RESUMEN
Selective degradation of damaged mitochondria by autophagy (mitophagy) is proposed to play an important role in cellular homeostasis. However, the molecular mechanisms and the requirement of mitochondrial quality control by mitophagy for cellular physiology are poorly understood. Here, we demonstrated that primary human cells maintain highly active basal mitophagy initiated by mitochondrial superoxide signaling. Mitophagy was found to be mediated by PINK1/Parkin-dependent pathway involving p62 as a selective autophagy receptor (SAR). Importantly, this pathway was suppressed upon the induction of cellular senescence and in naturally aged cells, leading to a robust shutdown of mitophagy. Inhibition of mitophagy in proliferating cells was sufficient to trigger the senescence program, while reactivation of mitophagy was necessary for the anti-senescence effects of NAD precursors or rapamycin. Furthermore, reactivation of mitophagy by a p62-targeting small molecule rescued markers of cellular aging, which establishes mitochondrial quality control as a promising target for anti-aging interventions.
Asunto(s)
Senescencia Celular , Mitocondrias , Mitofagia , Ubiquitina-Proteína Ligasas , Mitofagia/efectos de los fármacos , Humanos , Senescencia Celular/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteínas Quinasas/metabolismo , Fenotipo , Autofagia/efectos de los fármacos , Proteína Sequestosoma-1/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Superóxidos/metabolismo , Proteínas de Unión al ARNRESUMEN
Digested dietary fats are taken up by enterocytes where they are assembled into pre-chylomicrons in the endoplasmic reticulum followed by transport to the Golgi for maturation and subsequent secretion to the circulation1. The role of mitochondria in dietary lipid processing is unclear. Here we show that mitochondrial dysfunction in enterocytes inhibits chylomicron production and the transport of dietary lipids to peripheral organs. Mice with specific ablation of the mitochondrial aspartyl-tRNA synthetase DARS2 (ref. 2), the respiratory chain subunit SDHA3 or the assembly factor COX10 (ref. 4) in intestinal epithelial cells showed accumulation of large lipid droplets (LDs) in enterocytes of the proximal small intestine and failed to thrive. Feeding a fat-free diet suppressed the build-up of LDs in DARS2-deficient enterocytes, which shows that the accumulating lipids derive mostly from digested fat. Furthermore, metabolic tracing studies revealed an impaired transport of dietary lipids to peripheral organs in mice lacking DARS2 in intestinal epithelial cells. DARS2 deficiency caused a distinct lack of mature chylomicrons concomitant with a progressive dispersal of the Golgi apparatus in proximal enterocytes. This finding suggests that mitochondrial dysfunction results in impaired trafficking of chylomicrons from the endoplasmic reticulum to the Golgi, which in turn leads to storage of dietary lipids in large cytoplasmic LDs. Taken together, these results reveal a role for mitochondria in dietary lipid transport in enterocytes, which might be relevant for understanding the intestinal defects observed in patients with mitochondrial disorders5.
Asunto(s)
Grasas de la Dieta , Enterocitos , Metabolismo de los Lípidos , Mitocondrias , Animales , Ratones , Aspartato-ARNt Ligasa/metabolismo , Quilomicrones/metabolismo , Grasas de la Dieta/metabolismo , Complejo II de Transporte de Electrones/metabolismo , Retículo Endoplásmico/metabolismo , Enterocitos/metabolismo , Enterocitos/patología , Células Epiteliales/metabolismo , Aparato de Golgi/metabolismo , Intestinos , Gotas Lipídicas/metabolismo , Mitocondrias/metabolismo , Mitocondrias/patologíaRESUMEN
Senescent cells drive age-related tissue dysfunction partially through the induction of a chronic senescence-associated secretory phenotype (SASP)1. Mitochondria are major regulators of the SASP; however, the underlying mechanisms have not been elucidated2. Mitochondria are often essential for apoptosis, a cell fate distinct from cellular senescence. During apoptosis, widespread mitochondrial outer membrane permeabilization (MOMP) commits a cell to die3. Here we find that MOMP occurring in a subset of mitochondria is a feature of cellular senescence. This process, called minority MOMP (miMOMP), requires BAX and BAK macropores enabling the release of mitochondrial DNA (mtDNA) into the cytosol. Cytosolic mtDNA in turn activates the cGAS-STING pathway, a major regulator of the SASP. We find that inhibition of MOMP in vivo decreases inflammatory markers and improves healthspan in aged mice. Our results reveal that apoptosis and senescence are regulated by similar mitochondria-dependent mechanisms and that sublethal mitochondrial apoptotic stress is a major driver of the SASP. We provide proof-of-concept that inhibition of miMOMP-induced inflammation may be a therapeutic route to improve healthspan.
Asunto(s)
Apoptosis , Senescencia Celular , Citosol , ADN Mitocondrial , Mitocondrias , Animales , Ratones , Citosol/metabolismo , ADN Mitocondrial/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Necrosis por Permeabilidad de la Transmembrana Mitocondrial , Prueba de Estudio Conceptual , Inflamación/metabolismo , Fenotipo , Longevidad , Envejecimiento SaludableRESUMEN
The mitochondrial intermembrane space protein AIFM1 has been reported to mediate the import of MIA40/CHCHD4, which forms the import receptor in the mitochondrial disulfide relay. Here, we demonstrate that AIFM1 and MIA40/CHCHD4 cooperate beyond this MIA40/CHCHD4 import. We show that AIFM1 and MIA40/CHCHD4 form a stable long-lived complex in vitro, in different cell lines, and in tissues. In HEK293 cells lacking AIFM1, levels of MIA40 are unchanged, but the protein is present in the monomeric form. Monomeric MIA40 neither efficiently interacts with nor mediates the import of specific substrates. The import defect is especially severe for NDUFS5, a subunit of complex I of the respiratory chain. As a consequence, NDUFS5 accumulates in the cytosol and undergoes rapid proteasomal degradation. Lack of mitochondrial NDUFS5 in turn results in stalling of complex I assembly. Collectively, we demonstrate that AIFM1 serves two overlapping functions: importing MIA40/CHCHD4 and constituting an integral part of the disulfide relay that ensures efficient interaction of MIA40/CHCHD4 with specific substrates.
Asunto(s)
Factor Inductor de la Apoptosis , Complejo I de Transporte de Electrón , Proteínas de Transporte de Membrana Mitocondrial , Factor Inductor de la Apoptosis/metabolismo , Disulfuros/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Células HEK293 , Humanos , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Oxidación-Reducción , Transporte de ProteínasRESUMEN
Metabolic plasticity is the ability of a biological system to adapt its metabolic phenotype to different environmental stressors. We used a whole-body and tissue-specific phenotypic, functional, proteomic, metabolomic and transcriptomic approach to systematically assess metabolic plasticity in diet-induced obese mice after a combined nutritional and exercise intervention. Although most obesity and overnutrition-related pathological features were successfully reverted, we observed a high degree of metabolic dysfunction in visceral white adipose tissue, characterized by abnormal mitochondrial morphology and functionality. Despite two sequential therapeutic interventions and an apparent global healthy phenotype, obesity triggered a cascade of events in visceral adipose tissue progressing from mitochondrial metabolic and proteostatic alterations to widespread cellular stress, which compromises its biosynthetic and recycling capacity. In humans, weight loss after bariatric surgery showed a transcriptional signature in visceral adipose tissue similar to our mouse model of obesity reversion. Overall, our data indicate that obesity prompts a lasting metabolic fingerprint that leads to a progressive breakdown of metabolic plasticity in visceral adipose tissue.
Asunto(s)
Resistencia a la Insulina , Tejido Adiposo/metabolismo , Animales , Homeostasis , Grasa Intraabdominal/metabolismo , Ratones , Obesidad/genética , Obesidad/metabolismo , ProteómicaRESUMEN
Proliferating cells undergo metabolic changes in synchrony with cell cycle progression and cell division. Mitochondria provide fuel, metabolites, and ATP during different phases of the cell cycle, however it is not completely understood how mitochondrial function and the cell cycle are coordinated. CLUH (clustered mitochondria homolog) is a post-transcriptional regulator of mRNAs encoding mitochondrial proteins involved in oxidative phosphorylation and several metabolic pathways. Here, we show a role of CLUH in regulating the expression of astrin, which is involved in metaphase to anaphase progression, centrosome integrity, and mTORC1 inhibition. We find that CLUH binds both the SPAG5 mRNA and its product astrin, and controls the synthesis and the stability of the full-length astrin-1 isoform. We show that CLUH interacts with astrin-1 specifically during interphase. Astrin-depleted cells show mTORC1 hyperactivation and enhanced anabolism. On the other hand, cells lacking CLUH show decreased astrin levels and increased mTORC1 signaling, but cannot sustain anaplerotic and anabolic pathways. In absence of CLUH, cells fail to grow during G1, and progress faster through the cell cycle, indicating dysregulated matching of growth, metabolism, and cell cycling. Our data reveal a role of CLUH in coupling growth signaling pathways and mitochondrial metabolism with cell cycle progression.
Asunto(s)
Mitocondrias , Proteínas Mitocondriales , Azul Alcián , Ciclo Celular , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Metafase , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Fenazinas , Fenotiazinas , ARN Mensajero/metabolismo , ResorcinolesRESUMEN
The mitochondrial integrated stress response (mitoISR) has emerged as a major adaptive pathway to respiratory chain deficiency, but both the tissue specificity of its regulation, and how mitoISR adapts to different levels of mitochondrial dysfunction are largely unknown. Here, we report that diverse levels of mitochondrial cardiomyopathy activate mitoISR, including high production of FGF21, a cytokine with both paracrine and endocrine function, shown to be induced by respiratory chain dysfunction. Although being fully dispensable for the cell-autonomous and systemic responses to severe mitochondrial cardiomyopathy, in the conditions of mild-to-moderate cardiac OXPHOS dysfunction, FGF21 regulates a portion of mitoISR. In the absence of FGF21, a large part of the metabolic adaptation to mitochondrial dysfunction (one-carbon metabolism, transsulfuration, and serine and proline biosynthesis) is strongly blunted, independent of the primary mitoISR activator ATF4. Collectively, our work highlights the complexity of mitochondrial stress responses by revealing the importance of the tissue specificity and dose dependency of mitoISR.
RESUMEN
Assembly of the dimeric complex III (CIII2) in the mitochondrial inner membrane is an intricate process in which several accessory proteins are involved as assembly factors. Despite numerous studies, this process has yet to be fully understood. Here we report the identification of human OCIAD2 (ovarian carcinoma immunoreactive antigen-like protein 2) as an assembly factor for CIII2. OCIAD2 was found to be deregulated in several carcinomas and also in some neurogenerative disorders; however, its nonpathological role had not been elucidated. We have shown that OCIAD2 localizes to mitochondria and interacts with electron transport chain (ETC) proteins. Complete loss of OCIAD2 using gene editing in HEK293 cells resulted in abnormal mitochondrial morphology, a substantial decrease of both CIII2 and supercomplex III2+IV, and a reduction in CIII enzymatic activity. Identification of OCIAD2 as a protein required for assembly of functional CIII2 provides a new insight into the biogenesis and architecture of the ETC. Elucidating the mechanism of OCIAD2 action is important both for the understanding of cellular metabolism and for an understanding of its role in malignant transformation.
Asunto(s)
Carcinoma , Neoplasias Ováricas , Carcinoma/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Femenino , Células HEK293 , Humanos , Mitocondrias/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Ováricas/metabolismoRESUMEN
To ensure correct function, mitochondria have developed several mechanisms of protein quality control (QC). Protein homeostasis highly relies on chaperones and proteases to maintain proper folding and remove damaged proteins that might otherwise form cell-toxic aggregates. Besides quality control, mitochondrial proteases modulate and regulate many essential functions, such as trafficking, processing and activation of mitochondrial proteins, mitochondrial dynamics, mitophagy and apoptosis. Therefore, the impaired function of mitochondrial proteases is associated with various pathological conditions, including cancer, metabolic syndromes and neurodegenerative disorders. This review recapitulates and discusses the emerging roles of two major proteases of the mitochondrial matrix, LON and ClpXP. Although commonly acknowledge for their protein quality control role, recent advances have uncovered several highly regulated processes controlled by the LON and ClpXP connected to mitochondrial gene expression and respiratory chain function maintenance. Furthermore, both proteases have been lately recognized as potent targets for anticancer therapies, and we summarize those findings.
Asunto(s)
Neoplasias , Péptido Hidrolasas , Humanos , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Endopeptidasas/metabolismoRESUMEN
In response to disturbed mitochondrial gene expression and protein synthesis, an adaptive transcriptional response sharing a signature of the integrated stress response (ISR) is activated. We report an intricate interplay between three transcription factors regulating the mitochondrial stress response: CHOP, C/EBPß, and ATF4. We show that CHOP acts as a rheostat that attenuates prolonged ISR, prevents unfavorable metabolic alterations, and postpones the onset of mitochondrial cardiomyopathy. Upon mitochondrial dysfunction, CHOP interaction with C/EBPß is needed to adjust ATF4 levels, thus preventing overactivation of the ATF4-regulated transcriptional program. Failure of this interaction switches ISR from an acute to a chronic state, leading to early respiratory chain deficiency, energy crisis, and premature death. Therefore, contrary to its previously proposed role as a transcriptional activator of mitochondrial unfolded protein response, our results highlight a role of CHOP in the fine-tuning of mitochondrial ISR in mammals.
RESUMEN
The human mitochondrial ClpXP protease complex (HsClpXP) has recently attracted major attention as a target for novel anti-cancer therapies. Despite its important role in disease progression, the cellular role of HsClpXP is poorly characterized and only few small molecule inhibitors have been reported. Herein, we screened previously established S. aureus ClpXP inhibitors against the related human protease complex and identified potent small molecules against human ClpXP. The hit compounds showed anti-cancer activity in a panoply of leukemia, liver and breast cancer cell lines. We found that the bacterial ClpXP inhibitor 334 impairs the electron transport chain (ETC), enhances the production of mitochondrial reactive oxygen species (mtROS) and thereby promotes protein carbonylation, aberrant proteostasis and apoptosis. In addition, 334 induces cell death in re-isolated patient-derived xenograft (PDX) leukemia cells, potentiates the effect of DNA-damaging cytostatics and re-sensitizes resistant cancers to chemotherapy in non-apoptotic doses.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas Mitocondriales/antagonistas & inhibidores , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Staphylococcus aureusRESUMEN
Mitochondria are highly dynamic and stress-responsive organelles that are renewed, maintained and removed by a number of different mechanisms. Recent findings bring more evidence for the focused, defined, and regulatory function of the intramitochondrial proteases extending far beyond the traditional concepts of damage control and stress responses. Until recently, the macrodegradation processes, such as mitophagy, were promoted as the major regulator of OXPHOS remodelling and turnover. However, the spatiotemporal dynamics of the OXPHOS system can be greatly modulated by the intrinsic mitochondrial mechanisms acting apart from changes in the global mitochondrial dynamics. This, in turn, may substantially contribute to the shaping of the metabolic status of the cell.
Asunto(s)
Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Mitofagia , Fosforilación Oxidativa , Proteolisis , HumanosRESUMEN
The mammalian mitochondrial proteome consists of more than 1100 annotated proteins and their proteostasis is regulated by only a few ATP-dependent protease complexes. Technical advances in protein mass spectrometry allowed for detailed description of the mitoproteome from different species and tissues and their changes under specific conditions. However, protease-substrate relations within mitochondria are still poorly understood. Here, we combined Terminal Amine Isotope Labeling of Substrates (TAILS) N termini profiling of heart mitochondria proteomes isolated from wild type and Clpp-/- mice with a classical substrate-trapping screen using FLAG-tagged proteolytically active and inactive CLPP variants to identify new ClpXP substrates in mammalian mitochondria. Using TAILS, we identified N termini of more than 200 mitochondrial proteins. Expected N termini confirmed sequence determinants for mitochondrial targeting signal (MTS) cleavage and subsequent N-terminal processing after import, but the majority were protease-generated neo-N termini mapping to positions within the proteins. Quantitative comparison revealed widespread changes in protein processing patterns, including both strong increases or decreases in the abundance of specific neo-N termini, as well as an overall increase in the abundance of protease-generated neo-N termini in CLPP-deficient mitochondria that indicated altered mitochondrial proteostasis. Based on the combination of altered processing patterns, protein accumulation and stabilization in CLPP-deficient mice and interaction with CLPP, we identified OAT, HSPA9 and POLDIP2 and as novel bona fide ClpXP substrates. Finally, we propose that ClpXP participates in the cooperative degradation of UQCRC1. Together, our data provide the first landscape of the heart mitochondria N terminome and give further insights into regulatory and assisted proteolysis mediated by ClpXP.
Asunto(s)
Endopeptidasa Clp/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteolisis , Proteoma/metabolismo , Secuencia de Aminoácidos , Animales , Endopeptidasa Clp/deficiencia , Ratones , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Procesamiento Proteico-Postraduccional , Reproducibilidad de los Resultados , Especificidad por SustratoRESUMEN
Regulation of the turnover of complex I (CI), the largest mitochondrial respiratory chain complex, remains enigmatic despite huge advancement in understanding its structure and the assembly. Here, we report that the NADH-oxidizing N-module of CI is turned over at a higher rate and largely independently of the rest of the complex by mitochondrial matrix protease ClpXP, which selectively removes and degrades damaged subunits. The observed mechanism seems to be a safeguard against the accumulation of dysfunctional CI arising from the inactivation of the N-module subunits due to attrition caused by its constant activity under physiological conditions. This CI salvage pathway maintains highly functional CI through a favorable mechanism that demands much lower energetic cost than de novo synthesis and reassembly of the entire CI. Our results also identify ClpXP activity as an unforeseen target for therapeutic interventions in the large group of mitochondrial diseases characterized by the CI instability.
Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Animales , Complejo I de Transporte de Electrón/genética , Endopeptidasa Clp/genética , Endopeptidasa Clp/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismo , Mioblastos/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
Most manipulations that extend lifespan also increase resistance to various stress factors and environmental cues in a range of animals from yeast to mammals. However, the underlying molecular mechanisms regulating stress resistance during aging are still largely unknown. Here we identify Krüppel-like factor 1 (KLF-1) as a mediator of a cytoprotective response that dictates longevity induced by reduced mitochondrial function. A redox-regulated KLF-1 activation and transfer to the nucleus coincides with the peak of somatic mitochondrial biogenesis that occurs around a transition from larval stage L3 to D1. We further show that KLF-1 activates genes involved in the xenobiotic detoxification programme and identified cytochrome P450 oxidases, the KLF-1 main effectors, as longevity-assurance factors of mitochondrial mutants. Collectively, these findings underline the importance of the xenobiotic detoxification in the mitohormetic, longevity assurance pathway and identify KLF-1 as a central factor in orchestrating this response.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Mitocondrias/genética , Xenobióticos/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Femenino , Inactivación Metabólica , Factores de Transcripción de Tipo Kruppel/genética , Longevidad , Masculino , Mitocondrias/metabolismoRESUMEN
Mitochondria are fundamental for cellular metabolism as they are both a source and a target of nutrient intermediates originating from converging metabolic pathways, and their role in the regulation of systemic metabolism is increasingly recognized. Thus, maintenance of mitochondrial homeostasis is indispensable for a functional energy metabolism of the whole organism. Here, we report that loss of the mitochondrial matrix protease CLPP results in a lean phenotype with improved glucose homeostasis. Whole-body CLPP-deficient mice are protected from diet-induced obesity and insulin resistance, which was not present in mouse models with either liver- or muscle-specific depletion of CLPP However, CLPP ablation also leads to a decline in brown adipocytes function leaving mice unable to cope with a cold-induced stress due to non-functional adaptive thermogenesis. These results demonstrate a critical role for CLPP in different metabolic stress conditions such as high-fat diet feeding and cold exposure providing tools to understand pathologies with deregulated Clpp expression and novel insights into therapeutic approaches against metabolic dysfunctions linked to mitochondrial diseases.
Asunto(s)
Endopeptidasa Clp/genética , Homeostasis , Síndrome Metabólico/metabolismo , Termogénesis , Adipocitos Marrones/metabolismo , Adipogénesis , Animales , Frío , Dieta Alta en Grasa , Metabolismo Energético , Eliminación de Gen , Glucosa/metabolismo , Resistencia a la Insulina , Ratones , Ratones Noqueados , Mitocondrias , Estrés FisiológicoRESUMEN
MtDNA mutations are one of the hallmarks of ageing and age-related diseases. It is well established that somatic point mutations accumulate in mtDNA of multiple organs and tissues with increasing age and heteroplasmy is universal in mammals. However, the origin of these mutations remains controversial. The long-lasting hypothesis stating that mtDNA mutations emanate from oxidative damage via a self-perpetuating mechanism has been extensively challenged in recent years. Contrary to this initial ascertainment, mtDNA appears to be well protected from action of reactive oxygen species (ROS) through robust protein coating and endomitochondrial microcompartmentalization. Extensive development of scrupulous high-throughput DNA sequencing methods suggests that an imperfect replication process, rather than oxidative lesions are the main sources of mtDNA point mutations, indicating that mtDNA polymerase γ (POLG) might be responsible for the majority of mtDNA mutagenic events. Here, we summarize the recent knowledge in prevention and defence of mtDNA oxidative lesions and discuss the plausible mechanisms of mtDNA point mutation generation and fixation.
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Envejecimiento/fisiología , ADN Mitocondrial/genética , Mutación/genética , Estrés Oxidativo/fisiología , Envejecimiento/genética , Animales , Humanos , Estrés Oxidativo/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset, autosomal dominant familial Parkinson`s disease (PD) and variation at the LRRK2 locus contributes to the risk for idiopathic PD. LRRK2 can function as a protein kinase and mutations lead to increased kinase activity. To elucidate the pathophysiological mechanism of the R1441C mutation in the GTPase domain of LRRK2, we expressed human wild-type or R1441C LRRK2 in dopaminergic neurons of Drosophila and observe reduced locomotor activity, impaired survival and an age-dependent degeneration of dopaminergic neurons thereby creating a new PD-like model. To explore the function of LRRK2 variants in vivo, we performed mass spectrometry and quantified 3,616 proteins in the fly brain. We identify several differentially-expressed cytoskeletal, mitochondrial and synaptic vesicle proteins (SV), including synaptotagmin-1, syntaxin-1A and Rab3, in the brain of this LRRK2 fly model. In addition, a global phosphoproteome analysis reveals the enhanced phosphorylation of several SV proteins, including synaptojanin-1 (pThr1131) and the microtubule-associated protein futsch (pSer4106) in the brain of R1441C hLRRK2 flies. The direct phosphorylation of human synaptojanin-1 by R1441C hLRRK2 could further be confirmed by in vitro kinase assays. A protein-protein interaction screen in the fly brain confirms that LRRK2 robustly interacts with numerous SV proteins, including synaptojanin-1 and EndophilinA. Our proteomic, phosphoproteomic and interactome study in the Drosophila brain provides a systematic analyses of R1441C hLRRK2-induced pathobiological mechanisms in this model. We demonstrate for the first time that the R1441C mutation located within the LRRK2 GTPase domain induces the enhanced phosphorylation of SV proteins in the brain.
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Encéfalo/metabolismo , Neuronas Dopaminérgicas/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Enfermedad de Parkinson/genética , Proteoma/genética , Animales , Animales Modificados Genéticamente , Encéfalo/patología , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/patología , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulación de la Expresión Génica , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/biosíntesis , Mutación , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Monoéster Fosfórico Hidrolasas/biosíntesis , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , Mapas de Interacción de Proteínas , Vesículas Sinápticas/genética , Sinaptotagmina I/biosíntesis , Sinaptotagmina I/genética , Sintaxina 1/biosíntesis , Sintaxina 1/genética , Proteínas de Unión al GTP rab3/biosíntesis , Proteínas de Unión al GTP rab3/genéticaRESUMEN
Despite being one of the most studied proteases in bacteria, very little is known about the role of ClpXP in mitochondria. We now present evidence that mammalian CLPP has an essential role in determining the rate of mitochondrial protein synthesis by regulating the level of mitoribosome assembly. Through a proteomic approach and the use of a catalytically inactive CLPP, we produced the first comprehensive list of possible mammalian ClpXP substrates involved in the regulation of mitochondrial translation, oxidative phosphorylation, and a number of metabolic pathways. We further show that the defect in mitoribosomal assembly is a consequence of the accumulation of ERAL1, a putative 12S rRNA chaperone, and novel ClpXP substrate. The presented data suggest that the timely removal of ERAL1 from the small ribosomal subunit is essential for the efficient maturation of the mitoribosome and a normal rate of mitochondrial translation.