from the cytosol via receptor/translocase complexes on the mitochondrial outer and inner membranes known as TOMs and TIMs. Many of the subunits of these protein complexes are essential for cell survival in model yeast and other unicellular eukaryotes. Defects in the mitochondrial import machineries are also associated with various metabolic, developmental, and neurodegenerative disorders in multicellular organisms. In addition to their canonical functions, these protein translocases also help maintain mitochondrial structure and dynamics, lipid metabolism, and stress response. This review focuses on the role of Tim50, the receptor component of one of the TIM complexes, in different cellular functions, with an emphasis on the Tim50 homologue in parasitic protozoan Trypanosoma brucei.

Asunto(s)

Homeostasis , Mitocondrias/enzimología , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Trypanosoma brucei brucei/enzimología , Animales , Humanos , Proteínas de Transporte de Membrana Mitocondrial/genética , Transporte de Proteínas

RESUMEN

BACKGROUND: Rhabdomyosarcoma (RMS) is a pediatric cancer with rhabdomyoblastic phenotype and mitochondria act as pivotal regulators of its growth and progression. While miR-7-5p (miR-7) is reported to have a tumor-suppressive role, little is yet known about its antitumor activity in RMS. METHODS: The effects of miR-7 on RMS were analyzed both in vitro and in vivo. Cell death modalities induced by miR-7 were identified. Influence on mitochondria was evaluated through RNA sequencing data, morphological observation and mitochondrial functional assays, including outer membrane permeability, bioenergetics and redox balance. Dual-luciferase assay and phenotype validation after transient gene silencing were performed to identify miR-7 targets in RMS. RESULTS: MiR-7 executed anti-tumor effect in RMS beyond proliferation inhibition. Morphologic features and molecular characteristics with apoptosis and necroptosis were found in miR-7-transfected RMS cells. Chemical inhibitors of apoptosis and necroptosis were able to prevent miR-7-induced cell death. Further, we identified that mitochondrial impairment mainly contributed to these phenomena and mitochondrial proteins SLC25A37 and TIMM50 were crucial targets for miR-7 to induce cell death in RMS. CONCLUSION: Our results extended the mechanism of miR-7 antitumor role in rhabdomyosarcoma cancer, and provided potential implications for its therapy.

Asunto(s)

Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Membrana/genética , MicroARNs/genética , Proteínas Mitocondriales/genética , Rabdomiosarcoma/genética , Animales , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , Mitocondrias/genética , Mitocondrias/patología , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Necroptosis/genética , Especies Reactivas de Oxígeno/metabolismo , Rabdomiosarcoma/patología , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

3-Methylglutaconic aciduria (3-MGA-uria) syndromes comprise a heterogeneous group of diseases associated with mitochondrial membrane defects. Whole-exome sequencing identified compound heterozygous mutations in TIMM50 (c.[341 G>A];[805 G>A]) in a boy with West syndrome, optic atrophy, neutropenia, cardiomyopathy, Leigh syndrome, and persistent 3-MGA-uria. A comprehensive analysis of the mitochondrial function was performed in fibroblasts of the patient to elucidate the molecular basis of the disease. TIMM50 protein was severely reduced in the patient fibroblasts, regardless of the normal mRNA levels, suggesting that the mutated residues might be important for TIMM50 protein stability. Severe morphological defects and ultrastructural abnormalities with aberrant mitochondrial cristae organization in muscle and fibroblasts were found. The levels of fully assembled OXPHOS complexes and supercomplexes were strongly reduced in fibroblasts from this patient. High-resolution respirometry demonstrated a significant reduction of the maximum respiratory capacity. A TIMM50-deficient HEK293T cell line that we generated using CRISPR/Cas9 mimicked the respiratory defect observed in the patient fibroblasts; notably, this defect was rescued by transfection with a plasmid encoding the TIMM50 wild-type protein. In summary, we demonstrated that TIMM50 deficiency causes a severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology, such as the maintenance of proper mitochondrial morphology, OXPHOS assembly, and mitochondrial respiratory capacity.

Asunto(s)

Proteínas de Transporte de Membrana/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Mutación , Biomarcadores , Transporte de Electrón , Metabolismo Energético , Fibroblastos/metabolismo , Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Lactante , Masculino , Mitocondrias/ultraestructura , Enfermedades Mitocondriales/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestructura , Fenotipo , Transporte de Proteínas , Espasmos Infantiles/diagnóstico , Espasmos Infantiles/genética , Secuenciación del Exoma

RESUMEN

TIMM50 (Translocase of the inner mitochondrial membrane 50), also called TIM50, plays an essential role in mitochondrial membrane transportation. The existing literature suggests that TIMM50 may perform as an oncogenetic protein in breast cancer. However, the molecular mechanism, especially in human non-small cell lung cancer (NSCLC), is uncertain to date. In the present study, using immunohistochemistry, we found that TIMM50 expression significantly correlated with larger tumor size (P = 0.049), advanced TNM stage (P = 0.001), positive regional lymph node metastasis (P = 0.007), and poor overall survival (P = 0.001). Proliferation and invasion assay showed that TIMM50 dramatically promoted the ability of proliferation and invasion of NSCLC cells. Subsequent Western blotting results revealed that TIMM50 enhanced the expression of Cyclin D1 and Snail, and inhibited the expression of E-cadherin. Moreover, TIMM50 facilitated the expression of phosphorylated ERK and P90RSK. Incorporation of ERK inhibitor counteracted the upregulating expression of CyclinD1, and Snail, and downregulating expression of E-cadherin expression induced by TIMM50 overexpression. In conclusion, our data indicated that TIMM50 facilitated tumor proliferation and invasion of NSCLC through enhancing phosphorylation of its downstream ERK/P90RSK signaling pathway. We speculated that TIMM50 might be a useful prognosis marker of NSCLC patients.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/patología , Proteínas de Transporte de Membrana/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/secundario , Adulto , Anciano , Apoptosis , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/secundario , Estudios de Casos y Controles , Proliferación Celular , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Metástasis Linfática , Masculino , Proteínas de Transporte de Membrana/genética , Persona de Mediana Edad , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Invasividad Neoplásica , Fosforilación , Pronóstico , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Transducción de Señal , Tasa de Supervivencia , Células Tumorales Cultivadas

RESUMEN

TIMM50 is an essential component of the TIM23 complex, the mitochondrial inner membrane machinery that imports cytosolic proteins containing a mitochondrial targeting presequence into the mitochondrial inner compartment. Whole exome sequencing (WES) identified compound heterozygous pathogenic mutations in TIMM50 in an infant patient with rapidly progressive, severe encephalopathy. Patient fibroblasts presented low levels of TIMM50 and other components of the TIM23 complex, lower mitochondrial membrane potential, and impaired TIM23-dependent protein import. As a consequence, steady-state levels of several components of mitochondrial respiratory chain were decreased, resulting in decreased respiration and increased ROS production. Growth of patient fibroblasts in galactose shifted energy production metabolism toward oxidative phosphorylation (OxPhos), producing an apparent improvement in most of the above features but also increased apoptosis. Complementation of patient fibroblasts with TIMM50 improved or restored these features to control levels. Moreover, RNASEH1 and ISCU mutant fibroblasts only shared a few of these features with TIMM50 mutant fibroblasts. Our results indicate that mutations in TIMM50 cause multiple mitochondrial bioenergetic dysfunction and that functional TIMM50 is essential for cell survival in OxPhos-dependent conditions.

Asunto(s)

Encefalopatías/fisiopatología , Proteínas de Transporte de Membrana/genética , Enfermedades Mitocondriales/fisiopatología , Mutación , Fosforilación Oxidativa , Encefalopatías/diagnóstico , Encefalopatías/patología , Supervivencia Celular , Células Cultivadas , Femenino , Fibroblastos/patología , Prueba de Complementación Genética , Humanos , Lactante , Italia , Enfermedades Mitocondriales/diagnóstico , Enfermedades Mitocondriales/patología , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales

RESUMEN

Mitochondrial encephalopathies are a heterogeneous group of disorders that, usually carry grave prognosis. Recently a homozygous mutation, Gly372Ser, in the TIMM50 gene, was reported in an abstract form, in three sibs who suffered from intractable epilepsy and developmental delay accompanied by 3-methylglutaconic aciduria. We now report on four patients from two unrelated families who presented with severe intellectual disability and seizure disorder, accompanied by slightly elevated lactate level, 3-methylglutaconic aciduria and variable deficiency of mitochondrial complex V. Using exome analysis we identified two homozygous missense mutations, Arg217Trp and Thr252Met, in the TIMM50 gene. The TIMM50 protein is a subunit of TIM23 complex, the mitochondrial import machinery. It serves as the major receptor in the intermembrane space, binding to proteins which cross the mitochondrial inner membrane on their way to the matrix. The mutations, which affected evolutionary conserved residues and segregated with the disease in the families, were neither present in large cohorts of control exome analyses nor in our ethnic specific exome cohort. Given the phenotypic similarity, we conclude that missense mutations in TIMM50 are likely manifesting by severe intellectual disability and epilepsy accompanied by 3-methylglutaconic aciduria and variable mitochondrial complex V deficiency. 3-methylglutaconic aciduria is emerging as an important biomarker for mitochondrial dysfunction, in particular for mitochondrial membrane defects.

Asunto(s)

Adenosina Trifosfatasas/deficiencia , Epilepsia/genética , Proteínas de la Membrana/deficiencia , Proteínas de Transporte de Membrana/genética , Errores Innatos del Metabolismo/genética , Encefalomiopatías Mitocondriales/genética , Adenosina Trifosfatasas/genética , Proteínas Portadoras/genética , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Proteínas de la Membrana/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , ATPasas de Translocación de Protón Mitocondriales , Mutación , Polimorfismo de Nucleótido Simple , Embarazo