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Mitochondrial ribosomes (mitoribosomes) have undergone substantial evolutionary structural remodeling accompanied by loss of ribosomal RNA, while acquiring unique protein subunits located on the periphery. We generated CRISPR-mediated knockouts of all 14 unique (mitochondria-specific/supernumerary) human mitoribosomal proteins (snMRPs) in the small subunit to study the effect on mitoribosome assembly and protein synthesis, each leading to a unique mitoribosome assembly defect with variable impact on mitochondrial protein synthesis. Surprisingly, the stability of mS37 was reduced in all our snMRP knockouts of the small and large ribosomal subunits and patient-derived lines with mitoribosome assembly defects. A redox-regulated CX9C motif in mS37 was essential for protein stability, suggesting a potential mechanism to regulate mitochondrial protein synthesis. Together, our findings support a modular assembly of the human mitochondrial small ribosomal subunit mediated by essential supernumerary subunits and identify a redox regulatory role involving mS37 in mitochondrial protein synthesis in health and disease.
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Mitochondrial translation occurs on the mitochondrial ribosome, also known as the mitoribosome. The assembly of mitoribosomes is a highly coordinated process. During mitoribosome biogenesis, various assembly factors transiently associate with the nascent ribosome, facilitating the accurate and efficient construction of the mitoribosome. However, the specific factors involved in the assembly process, the precise mechanisms, and the cellular compartments involved in this vital process are not yet fully understood. In this study, we discovered a crucial role for GTP-binding protein 8 (GTPBP8) in the assembly of the mitoribosomal large subunit (mt-LSU) and mitochondrial translation. GTPBP8 is identified as a novel GTPase located in the matrix and peripherally bound to the inner mitochondrial membrane. Importantly, GTPBP8 is specifically associated with the mt-LSU during its assembly. Depletion of GTPBP8 leads to an abnormal accumulation of mt-LSU, indicating that GTPBP8 is critical for proper mt-LSU assembly. Furthermore, the absence of GTPBP8 results in reduced levels of fully assembled 55S monosomes. This impaired assembly leads to compromised mitochondrial translation and, consequently, impaired mitochondrial function. The identification of GTPBP8 as an important player in these processes provides new insights into the molecular mechanisms underlying mitochondrial protein synthesis and its regulation.
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Mitocondrias , Membranas Mitocondriales , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Ribosomas Mitocondriales/química , Ribosomas Mitocondriales/metabolismo , Biosíntesis de Proteínas , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismoRESUMEN
The involvement of mitochondrial dysfunction in cystatin B (CSTB) deficiency has been suggested, but its role in the onset of neurodegeneration, myoclonus, and ataxia in the CSTB-deficient mouse model (Cstb-/-) is yet unknown. CSTB is an inhibitor of lysosomal and nuclear cysteine cathepsins. In humans, partial loss-of-function mutations cause the progressive myoclonus epilepsy neurodegenerative disorder, EPM1. Here we applied proteome analysis and respirometry on cerebellar synaptosomes from early symptomatic (Cstb-/-) mice to identify the molecular mechanisms involved in the onset of CSTB-deficiency associated neural pathogenesis. Proteome analysis showed that CSTB deficiency is associated with differential expression of mitochondrial and synaptic proteins, and respirometry revealed a progressive impairment in mitochondrial function coinciding with the onset of myoclonus and neurodegeneration in (Cstb-/-) mice. This mitochondrial dysfunction was not associated with alterations in mitochondrial DNA copy number or membrane ultrastructure. Collectively, our results show that CSTB deficiency generates a defect in synaptic mitochondrial bioenergetics that coincides with the onset and progression of the clinical phenotypes, and thus is likely a contributor to the pathogenesis of EPM1.
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Delays in appropriate antibiotic therapy are a key determinant for deleterious outcomes among patients with vancomycin-resistant Enterococcus (VRE) bloodstream infections (BSIs). This was a multi-center pre/post-implementation study, assessing the impact of a molecular rapid diagnostic test (Verigene® GP-BC, Luminex Corporation, Northbrook, IL, USA) on outcomes of adult patients with VRE BSIs. The primary outcome was time to optimal therapy (TOT). Multivariable logistic and Cox proportional hazard regression models were used to determine the independent associations of post-implementation, TOT, early vs. delayed therapy, and mortality. A total of 104 patients with VRE BSIs were included: 50 and 54 in the pre- and post-implementation periods, respectively. The post- vs. pre-implementation group was associated with a 1.8-fold faster rate to optimized therapy (adjusted risk ratio, 1.841 [95% CI 1.234-2.746]), 6-fold higher likelihood to receive early effective therapy (<24 h, adjusted odds ratio, 6.031 [2.526-14.401]), and a 67% lower hazards for 30-day in-hospital mortality (adjusted hazard ratio, 0.322 [0.124-1.831]), after adjusting for age, sex, and severity scores. Inversely, delayed therapy was associated with a 10-fold higher risk of in-hospital mortality (aOR 10.488, [2.497-44.050]). Reduced TOT and in-hospital mortality were also observed in subgroups of immunosuppressed patients in post-implementation. These findings demonstrate that the addition of molecular rapid diagnostic tests (mRDT) to clinical microbiology and antimicrobial stewardship practices are associated with a clinically significant reduction in TOT, which is associated with lower mortality for patients with VRE BSIs, underscoring the importance of mRDTs in the management of VRE infections.
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Pathogenic variants that disrupt human mitochondrial protein synthesis are associated with a clinically heterogeneous group of diseases. Despite an impairment in oxidative phosphorylation being a common phenotype, the underlying molecular pathogenesis is more complex than simply a bioenergetic deficiency. Currently, we have limited mechanistic understanding on the scope by which a primary defect in mitochondrial protein synthesis contributes to organelle dysfunction. Since the proteins encoded in the mitochondrial genome are hydrophobic and need co-translational insertion into a lipid bilayer, responsive quality control mechanisms are required to resolve aberrations that arise with the synthesis of truncated and misfolded proteins. Here, we show that defects in the OXA1L-mediated insertion of MT-ATP6 nascent chains into the mitochondrial inner membrane are rapidly resolved by the AFG3L2 protease complex. Using pathogenic MT-ATP6 variants, we then reveal discrete steps in this quality control mechanism and the differential functional consequences to mitochondrial gene expression. The inherent ability of a given cell type to recognize and resolve impairments in mitochondrial protein synthesis may in part contribute at the molecular level to the wide clinical spectrum of these disorders.
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Fosforilación Oxidativa , Biosíntesis de Proteínas , Proteínas Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/genética , Mutación , FenotipoRESUMEN
High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and assessment of respiratory complexes using diversified substrate-uncoupler-inhibitor titration (SUIT) protocols. Here, the usage of two high-resolution respirometry devices is demonstrated, and a basic collection of protocols applicable for the analysis of cultured cells, skeletal and heart muscle fibers, and soft tissues such as the brain and liver are presented. Protocols for cultured cells and tissues are provided for a chamber-based respirometer and cultured cells for a microplate-based respirometer, both encompassing standard respiration protocols. For comparative purposes, CRISPR-engineered HEK293 cells deficient in mitochondrial translation causing multiple respiratory system deficiency are used with both devices to demonstrate cellular defects in respiration. Both respirometers allow for comprehensive measurement of cellular respiration with their respective technical merits and suitability dependent on the research question and model under study.
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Respiración de la Célula , Consumo de Oxígeno , Respiración de la Célula/fisiología , Metabolismo Energético/fisiología , Células HEK293 , Humanos , Mitocondrias Musculares/metabolismo , Fosforilación Oxidativa , Consumo de Oxígeno/fisiologíaRESUMEN
As ancient bacterial endosymbionts of eukaryotic cells, mitochondria have retained their own circular DNA as well as protein translation system including mitochondrial ribosomes (mitoribosomes). In recent years, methodological advancements in cryoelectron microscopy and mass spectrometry have revealed the extent of the evolutionary divergence of mitoribosomes from their bacterial ancestors and their adaptation to the synthesis of 13 mitochondrial DNA encoded oxidative phosphorylation complex subunits. In addition to the structural data, the first assembly pathway maps of mitoribosomes have started to emerge and concomitantly also the assembly factors involved in this process to achieve fully translational competent particles. These transiently associated factors assist in the intricate assembly process of mitoribosomes by enhancing protein incorporation, ribosomal RNA folding and modification, and by blocking premature or non-native protein binding, for example. This review focuses on summarizing the current understanding of the known mammalian mitoribosome assembly factors and discussing their possible roles in the assembly of small or large mitoribosomal subunits.
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Genoma Mitocondrial , Mamíferos/genética , Mamíferos/fisiología , Ribosomas Mitocondriales/fisiología , AnimalesRESUMEN
We investigated the clinical, genetic, and pathological characteristics of a previously unknown severe juvenile brain disorder in several litters of Parson Russel Terriers. The disease started with epileptic seizures at 6-12 weeks of age and progressed rapidly to status epilepticus and death or euthanasia. Histopathological changes at autopsy were restricted to the brain. There was severe acute neuronal degeneration and necrosis diffusely affecting the grey matter throughout the brain with extensive intraneuronal mitochondrial crowding and accumulation of amyloid-ß (Aß). Combined homozygosity mapping and genome sequencing revealed an in-frame 6-bp deletion in the nuclear-encoded pitrilysin metallopeptidase 1 (PITRM1) encoding for a mitochondrial protease involved in mitochondrial targeting sequence processing and degradation. The 6-bp deletion results in the loss of two amino acid residues in the N-terminal part of PITRM1, potentially affecting protein folding and function. Assessment of the mitochondrial function in the affected brain tissue showed a significant deficiency in respiratory chain function. The functional consequences of the mutation were modeled in yeast and showed impaired growth in permissive conditions and an impaired respiration capacity. Loss-of-function variants in human PITRM1 result in a childhood-onset progressive amyloidotic neurological syndrome characterized by spinocerebellar ataxia with behavioral, psychiatric and cognitive abnormalities. Homozygous Pitrm1-knockout mice are embryonic lethal, while heterozygotes show a progressive, neurodegenerative phenotype characterized by impairment in motor coordination and Aß deposits. Our study describes a novel early-onset PITRM1-related neurodegenerative canine brain disorder with mitochondrial dysfunction, Aß accumulation, and lethal epilepsy. The findings highlight the essential role of PITRM1 in neuronal survival and strengthen the connection between mitochondrial dysfunction and neurodegeneration.
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Enfermedades de los Perros/genética , Epilepsia/veterinaria , Metaloendopeptidasas/genética , Mitocondrias/metabolismo , Enfermedades Neurodegenerativas/veterinaria , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/enzimología , Encéfalo/metabolismo , Encéfalo/patología , Enfermedades de los Perros/patología , Perros , Epilepsia/genética , Femenino , Masculino , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Consumo de Oxígeno , Linaje , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismoRESUMEN
Progressive myoclonus epilepsies (PMEs) comprise a group of clinically and genetically heterogeneous rare diseases. Over 70% of PME cases can now be molecularly solved. Known PME genes encode a variety of proteins, many involved in lysosomal and endosomal function. We performed whole-exome sequencing (WES) in 84 (78 unrelated) unsolved PME-affected individuals, with or without additional family members, to discover novel causes. We identified likely disease-causing variants in 24 out of 78 (31%) unrelated individuals, despite previous genetic analyses. The diagnostic yield was significantly higher for individuals studied as trios or families (14/28) versus singletons (10/50) (OR = 3.9, p value = 0.01, Fisher's exact test). The 24 likely solved cases of PME involved 18 genes. First, we found and functionally validated five heterozygous variants in NUS1 and DHDDS and a homozygous variant in ALG10, with no previous disease associations. All three genes are involved in dolichol-dependent protein glycosylation, a pathway not previously implicated in PME. Second, we independently validate SEMA6B as a dominant PME gene in two unrelated individuals. Third, in five families, we identified variants in established PME genes; three with intronic or copy-number changes (CLN6, GBA, NEU1) and two very rare causes (ASAH1, CERS1). Fourth, we found a group of genes usually associated with developmental and epileptic encephalopathies, but here, remarkably, presenting as PME, with or without prior developmental delay. Our systematic analysis of these cases suggests that the small residuum of unsolved cases will most likely be a collection of very rare, genetically heterogeneous etiologies.
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Dolicoles/metabolismo , Mutación/genética , Epilepsias Mioclónicas Progresivas/genética , Adolescente , Adulto , Edad de Inicio , Niño , Preescolar , Estudios de Cohortes , Variaciones en el Número de Copia de ADN/genética , Femenino , Glicosilación , Humanos , Intrones/genética , Masculino , Persona de Mediana Edad , Epilepsias Mioclónicas Progresivas/clasificación , Secuenciación del Exoma , Adulto JovenRESUMEN
Mitochondrial acyl-coenzyme A species are emerging as important sources of protein modification and damage. Succinyl-CoA ligase (SCL) deficiency causes a mitochondrial encephalomyopathy of unknown pathomechanism. Here, we show that succinyl-CoA accumulates in cells derived from patients with recessive mutations in the tricarboxylic acid cycle (TCA) gene succinyl-CoA ligase subunit-ß (SUCLA2), causing global protein hyper-succinylation. Using mass spectrometry, we quantify nearly 1,000 protein succinylation sites on 366 proteins from patient-derived fibroblasts and myotubes. Interestingly, hyper-succinylated proteins are distributed across cellular compartments, and many are known targets of the (NAD+)-dependent desuccinylase SIRT5. To test the contribution of hyper-succinylation to disease progression, we develop a zebrafish model of the SCL deficiency and find that SIRT5 gain-of-function reduces global protein succinylation and improves survival. Thus, increased succinyl-CoA levels contribute to the pathology of SCL deficiency through post-translational modifications.
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Acilcoenzima A/metabolismo , Enfermedades Mitocondriales/patología , Succinato-CoA Ligasas/genética , Animales , Células Cultivadas , Femenino , Humanos , Lactante , Lisina/metabolismo , Masculino , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Mutación , Proteómica , Sirtuinas/deficiencia , Sirtuinas/genética , Sirtuinas/metabolismo , Succinato-CoA Ligasas/deficiencia , Succinato-CoA Ligasas/metabolismo , Análisis de Supervivencia , Pez CebraRESUMEN
Rapid diagnostic testing (RDT) combined with an antimicrobial stewardship program (ASP) has shown improved outcomes in bloodstream infections (BSIs). We assessed the impact of RDT, surveillance software, and ASP pharmacist staffing on time to optimal therapy (TOT) in Gram-negative BSIs. Adults with Gram-negative BSIs were included in this retrospective evaluation across 2 study periods. The preimplementation group (nâ¯=â¯121) had longer TOT than the postimplementation group (nâ¯=â¯120) (59.6⯱â¯36.2â¯h versus 29.0⯱â¯24.2â¯h, Pâ¯<â¯0.001). Escalation (51.1⯱â¯26.4â¯h versus 16.9⯱â¯15.7â¯h, Pâ¯<â¯0.001) and de-escalation (63.1⯱â¯39.5â¯h versus 39.2⯱â¯25.6â¯h, Pâ¯<â¯0.01) of therapy were shorter in the postimplementation group. TOT for patients with multidrug-resistant organisms (MDROs) was shorter in the postimplementation group (61.8⯱â¯37.2â¯h versus 21.9⯱â¯18.8â¯h, Pâ¯<â¯0.001). TOT was shorter during fully staffed clinical pharmacist hours (30.6⯱â¯58.9â¯h versus 19.7⯱â¯31.7â¯h, pâ¯=â¯0.014). Implementation of RDT and surveillance software with an ASP decreased TOT for Gram-negative BSIs, including MDROs.
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Antiinfecciosos/uso terapéutico , Bacteriemia/tratamiento farmacológico , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Programas Informáticos , Recursos Humanos , Anciano , Anciano de 80 o más Años , Programas de Optimización del Uso de los Antimicrobianos , Técnicas y Procedimientos Diagnósticos , Farmacorresistencia Bacteriana Múltiple , Femenino , Hospitales Comunitarios , Humanos , Pacientes Internos , Masculino , Persona de Mediana Edad , Farmacéuticos , Vigilancia en Salud Pública , Estudios Retrospectivos , Factores de TiempoRESUMEN
Mutations of mitochondrial DNA (mtDNA) often underlie mitochondrial disease, one of the most common inherited metabolic disorders. Since the sequencing of the human mitochondrial genome and the discovery of pathogenic mutations in mtDNA more than 30 years ago, a movement towards generating methods for robust manipulation of mtDNA has ensued, although with relatively few advances and some controversy. While developments in the transformation of mammalian mtDNA have stood still for some time, recent demonstrations of programmable nuclease-based technology suggest that clinical manipulation of mtDNA heteroplasmy may be on the horizon for these largely untreatable disorders. Here we review historical and recent developments in mitochondrially targeted nuclease technology and the clinical outlook for treatment of hereditary mitochondrial disease.
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ADN Mitocondrial/genética , Heteroplasmia/genética , Mitocondrias/genética , Animales , Humanos , Enfermedades Mitocondriales/genética , Mutación/genéticaRESUMEN
Purpose: Both cardiomyocytes and cardiac fibroblasts (CF) play essential roles in cardiac development, function, and remodeling. Properties of 3D co-cultures are incompletely understood. Hence, 3D co-culture of cardiomyocytes and CF was characterized, and selected features compared with single-type and 2D culture conditions. Methods: Human cardiomyocytes derived from induced-pluripotent stem cells (hiPSC-CMs) were obtained from Cellular Dynamics or Ncardia, and primary human cardiac fibroblasts from ScienCell. Cardiac spheroids were investigated using cryosections and whole-mount confocal microscopy, video motion analysis, scanning-, and transmission-electron microscopy (SEM, TEM), action potential recording, and quantitative PCR (qPCR). Results: Spheroids formed in hanging drops or in non-adhesive wells showed spontaneous contractions for at least 1 month with frequent media changes. SEM of mechanically opened spheroids revealed a dense inner structure and no signs of blebbing. TEM of co-culture spheroids at 1 month showed myofibrils, intercalated disc-like structures and mitochondria. Ultrastructural features were comparable to fetal human myocardium. We then assessed immunostained 2D cultures, cryosections of spheroids, and whole-mount preparations by confocal microscopy. CF in co-culture spheroids assumed a small size and shape similar to the situation in ventricular tissue. Spheroids made only of CF and cultured for 3 weeks showed no stress fibers and strongly reduced amounts of alpha smooth muscle actin compared to early spheroids and 2D cultures as shown by confocal microscopy, western blotting, and qPCR. The addition of CF to cardiac spheroids did not lead to arrhythmogenic effects as measured by sharp-electrode electrophysiology. Video motion analysis showed a faster spontaneous contraction rate in co-culture spheroids compared to pure hiPSC-CMs, but similar contraction amplitudes and kinetics. Spontaneous contraction rates were not dependent on spheroid size. Applying increasing pacing frequencies resulted in decreasing contraction amplitudes without positive staircase effect. Gene expression analysis of selected cytoskeleton and myofibrillar proteins showed more tissue-like expression patterns in co-culture spheroids than with cardiomyocytes alone or in 2D culture. Conclusion: We demonstrate that the use of 3D co-culture of hiPSC-CMs and CF is superior over 2D culture conditions for co-culture models and more closely mimicking the native state of the myocardium with relevance to drug development as well as for personalized medicine.
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Mitochondrial dysfunction elicits stress responses that safeguard cellular homeostasis against metabolic insults. Mitochondrial integrated stress response (ISRmt) is a major response to mitochondrial (mt)DNA expression stress (mtDNA maintenance, translation defects), but the knowledge of dynamics or interdependence of components is lacking. We report that in mitochondrial myopathy, ISRmt progresses in temporal stages and development from early to chronic and is regulated by autocrine and endocrine effects of FGF21, a metabolic hormone with pleiotropic effects. Initial disease signs induce transcriptional ISRmt (ATF5, mitochondrial one-carbon cycle, FGF21, and GDF15). The local progression to 2nd metabolic ISRmt stage (ATF3, ATF4, glucose uptake, serine biosynthesis, and transsulfuration) is FGF21 dependent. Mitochondrial unfolded protein response marks the 3rd ISRmt stage of failing tissue. Systemically, FGF21 drives weight loss and glucose preference, and modifies metabolism and respiratory chain deficiency in a specific hippocampal brain region. Our evidence indicates that FGF21 is a local and systemic messenger of mtDNA stress in mice and humans with mitochondrial disease.
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ADN Mitocondrial/metabolismo , Factores de Crecimiento de Fibroblastos/fisiología , Mitocondrias/metabolismo , Miopatías Mitocondriales/metabolismo , Estrés Fisiológico/fisiología , Factores de Transcripción Activadores/metabolismo , Animales , Línea Celular , ADN Mitocondrial/genética , Escherichia coli , Femenino , Factores de Crecimiento de Fibroblastos/genética , Factor 15 de Diferenciación de Crecimiento/metabolismo , Humanos , Masculino , Ratones , Mitocondrias/genética , Miopatías Mitocondriales/genética , Eliminación de Secuencia , Estrés Fisiológico/genéticaRESUMEN
Hartsfield syndrome is a rare clinical entity characterized by holoprosencephaly and ectrodactyly with the variable feature of cleft lip/palate. In addition to these symptoms patients with Hartsfield syndrome can show developmental delay of variable severity, isolated hypogonadotropic hypogonadism, central diabetes insipidus, vertebral anomalies, eye anomalies, and cardiac malformations. Pathogenic variants in FGFR1 have been described to cause phenotypically different FGFR1-related disorders such as Hartsfield syndrome, hypogonadotropic hypogonadism with or without anosmia, Jackson-Weiss syndrome, osteoglophonic dysplasia, Pfeiffer syndrome, and trigonocephaly Type 1. Here, we report three patients with Hartsfield syndrome from two unrelated families. Exome sequencing revealed two siblings harboring a novel de novo heterozygous synonymous variant c.1029G>A, p.Ala343Ala causing a cryptic splice donor site in exon 8 of FGFR1 likely due to gonadal mosaicism in one parent. The third case was a sporadic patient with a novel de novo heterozygous missense variant c.1868A>G, p.(Asp623Gly).
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Labio Leporino/diagnóstico , Labio Leporino/genética , Fisura del Paladar/diagnóstico , Fisura del Paladar/genética , Dedos/anomalías , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Deformidades Congénitas de la Mano/diagnóstico , Deformidades Congénitas de la Mano/genética , Holoprosencefalia/diagnóstico , Holoprosencefalia/genética , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Mutación Missense , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Mutación Silenciosa , Análisis Mutacional de ADN , Femenino , Estudios de Asociación Genética/métodos , Humanos , Masculino , Linaje , FenotipoRESUMEN
O6-methylguanine-DNA methyltransferase (MGMT) is an enzyme that removes alkyl groups at the O6-position of guanine in DNA. MGMT expression is reduced or absent in many tumor types derived from a diverse range of tissues, most notably in glioma. Low MGMT expression confers significant sensitivity to DNA alkylating agents such as temozolomide, providing a natural therapeutic index over normal tissue. In this study, we sought to identify novel approaches that could maximally exploit the therapeutic index between tumor cells and normal tissues based on MGMT expression, as a means to enhance selective tumor cell killing. Temozolomide, unlike other alkylators, activated the ataxia telangiectasia and Rad3-related (ATR)-checkpoint kinase 1 (Chk1) axis in a manner that was highly dependent on MGMT status. Temozolomide induced growth delay, DNA double-strand breaks, and G2-M cell-cycle arrest, which led to ATR-dependent phosphorylation of Chk1; this effect was dependent on reduced MGMT expression. Treatment of MGMT-deficient cells with temozolomide increased sensitivity to ATR inhibitors both in vitro and in vivo across numerous tumor cell types. Taken together, this study reveals a novel approach for selectively targeting MGMT-deficient cells with ATR inhibitors and temozolomide. As ATR inhibitors are currently being tested in clinical trials, and temozolomide is a commonly used chemotherapeutic, this approach is clinically actionable. Furthermore, this interaction potently exploits a DNA-repair defect found in many cancers. SIGNIFICANCE: Monofunctional alkylating agents sensitize MGMT-deficient tumor cells to ATR inhibitors.
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Antineoplásicos Alquilantes/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Isoxazoles/farmacología , Pirazinas/farmacología , Temozolomida/farmacología , Proteínas Supresoras de Tumor/metabolismo , Animales , Antineoplásicos Alquilantes/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN , Sinergismo Farmacológico , Femenino , Humanos , Isoxazoles/administración & dosificación , Ratones Desnudos , Pirazinas/administración & dosificación , Temozolomida/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Dysfunction of mitochondrial translation is an increasingly important molecular cause of human disease, but structural defects of mitochondrial ribosomal subunits are rare. We used next-generation sequencing to identify a homozygous variant in the mitochondrial small ribosomal protein 14 (MRPS14, uS14m) in a patient manifesting with perinatal hypertrophic cardiomyopathy, growth retardation, muscle hypotonia, elevated lactate, dysmorphy and mental retardation. In skeletal muscle and fibroblasts from the patient, there was biochemical deficiency in complex IV of the respiratory chain. In fibroblasts, mitochondrial translation was impaired, and ectopic expression of a wild-type MRPS14 cDNA functionally complemented this defect. Surprisingly, the mutant uS14m was stable and did not affect assembly of the small ribosomal subunit. Instead, structural modeling of the uS14m mutation predicted a disruption to the ribosomal mRNA channel.Collectively, our data demonstrate pathogenic mutations in MRPS14 can manifest as a perinatal-onset mitochondrial hypertrophic cardiomyopathy with a novel molecular pathogenic mechanism that impairs the function of mitochondrial ribosomes during translation elongation or mitochondrial mRNA recruitment rather than assembly.
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Cardiomiopatía Hipertrófica/genética , Mitocondrias/genética , Enfermedades Mitocondriales/genética , Proteínas Ribosómicas/genética , Acidosis Láctica/genética , Acidosis Láctica/metabolismo , Acidosis Láctica/patología , Secuencia de Aminoácidos/genética , Cardiomiopatía Hipertrófica/metabolismo , Cardiomiopatía Hipertrófica/patología , Niño , Preescolar , Complejo IV de Transporte de Electrones/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Homocigoto , Humanos , Lactante , Recién Nacido , Mitocondrias/metabolismo , Enfermedades Mitocondriales/patología , Ribosomas Mitocondriales/metabolismo , Ribosomas Mitocondriales/patología , Mutación , LinajeRESUMEN
Optic atrophy 1 (OPA1) is a mitochondrial inner membrane protein that has an important role in mitochondrial fusion and structural integrity. Dysfunctional OPA1 mutations cause atrophy of the optic nerve leading to blindness. Here, we show that OPA1 has an important role in the innate immune system. Using conditional knockout mice lacking Opa1 in neutrophils (Opa1N∆), we report that lack of OPA1 reduces the activity of mitochondrial electron transport complex I in neutrophils. This then causes a decline in adenosine-triphosphate (ATP) production through glycolysis due to lowered NAD+ availability. Additionally, we show that OPA1-dependent ATP production in these cells is required for microtubule network assembly and for the formation of neutrophil extracellular traps. Finally, we show that Opa1N∆ mice exhibit a reduced antibacterial defense capability against Pseudomonas aeruginosa.
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Adenosina Trifosfato/metabolismo , Trampas Extracelulares/metabolismo , GTP Fosfohidrolasas/inmunología , GTP Fosfohidrolasas/metabolismo , Glucólisis/fisiología , Neutrófilos/metabolismo , Actinas/metabolismo , Animales , Antibacterianos/farmacología , Médula Ósea , Línea Celular Tumoral , Complejo I de Transporte de Electrón/efectos de los fármacos , Complejo I de Transporte de Electrón/metabolismo , GTP Fosfohidrolasas/genética , Perfilación de la Expresión Génica , Humanos , Inmunidad Innata , Pulmón/inmunología , Pulmón/microbiología , Ratones , Ratones Noqueados , Microtúbulos/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Neutrófilos/citología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/patogenicidad , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Mutations in mitochondrial ATP synthase 6 (MT-ATP6) are a frequent cause of NARP (neurogenic muscle weakness, ataxia, and retinitis pigmentosa) or Leigh syndromes, especially a point mutation at nucleotide position 8993. M.8969G>A is a rare MT-ATP6 mutation, previously reported only in three individuals, causing multisystem disorders with mitochondrial myopathy, lactic acidosis, and sideroblastic anemia or IgA nephropathy. We present two siblings with the m.8969G>A mutation and a novel, substantially milder phenotype with lactic acidosis, poor growth, and intellectual disability. Our findings expand the phenotypic spectrum and show that mtDNA mutations should be taken account also with milder, stable phenotypes.