RESUMO
The human mitochondrial genome encodes thirteen core subunits of the oxidative phosphorylation system, and defects in mitochondrial gene expression lead to severe neuromuscular disorders. However, the mechanisms of mitochondrial gene expression remain poorly understood due to a lack of experimental approaches to analyze these processes. Here, we present an in vitro system to silence translation in purified mitochondria. In vitro import of chemically synthesized precursor-morpholino hybrids allows us to target translation of individual mitochondrial mRNAs. By applying this approach, we conclude that the bicistronic, overlapping ATP8/ATP6 transcript is translated through a single ribosome/mRNA engagement. We show that recruitment of COX1 assembly factors to translating ribosomes depends on nascent chain formation. By defining mRNA-specific interactomes for COX1 and COX2, we reveal an unexpected function of the cytosolic oncofetal IGF2BP1, an RNA-binding protein, in mitochondrial translation. Our data provide insight into mitochondrial translation and innovative strategies to investigate mitochondrial gene expression.
Assuntos
Regulação da Expressão Gênica , Inativação Gênica , Genes Mitocondriais , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/genética , Células HEK293 , Humanos , Proteínas Mitocondriais/metabolismo , Oligonucleotídeos/química , Fosforilação Oxidativa , Biossíntese de Proteínas , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mitocondrial/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Cellular proteostasis requires transport of polypeptides across membranes. Although defective transport processes trigger cytosolic rescue and quality control mechanisms that clear translocases and membranes from unproductive cargo, proteins that are synthesized within mitochondria are not accessible to these mechanisms. Mitochondrial-encoded proteins are inserted cotranslationally into the inner membrane by the conserved insertase OXA1L. Here, we identify TMEM126A as a OXA1L-interacting protein. TMEM126A associates with mitochondrial ribosomes and translation products. Loss of TMEM126A leads to the destabilization of mitochondrial translation products, triggering an inner membrane quality control process, in which newly synthesized proteins are degraded by the mitochondrial iAAA protease. Our data reveal that TMEM126A cooperates with OXA1L in protein insertion into the membrane. Upon loss of TMEM126A, the cargo-blocked OXA1L insertase complexes undergo proteolytic clearance by the iAAA protease machinery together with its cargo.
Assuntos
Mitocôndrias , Membranas Mitocondriais , Mitocôndrias/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Peptídeo Hidrolases/metabolismoRESUMO
Mitochondrial ribosomes translate membrane integral core subunits of the oxidative phosphorylation system encoded by mtDNA. These translation products associate with nuclear-encoded, imported proteins to form enzyme complexes that produce ATP. Here, we show that human mitochondrial ribosomes display translational plasticity to cope with the supply of imported nuclear-encoded subunits. Ribosomes expressing mitochondrial-encoded COX1 mRNA selectively engage with cytochrome c oxidase assembly factors in the inner membrane. Assembly defects of the cytochrome c oxidase arrest mitochondrial translation in a ribosome nascent chain complex with a partially membrane-inserted COX1 translation product. This complex represents a primed state of the translation product that can be retrieved for assembly. These findings establish a mammalian translational plasticity pathway in mitochondria that enables adaptation of mitochondrial protein synthesis to the influx of nuclear-encoded subunits.
Assuntos
Ciclo-Oxigenase 1/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Transporte Ativo do Núcleo Celular , Linhagem Celular Tumoral , Ciclo-Oxigenase 1/biossíntese , Ciclo-Oxigenase 1/genética , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Complexo IV da Cadeia de Transporte de Elétrons/genética , Células HEK293 , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Fosforilação Oxidativa , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mitocondrial , Ribossomos/metabolismoRESUMO
While antibiotics are intended to specifically target bacteria, most are known to affect host cell physiology. In addition, some antibiotic classes are reported as immunosuppressive for reasons that remain unclear. Here, we show that Linezolid, a ribosomal-targeting antibiotic (RAbo), effectively blocked the course of a T cell-mediated autoimmune disease. Linezolid and other RAbos were strong inhibitors of T helper-17 cell effector function in vitro, showing that this effect was independent of their antibiotic activity. Perturbing mitochondrial translation in differentiating T cells, either with RAbos or through the inhibition of mitochondrial elongation factor G1 (mEF-G1) progressively compromised the integrity of the electron transport chain. Ultimately, this led to deficient oxidative phosphorylation, diminishing nicotinamide adenine dinucleotide concentrations and impairing cytokine production in differentiating T cells. In accordance, mice lacking mEF-G1 in T cells were protected from experimental autoimmune encephalomyelitis, demonstrating that this pathway is crucial in maintaining T cell function and pathogenicity.
Assuntos
Antibacterianos/uso terapêutico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Linezolida/uso terapêutico , Mitocôndrias/metabolismo , Peptídeos Cíclicos/uso terapêutico , Ribossomos/metabolismo , Células Th17/fisiologia , Animais , Autoimunidade/efeitos dos fármacos , Diferenciação Celular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Terapia de Alvo Molecular , Esclerose Múltipla/tratamento farmacológico , NAD/metabolismo , Fosforilação Oxidativa , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/metabolismoRESUMO
Mitochondrial-encoded subunits of the oxidative phosphorylation system assemble with nuclear-encoded subunits into enzymatic complexes. Recent findings showed that mitochondrial translation is linked to other mitochondrial functions, as well as to cellular processes. The supply of mitochondrial-encoded proteins is coordinated by the coupling of mitochondrial protein synthesis with assembly of respiratory chain complexes. MicroRNAs imported from the cytoplasm into mitochondria were, surprisingly, found to act as regulators of mitochondrial translation. In turn, translation in mitochondria controls cellular proliferation, and mitochondrial ribosomal subunits contribute to the cytoplasmic stress response. Thus, translation in mitochondria is apparently integrated into cellular processes.
Assuntos
MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/biossíntese , Biossíntese de Proteínas/fisiologia , RNA/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , Transporte de Elétrons/fisiologia , Humanos , MicroRNAs/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , RNA/genética , RNA MitocondrialRESUMO
Mitochondrial respiratory-chain complexes assemble from subunits of dual genetic origin assisted by specialized assembly factors. Whereas core subunits are translated on mitochondrial ribosomes, others are imported after cytosolic translation. How imported subunits are ushered to assembly intermediates containing mitochondria-encoded subunits is unresolved. Here, we report a comprehensive dissection of early cytochrome c oxidase assembly intermediates containing proteins required for normal mitochondrial translation and reveal assembly factors promoting biogenesis of human respiratory-chain complexes. We find that TIM21, a subunit of the inner-membrane presequence translocase, is also present in the major assembly intermediates containing newly mitochondria-synthesized and imported respiratory-chain subunits, which we term MITRAC complexes. Human TIM21 is dispensable for protein import but required for integration of early-assembling, presequence-containing subunits into respiratory-chain intermediates. We establish an unexpected molecular link between the TIM23 transport machinery and assembly of respiratory-chain complexes that regulate mitochondrial protein synthesis in response to their assembly state.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Citosol/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana Transportadoras/química , Mitocôndrias/química , Mitocôndrias/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/química , Biossíntese de ProteínasRESUMO
In humans, up to 1,500 mitochondrial precursor proteins are synthesized at cytosolic ribosomes and must be imported into the organelle. This is not only essential for mitochondrial but also for many cytosolic functions. The majority of mitochondrial precursor proteins are imported over the translocase of the outer membrane (TOM). In recent years, high-resolution structure analyses from different organisms shed light on the composition and arrangement of the TOM complex. Although significant similarities have been found, differences were also observed, which have been favored during evolution and could reflect the manifold functions of TOM with cellular signaling and its response to altered metabolic situations. A key component within these regulatory mechanisms is TOMM70, which is involved in protein import, forms contacts to the ER and the nucleus, but is also involved in cellular defense mechanisms during infections.
RESUMO
The mitochondrial genome encodes for thirteen core subunits of the oxidative phosphorylation system. These proteins assemble with imported proteins in a modular manner into stoichiometric enzyme complexes. Assembly factors assist in these biogenesis processes by providing co-factors or stabilizing transient assembly stages. However, how expression of the mitochondrial-encoded subunits is regulated to match the availability of nuclear-encoded subunits is still unresolved. Here, we address the function of MITRAC15/COA1, a protein that participates in complex I biogenesis and complex IV biogenesis. Our analyses of a MITRAC15 knockout mutant reveal that MITRAC15 is required for translation of the mitochondrial-encoded complex I subunit ND2. We find that MITRAC15 is a constituent of a ribosome-nascent chain complex during ND2 translation. Chemical crosslinking analyses demonstrate that binding of the ND2-specific assembly factor ACAD9 to the ND2 polypeptide occurs at the C-terminus and thus downstream of MITRAC15. Our analyses demonstrate that expression of the founder subunit ND2 of complex I undergoes regulation. Moreover, a ribosome-nascent chain complex with MITRAC15 is at the heart of this process.
Assuntos
Biossíntese de Proteínas , Ribossomos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Mitochondrial gene expression uses a non-universal genetic code in mammals. Besides reading the conventional AUG codon, mitochondrial (mt-)tRNAMet mediates incorporation of methionine on AUA and AUU codons during translation initiation and on AUA codons during elongation. We show that the RNA methyltransferase NSUN3 localises to mitochondria and interacts with mt-tRNAMet to methylate cytosine 34 (C34) at the wobble position. NSUN3 specifically recognises the anticodon stem loop (ASL) of the tRNA, explaining why a mutation that compromises ASL basepairing leads to disease. We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m5C34 of mt-tRNAMet to generate an f5C34 modification. In vitro codon recognition studies with mitochondrial translation factors reveal preferential utilisation of m5C34 mt-tRNAMet in initiation. Depletion of either NSUN3 or ABH1 strongly affects mitochondrial translation in human cells, implying that modifications generated by both enzymes are necessary for mt-tRNAMet function. Together, our data reveal how modifications in mt-tRNAMet are generated by the sequential action of NSUN3 and ABH1, allowing the single mitochondrial tRNAMet to recognise the different codons encoding methionine.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Códon/metabolismo , Proteínas de Membrana/metabolismo , Metiltransferases/metabolismo , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Biossíntese de Proteínas , RNA de Transferência de Metionina/metabolismo , Animais , Humanos , Mamíferos , Análise de Sequência de DNARESUMO
The three mitochondrial-encoded proteins, COX1, COX2, and COX3, form the core of the cytochrome c oxidase. Upon synthesis, COX2 engages with COX20 in the inner mitochondrial membrane, a scaffold protein that recruits metallochaperones for copper delivery to the CuA-Site of COX2. Here we identified the human protein, TMEM177 as a constituent of the COX20 interaction network. Loss or increase in the amount of TMEM177 affects COX20 abundance leading to reduced or increased COX20 levels respectively. TMEM177 associates with newly synthesized COX2 and SCO2 in a COX20-dependent manner. Our data shows that by unbalancing the amount of TMEM177, newly synthesized COX2 accumulates in a COX20-associated state. We conclude that TMEM177 promotes assembly of COX2 at the level of CuA-site formation.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Membrana/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas Mitocondriais/genéticaRESUMO
RATIONALE: Changes in redox potentials of cardiac myocytes are linked to several cardiovascular diseases. Redox alterations are currently mostly described qualitatively using chemical sensors, which however do not allow quantifying redox potentials, lack specificity, and the possibility to analyze subcellular domains. Recent advances to quantitatively describe defined redox changes include the application of genetically encoded redox biosensors. OBJECTIVE: Establishment of mouse models, which allow the quantification of the glutathione redox potential (EGSH) in the cytoplasm and the mitochondrial matrix of isolated cardiac myocytes and in Langendorff-perfused hearts based on the use of the redox-sensitive green fluorescent protein 2, coupled to the glutaredoxin 1 (Grx1-roGFP2). METHODS AND RESULTS: We generated transgenic mice with cardiac myocyte-restricted expression of Grx1-roGFP2 targeted either to the mitochondrial matrix or to the cytoplasm. The response of the roGFP2 toward H2O2, diamide, and dithiothreitol was titrated and used to determine the EGSH in isolated cardiac myocytes and in Langendorff-perfused hearts. Distinct EGSH were observed in the cytoplasm and the mitochondrial matrix. Stimulation of the cardiac myocytes with isoprenaline, angiotensin II, or exposure to hypoxia/reoxygenation additionally underscored that these compartments responded independently. A compartment-specific response was also observed 3 to 14 days after myocardial infarction. CONCLUSIONS: We introduce redox biosensor mice as a new tool, which allows quantification of defined alterations of EGSH in the cytoplasm and the mitochondrial matrix in cardiac myocytes and can be exploited to answer questions in basic and translational cardiovascular research.
Assuntos
Técnicas Biossensoriais/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Células Cultivadas , Coração/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Oxirredução , Consumo de Oxigênio/fisiologiaRESUMO
Mitochondria provide the main portion of cellular energy in form of ATP produced by the F1Fo ATP synthase, which uses the electrochemical gradient, generated by the mitochondrial respiratory chain (MRC). In human mitochondria, the MRC is composed of four multisubunit enzyme complexes, with the cytochrome c oxidase (COX, also known as complex IV) as the terminal enzyme. COX comprises 14 structural subunits, of nuclear or mitochondrial origin. Hence, mitochondria are faced with the predicament of organizing and controlling COX assembly with subunits that are synthesized by different translation machineries and that reach the inner membrane by alternative transport routes. An increasing number of COX assembly factors have been identified in recent years. Interestingly, mutations in several of these factors have been associated with human disorders leading to COX deficiency. Recently, studies have provided mechanistic insights into crosstalk between assembly intermediates, import processes and the synthesis of COX subunits in mitochondria, thus linking conceptually separated functions. This Cell Science at a Glance article and the accompanying poster will focus on COX assembly and discuss recent discoveries in the field, the molecular functions of known factors, as well as new players and control mechanisms. Furthermore, these findings will be discussed in the context of human COX-related disorders.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genéticaRESUMO
Cristae are invaginations of the mitochondrial inner membrane that are crucial for cellular energy metabolism. The formation of cristae requires the presence of a protein complex known as MICOS, which is conserved across eukaryotic species. One of the subunits of this complex, MIC10, is a transmembrane protein that supports cristae formation by oligomerization. In Drosophila melanogaster, three MIC10-like proteins with different tissue-specific expression patterns exist. We demonstrate that CG41128/MINOS1b/DmMIC10b is the major MIC10 orthologue in flies. Its loss destabilizes MICOS, disturbs cristae architecture, and reduces the life span and fertility of flies. We show that DmMIC10b has a unique ability to polymerize into bundles of filaments, which can remodel mitochondrial crista membranes. The formation of these filaments relies on conserved glycine and cysteine residues, and can be suppressed by the co-expression of other Drosophila MICOS proteins. These findings provide new insights into the regulation of MICOS in flies, and suggest potential mechanisms for the maintenance of mitochondrial ultrastructure.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila melanogaster , Membranas Mitocondriais , Citoesqueleto , Membranas Associadas à Mitocôndria , Proteínas de Drosophila/genéticaRESUMO
Mitochondria are the powerhouses of the cell as they produce the majority of ATP with their oxidative phosphorylation (OXPHOS) machinery. The OXPHOS system is composed of the F1 Fo ATP synthase and four mitochondrial respiratory chain complexes, the terminal enzyme of which is the cytochrome c oxidase (complex IV) that transfers electrons to oxygen, generating water. Complex IV comprises of 14 structural subunits of dual genetic origin: while the three core subunits are mitochondrial encoded, the remaining constituents are encoded by the nuclear genome. Hence, the assembly of complex IV requires the coordination of two spatially separated gene expression machinery. Recent efforts elucidated an increasing number of proteins involved in mitochondrial gene expression, which are linked to complex IV assembly. Additionally, several COX1 biogenesis factors have been intensively biochemically investigated and an increasing number of structural snapshots shed light on the organization of macromolecular complexes such as the mitoribosome or the cytochrome c oxidase. Here, we focus on COX1 translation regulation and highlight the advanced understanding of early steps during COX1 assembly and its link to mitochondrial translation regulation.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Proteínas de Saccharomyces cerevisiae , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Translation termination requires release factors that read a STOP codon in the decoding center and subsequently facilitate the hydrolysis of the nascent peptide chain from the peptidyl tRNA within the ribosome. In human mitochondria eleven open reading frames terminate in the standard UAA or UAG STOP codon, which can be recognized by mtRF1a, the proposed major mitochondrial release factor. However, two transcripts encoding for COX1 and ND6 terminate in the non-conventional AGA or AGG codon, respectively. How translation termination is achieved in these two cases is not known. We address this long-standing open question by showing that the non-canonical release factor mtRF1 is a specialized release factor that triggers COX1 translation termination, while mtRF1a terminates the majority of other mitochondrial translation events including the non-canonical ND6. Loss of mtRF1 leads to isolated COX deficiency and activates the mitochondrial ribosome-associated quality control accompanied by the degradation of COX1 mRNA to prevent an overload of the ribosome rescue system. Taken together, these results establish the role of mtRF1 in mitochondrial translation, which had been a mystery for decades, and lead to a comprehensive picture of translation termination in human mitochondria.
Assuntos
Ciclo-Oxigenase 1 , Proteínas Mitocondriais , Ribossomos Mitocondriais , Fatores de Terminação de Peptídeos , Humanos , Códon de Terminação/genética , Códon de Terminação/metabolismo , Ribossomos Mitocondriais/metabolismo , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Biossíntese de Proteínas , Controle de Qualidade , Ribossomos/genética , Ribossomos/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Ciclo-Oxigenase 1/genéticaRESUMO
The relevance of molecular mechanisms governing mitochondrial proteostasis to the differentiation and function of hematopoietic and immune cells is largely elusive. Through dissection of the network of proteins related to HCLS1-associated protein X-1, we defined a potentially novel functional CLPB/HAX1/(PRKD2)/HSP27 axis with critical importance for the differentiation of neutrophil granulocytes and, thus, elucidated molecular and metabolic mechanisms underlying congenital neutropenia in patients with HAX1 deficiency as well as bi- and monoallelic mutations in CLPB. As shown by stable isotope labeling by amino acids in cell culture (SILAC) proteomics, CLPB and HAX1 control the balance of mitochondrial protein synthesis and persistence crucial for proper mitochondrial function. Impaired mitochondrial protein dynamics are associated with decreased abundance of the serine-threonine kinase PRKD2 and HSP27 phosphorylated on serines 78 and 82. Cellular defects in HAX1-/- cells can be functionally reconstituted by HSP27. Thus, mitochondrial proteostasis emerges as a critical molecular and metabolic mechanism governing the differentiation and function of neutrophil granulocytes.
Assuntos
Neutrófilos , Proteostase , Proteínas Adaptadoras de Transdução de Sinal/genética , Granulócitos/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Neutrófilos/metabolismoRESUMO
The bacterial Ras-like protein Era has been reported previously to bind 16S rRNA within the 30S ribosomal subunit and to play a crucial role in ribosome assembly. An orthologue of this essential GTPase ERAL1 (Era G-protein-like 1) exists in higher eukaryotes and although its exact molecular function and cellular localization is unknown, its absence has been linked to apoptosis. In the present study we show that human ERAL1 is a mitochondrial protein important for the formation of the 28S small mitoribosomal subunit. We also show that ERAL1 binds in vivo to the rRNA component of the small subunit [12S mt (mitochondrial)-rRNA]. Bacterial Era associates with a 3' unstructured nonanucleotide immediately downstream of the terminal stem-loop (helix 45) of 16S rRNA. This site contains an AUCA sequence highly conserved across all domains of life, immediately upstream of the anti-Shine-Dalgarno sequence, which is conserved in bacteria. Strikingly, this entire region is absent from 12S mt-rRNA. We have mapped the ERAL1-binding site to a 33 nucleotide section delineating the 3' terminal stem-loop region of 12S mt-rRNA. This loop contains two adenine residues that are reported to be dimethylated on mitoribosome maturation. Furthermore, and also in contrast with the bacterial orthologue, loss of ERAL1 leads to rapid decay of nascent 12S mt-rRNA, consistent with a role as a mitochondrial RNA chaperone. Finally, whereas depletion of ERAL1 leads to apoptosis, cell death occurs prior to any appreciable loss of mitochondrial protein synthesis or reduction in the stability of mitochondrial mRNA.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , RNA Ribossômico 28S/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Northern Blotting , Linhagem Celular , Proteínas de Ligação ao GTP/genética , Células HeLa , Humanos , Proteínas Mitocondriais/biossíntese , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , RNA/genética , Interferência de RNA , Estabilidade de RNA , RNA Mitocondrial , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Ribossômico 28S/genética , Proteínas de Ligação a RNA/genéticaRESUMO
The mitochondrial proteome is built and maintained mainly by import of nuclear-encoded precursor proteins. Most of these precursors use N-terminal presequences as targeting signals that are removed by mitochondrial matrix proteases. The essential mitochondrial processing protease MPP cleaves presequences after import into the organelle thereby enabling protein folding and functionality. The cleaved presequences are subsequently degraded by peptidases. While most of these processes have been discovered in yeast, characterization of the human enzymes is still scarce. As the matrix presequence peptidase PreP has been reported to play a role in Alzheimer's disease, analysis of impaired peptide turnover in human cells is of huge interest. Here, we report the characterization of HEK293T PreP knockout cells. Loss of PreP causes severe defects in oxidative phosphorylation and changes in nuclear expression of stress response marker genes. The mitochondrial defects upon lack of PreP result from the accumulation of presequence peptides that trigger feedback inhibition of MPP and accumulation of nonprocessed precursor proteins. Also, the mitochondrial intermediate peptidase MIP that cleaves eight residues from a subset of precursors after MPP processing is compromised upon loss of PreP suggesting that PreP also degrades MIP generated octapeptides. Investigation of the PrePR183Q patient mutation associated with neurological disorders revealed that the mutation destabilizes the protein making it susceptible to enhanced degradation and aggregation upon heat shock. Taken together, our data reveal a functional coupling between precursor processing by MPP and MIP and presequence degradation by PreP in human mitochondria that is crucial to maintain a functional organellar proteome.
Assuntos
Retroalimentação Fisiológica , Metaloendopeptidases/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Serina Endopeptidases/genética , Sequência de Aminoácidos , Sequência de Bases , Sistemas CRISPR-Cas , Fracionamento Celular , Proliferação de Células , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Metaloendopeptidases/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/deficiência , Mutação , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fosforilação Oxidativa , Proteólise , Serina Endopeptidases/deficiência , Estresse Fisiológico/genética , Peptidase de Processamento MitocondrialRESUMO
Chronic pain is a prevalent medical problem, and its molecular basis remains poorly understood. Here, we demonstrate the significance of the transmembrane protein (Tmem) 160 for nerve injury-induced neuropathic pain. An extensive behavioral assessment suggests a pain modality- and entity-specific phenotype in male Tmem160 global knockout (KO) mice: delayed establishment of tactile hypersensitivity and alterations in self-grooming after nerve injury. In contrast, Tmem160 seems to be dispensable for other nerve injury-induced pain modalities, such as non-evoked and movement-evoked pain, and for other pain entities. Mechanistically, we show that global KO males exhibit dampened neuroimmune signaling and diminished TRPA1-mediated activity in cultured dorsal root ganglia. Neither these changes nor altered pain-related behaviors are observed in global KO female and male peripheral sensory neuron-specific KO mice. Our findings reveal Tmem160 as a sexually dimorphic factor contributing to the establishment, but not maintenance, of discrete nerve injury-induced pain behaviors in male mice.
Assuntos
Citocinas/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Neuralgia/imunologia , Neuralgia/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Comportamento Animal , Dor Crônica/imunologia , Dor Crônica/metabolismo , Feminino , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Inflamação , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuroimunomodulação , Testes Neuropsicológicos , Transdução de Sinais , Canal de Cátion TRPA1/metabolismoRESUMO
Human mitochondria express a genome that encodes thirteen core subunits of the oxidative phosphorylation system (OXPHOS). These proteins insert into the inner membrane co-translationally. Therefore, mitochondrial ribosomes engage with the OXA1L-insertase and membrane-associated proteins, which support membrane insertion of translation products and early assembly steps into OXPHOS complexes. To identify ribosome-associated biogenesis factors for the OXPHOS system, we purified ribosomes and associated proteins from mitochondria. We identified TMEM223 as a ribosome-associated protein involved in complex IV biogenesis. TMEM223 stimulates the translation of COX1 mRNA and is a constituent of early COX1 assembly intermediates. Moreover, we show that SMIM4 together with C12ORF73 interacts with newly synthesized cytochrome b to support initial steps of complex III biogenesis in complex with UQCC1 and UQCC2. Our analyses define the interactome of the human mitochondrial ribosome and reveal novel assembly factors for complex III and IV biogenesis that link early assembly stages to the translation machinery.