RESUMO
The reversible oxidation of methionine plays a crucial role in redox regulation of proteins. Methionine oxidation in proteins causes major structural modifications that can destabilize and abrogate their function. The highly conserved methionine sulfoxide reductases protect proteins from oxidative damage by reducing their oxidized methionines, thus restoring their stability and function. Deletion or mutation in conserved methionine sulfoxide reductases leads to aging and several human neurological disorders and also reduces yeast growth on nonfermentable carbon sources. Despite their importance in human health, limited information about their physiological substrates in humans and yeast is available. For the first time, we show that Mxr2 interacts in vivo with two core proteins of the cytoplasm to vacuole targeting (Cvt) autophagy pathway, Atg19, and Ape1 in Saccharomyces cerevisiae. Deletion of MXR2 induces instability and early turnover of immature Ape1 and Atg19 proteins and reduces the leucine aminopeptidase activity of Ape1 without affecting the maturation process of Ape1. Additonally, Mxr2 interacts with the immature Ape1, dependent on Met17 present within the propeptide of Ape1 as a single substitution mutation of Met17 to Leu abolishes this interaction. Importantly, Ape1 M17L mutant protein resists oxidative stress-induced degradation in WT and mxr2Δ cells. By identifying Atg19 and Ape1 as cytosolic substrates of Mxr2, our study maps the hitherto unexplored connection between Mxr2 and the Cvt autophagy pathway and sheds light on Mxr2-dependent oxidative regulation of the Cvt pathway.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Autofagia , Metionina/metabolismo , Metionina Sulfóxido Redutases/genética , Metionina Sulfóxido Redutases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Citoplasma/metabolismo , Vacúolos/metabolismo , Estresse Oxidativo , Estabilidade ProteicaRESUMO
Mitochondrial morphology and dynamics maintain mitochondrial integrity by regulating its size, shape, distribution, and connectivity, thereby modulating various cellular processes. Several studies have established a functional link between mitochondrial dynamics, mitophagy, and cell death, but further investigation is needed to identify specific proteins involved in mitochondrial dynamics. Any alteration in the integrity of mitochondria has severe ramifications that include disorders like cancer and neurodegeneration. In this study, we used budding yeast as a model organism and found that Pil1, the major component of the eisosome complex, also localizes to the periphery of mitochondria. Interestingly, the absence of Pil1 causes the branched tubular morphology of mitochondria to be abnormally fused or aggregated, whereas its overexpression leads to mitochondrial fragmentation. Most importantly, pil1Δ cells are defective in mitophagy and bulk autophagy, resulting in elevated levels of reactive oxygen species and protein aggregates. In addition, we show that pil1Δ cells are more prone to cell death. Yeast two-hybrid analysis and co-immunoprecipitations show the interaction of Pil1 with two major proteins in mitochondrial fission, Fis1 and Dnm1. Additionally, our data suggest that the role of Pil1 in maintaining mitochondrial shape is dependent on Fis1 and Dnm1, but it functions independently in mitophagy and cell death pathways. Together, our data suggest that Pil1, an eisosome protein, is a novel regulator of mitochondrial morphology, mitophagy, and cell death.
Assuntos
Dinâmica Mitocondrial , Mitofagia , Fosfoproteínas , Proteínas de Saccharomyces cerevisiae , Morte Celular , Mitocôndrias/genética , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mitofagia/fisiologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Germline mutations in RECQL4 helicase are associated with Rothmund-Thomson syndrome, which is characterized by a predisposition to cancer. RECQL4 localizes to the mitochondria, where it acts as an accessory factor during mitochondrial DNA replication. To understand the specific mitochondrial functions of RECQL4, we created isogenic cell lines, in which the mitochondrial localization of the helicase was either retained or abolished. The mitochondrial integrity was affected due to the absence of RECQL4 in mitochondria, leading to a decrease in F1F0-ATP synthase activity. In cells where RECQL4 does not localize to mitochondria, the membrane potential was decreased, whereas ROS levels increased due to the presence of high levels of catalytically inactive SOD2. Inactive SOD2 accumulated owing to diminished SIRT3 activity. Lack of the mitochondrial functions of RECQL4 led to aerobic glycolysis that, in turn, led to an increased invasive capability within these cells. Together, this study demonstrates for the first time that, owing to its mitochondrial functions, the accessory mitochondrial replication helicase RECQL4 prevents the invasive step in the neoplastic transformation process.
Assuntos
Transformação Celular Neoplásica/metabolismo , Glucose/metabolismo , Glicólise/fisiologia , Mitocôndrias/metabolismo , RecQ Helicases/metabolismo , Sirtuína 3/metabolismo , Superóxido Dismutase/metabolismo , Linhagem Celular , Replicação do DNA/genética , DNA Mitocondrial/genética , Células HCT116 , Humanos , Potencial da Membrana Mitocondrial/fisiologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Espécies Reativas de Oxigênio/metabolismo , RecQ Helicases/genética , Síndrome de Rothmund-Thomson/genéticaRESUMO
Noncanonical functions of several nuclear transcription factors in the mitochondria have been gaining exceptional traction over the years. These transcription factors include nuclear hormone receptors like estrogen, glucocorticoid, and thyroid hormone receptors: p53, IRF3, STAT3, STAT5, CREB, NF-kB, and MEF-2D. Mitochondria-localized nuclear transcription factors regulate mitochondrial processes like apoptosis, respiration and mitochondrial transcription albeit being nuclear in origin and having nuclear functions. Hence, the cell permits these multi-stationed transcription factors to orchestrate and fine-tune cellular metabolism at various levels of operation. Despite their ubiquitous distribution in different subcompartments of mitochondria, their targeting mechanism is poorly understood. Here, we review the current status of mitochondria-localized transcription factors and discuss the possible targeting mechanism besides the functional interplay between these factors.
Assuntos
Mitocôndrias/metabolismo , Fatores de Transcrição/fisiologia , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Humanos , Fator Regulador 3 de Interferon/fisiologia , NF-kappa B/fisiologia , Transporte Proteico , Receptores de Estrogênio/análise , Receptores de Glucocorticoides/análise , Receptores dos Hormônios Tireóideos/análise , Fatores de Transcrição STAT/fisiologiaRESUMO
Mge1, a yeast homologue of Escherichia coli GrpE, is an evolutionarily conserved homodimeric nucleotide exchange factor of mitochondrial Hsp70. Temperature-dependent reversible structural alteration from a dimeric to a monomeric form is critical for Mge1 to act as a thermosensor. However, very limited information about the structural component or amino acid residue(s) that contributes to thermal sensing of Mge1/GrpE is available. In this report, we have identified a single point mutation, His167 to Leu (H167L), within the hinge region of Mge1 that confers thermal resistance to yeast. Circular dichroism, cross-linking, and refolding studies with recombinant proteins show that the Mge1 H167L mutant has increased thermal stability compared to that of wild-type Mge1 and also augments Hsp70-mediated protein refolding activity. While thermal denaturation studies suggest flexibility in the mutant, ionic quenching studies and limited proteolysis analysis reveal a relatively more rigid structure compared to that of the wild type. Intriguingly, Thermus thermophilus GrpE has a leucine at the corresponding position akin to the Mge1 mutant, and thermophilus proteins are well-known for their rigidity and hydrophobicity. Taken together, our results show that the yeast Mge1 H167L mutant functionally and structurally mimics T. thermophilus GrpE.
Assuntos
Proteínas de Choque Térmico HSP70/genética , Temperatura Alta , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas Mitocondriais/genética , Chaperonas Moleculares/genética , Mutação Puntual , Proteínas de Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Dicroísmo Circular , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Desnaturação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Estabilidade Proteica , Desdobramento de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Mitochondria play an essential role in synthesis and export of iron-sulfur (Fe-S) clusters to other sections of a cell. Although the mechanism of Fe-S cluster synthesis is well elucidated, information on the identity of the proteins involved in the export pathway is limited. The present study identifies hMIA40 (human mitochondrial intermembrane space import and assembly protein 40), also known as CHCHD4 (coiled-coil-helix-coiled-coil-helix domain-containing 4), as a component of the mitochondrial Fe-S cluster export machinery. hMIA40 is an iron-binding protein with the ability to bind iron in vivo and in vitro. hMIA40 harbours CPC (Cys-Pro-Cys) motif-dependent Fe-S clusters that are sensitive to oxidation. Depletion of hMIA40 results in accumulation of iron in mitochondria concomitant with decreases in the activity and stability of Fe-S-containing cytosolic enzymes. Intriguingly, overexpression of either the mitochondrial export component or cytosolic the Fe-S cluster assembly component does not have any effect on the phenotype of hMIA40-depleted cells. Taken together, our results demonstrate an indispensable role for hMIA40 for the export of Fe-S clusters from mitochondria.
Assuntos
Ferro/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Enxofre/metabolismo , Motivos de Aminoácidos , Transporte Biológico Ativo/fisiologia , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora MitocondrialRESUMO
In response to apoptotic stimuli, cytochrome c, an inter-membrane space protein is released from mitochondria to activate the cascade of caspases that leads to apoptosis. Recent evidence suggests that cytochrome c interacts with tRNA in the cytoplasm and this interaction was shown to inhibit the caspase mediated apoptotic process. Interestingly, cytochrome c does not contain any putative RNA binding domain. In this report, we sought to define the structural component of cytochrome c that is involved in binding of tRNA. By using gel mobility shift assays, we show that holocytochrome c can interact with tRNA but not apocytochrome c that lacks the heme domain suggesting that heme is essential for the interaction of cytochrome c to tRNA. In addition, using in vitro cross linking and circular dichroism spectroscopic studies, we show that cytochrome c can undergo heme mediated oligomerization. Prevention of heme mediated oligomerization of cytochrome c by potassium ferricyanide treatment prevents the binding of tRNA and promotes caspase activation. Our studies provide a novel regulation of apoptosis by heme dependent tRNA interaction to cytochrome c.
Assuntos
Apoptose , Citocromos c/química , Citocromos c/metabolismo , Heme/metabolismo , RNA de Transferência/metabolismo , Animais , Caspases/metabolismo , Bovinos , Células HEK293 , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , RNA de Transferência/químicaRESUMO
Mitochondria empower the liver to regulate lipid homeostasis by enabling fatty acid oxidation during starvation and lipogenesis during nutrient-rich conditions. It is unknown if mitochondria can seamlessly regulate these two distinct processes or if two discrete populations of mitochondria achieve these two functions in the liver. For the first time in the liver, we report the isolation of two distinct populations of mitochondria from male Wistar rats on an ad-libitum diet: cytoplasmic mitochondria and lipid droplet-associated mitochondria. Our studies show that while lipid droplet mitochondria exhibit higher fatty acid oxidation and are marked by enhanced levels of pACC2, MFN2, and CPT1 activity, cytoplasmic mitochondria are associated with higher respiration capacity. Notably, lipid droplet-associated mitochondria isolated from a non-alcoholic fatty liver disease (NAFLD) rat model are compromised for fatty acid oxidation. We demonstrate the importance of functional segregation of mitochondria as any aberration in lipid droplet-associated mitochondria may lead to NAFLD.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Masculino , Ratos , Animais , Hepatopatia Gordurosa não Alcoólica/metabolismo , Ratos Wistar , Gotículas Lipídicas , Fígado/metabolismo , Mitocôndrias/metabolismo , Metabolismo dos Lipídeos , Metabolismo Energético , Ácidos Graxos/metabolismo , LipídeosRESUMO
Mitochondrial dysfunction mediated by CCCP (carbonyl cyanide m-chlorophenyl hydrazone), an inhibitor of mitochondrial oxidative phosphorylation, evokes the integrated stress response (ISR), which is analyzed here by eIF2α phosphorylation and expression profiles of ATF4 and CHOP proteins. Our findings suggest that the CCCP-induced ISR pathway is mediated by activation of HRI kinase, but not by GCN2, PERK, or PKR. Also, CCCP activates AMPK, a cellular energy sensor, and AKT, a regulator implicated in cell survival, and suppresses phosphorylation of mTORC1 substrates eIF4E-BP1 and S6K. CCCP also downregulates translation and promotes autophagy, leading to noncaspase-mediated cell death in HepG2 cells. All these events are neutralized by NAC, an anti-ROS, suggesting that CCCP-induced mitochondrial dysfunction promotes oxidative stress. ISRIB, an inhibitor of the ISR pathway, mitigates CCCP-induced expression of ATF4 and CHOP, activation of AKT, and autophagy, similar to NAC. However, it fails to reverse CCCP-induced AMPK activation, suggesting that CCCP-induced autophagy is dependent on ISR and independent of AMPK activation. ISRIB restores partly, inhibition in eIF4E-BP1 phosphorylation, promotes eIF2α phosphorylation, albeit slowly, and mitigates suppression of translation accordingly, in CCCP-treated cells. These findings are consistent with the idea that CCCP-induced oxidative stress leading to eIF2α phosphorylation and ATF4 expression, which is known to stimulate genes involved in autophagy, play a pro-survival role together with AKT activation and regulate mTOR-mediated eIF4E-BP1 phosphorylation.
Assuntos
Fator 4 Ativador da Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Fator de Iniciação 2 em Eucariotos/genética , Mitocôndrias/genética , Proteínas Quinases/genética , Quinases Proteína-Quinases Ativadas por AMP , Acetamidas/farmacologia , Autofagia/genética , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Cicloexilaminas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Fator de Transcrição CHOP/genéticaRESUMO
It has become amply clear that mitochondrial function defined by quality, quantity, dynamics, homeostasis, and regulated by mitophagy and mitochondrial biogenesis is a critical metric of human aging and disease. As a consequence, therapeutic interventions that can improve mitochondrial function can have a profound impact on human health and longevity. Kisspeptins are neuropeptides belonging to the family of metastasis suppressors that are known to regulate functions like fertility, reproduction, and metabolism. Using SKNSH cell line, hippocampus explant cultures and hippocampus of aging Wistar rat models, we show that Kisspeptin-10 (Kp) induces autophagy and mitophagy via calcium, Ca2+/CaM-dependent protein kinase kinase ß (CaMKKß), AMP-activated protein kinase (AMPK), and Unc-51 like autophagy activating kinase (ULK1) signaling pathway that is independent of mammalian target of rapamycin (mTOR). Intriguingly, Kp administration in vivo also results in the enhancement of mitochondrial number, complex I activity, and Adenosine Triphosphate (ATP) levels. This study uncovers potential effects of Kp in protecting mitochondrial health and as a possible therapeutic intervention to hippocampus associated impairments such as memory, cognitive aging, and other diseases linked to mitochondrial dysfunction.
Assuntos
Envelhecimento/genética , Kisspeptinas/genética , Neurônios/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Envelhecimento/patologia , Animais , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Encéfalo/metabolismo , Encéfalo/patologia , Cálcio/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina , Complexo I de Transporte de Elétrons/genética , Hipocampo/metabolismo , Hipocampo/patologia , Mitocôndrias , Mitofagia/genética , Neurônios/patologia , Biogênese de Organelas , Proteínas Quinases/genética , Ratos , Transdução de Sinais/genéticaRESUMO
Aging leads to the diminished pulsatile secretion of hypothalamic gonadotropin-releasing hormone (GnRH). Kisspeptin (Kp), the upstream regulator of the hypothalamic-pituitary-gonadal (HPG) axis, regulates GnRH synthesis and release through its cognate receptor, G-protein coupled receptor 54 (GPR54). In turn, GnRH regulates GPR54 expression. GnRH administration into the third ventricle has been shown to induce neurogenesis in different brain regions in old age. However, aging-associated changes in hypothalamic and extra-hypothalamic GPR54 expression were unclear. Therefore, the expression levels of GPR54 were evaluated in various brain regions of adult (age, 3-4 months) and old (age, 20-24 months) male Wistar rats in the present study. In the hypothalamus, mRNA and protein levels of Kp and GPR54 were identified to be significantly decreased in old age. Furthermore, GnRH1 expression in the hypothalamus was analyzed to observe the functional consequence of a reduced Kp-GPR54 system in the hypothalamus. It was found that hypothalamic GnRH1 levels were significantly decreased in old age. As GnRH regulates GPR54 levels, GPR54 was examined in extra-hypothalamic regions. GPR54 levels were found to be significantly decreased in the hippocampus and medulla and pons in old-age rats when compared to adult rats. Notably, GPR54 expression was observed in the frontal lobe, cortex, midbrain and cerebellum of adult and old-age rats; however, the difference between the two groups was not statistically significant. To the best of our knowledge, this is the first study that provides the quantitative distribution of GPR54 in different brain regions during aging. Thus, the reduced levels of Kp and its receptor, GPR54 in the hypothalamus could be cumulatively responsible for reduced levels of GnRH observed in old age.
RESUMO
Previously we showed that xenobiotic inducible cytochrome P450 (CYP) proteins are bimodally targeted to the endoplasmic reticulum and mitochondria. In this study, we investigated the mechanism of delivery of chimeric signal containing CYP proteins to the peripheral and channel-forming mitochondrial outer membrane translocases (TOMs). CYP+33/1A1 and CYP2B1 did not require peripheral TOM70, TOM20, or TOM22 for translocation through the channel-forming TOM40 protein. In contrast, CYP+5/1A1 and CYP2E1 were able to bypass TOM20 and TOM22 but required TOM70. CYP27, which contains a canonical cleavable mitochondrial signal, required all of the peripheral TOMs for its mitochondrial translocation. We investigated the underlying mechanisms of bypass of peripheral TOMs by CYPs with chimeric signals. The results suggested that interaction of CYPs with Hsp70, a cytosolic chaperone involved in the mitochondrial import, alone was sufficient for the recognition of chimeric signals by peripheral TOMs. However, sequential interaction of chimeric signal containing CYPs with Hsp70 and Hsp90 resulted in the bypass of peripheral TOMs, whereas CYP27A1 interacted only with Hsp70 and was not able to bypass peripheral TOMs. Our results also show that delivery of a chimeric signal containing client protein by Hsp90 required the cytosol-exposed NH(2)-terminal 143 amino acids of TOM40. TOM40 devoid of this domain was unable to import CYP proteins. These results suggest that compared with the unimodal mitochondrial targeting signals, the chimeric mitochondrial targeting signals are highly evolved and dynamic in nature.
Assuntos
Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Transporte Biológico Ativo , Sistema Enzimático do Citocromo P-450/genética , Genes Fúngicos , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Técnicas In Vitro , Proteínas de Membrana Transportadoras/genética , Proteínas de Transporte da Membrana Mitocondrial , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Modelos Biológicos , Mutação , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Coelhos , Ratos , Receptores de Superfície Celular , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
STAT3, a transcription factor involved in various physiological and pathological processes, is also present in mitochondria. Mitochondrial STAT3 regulates complex I activity and reactive oxygen species (ROS) production, yet the mechanisms governing its translocation to mitochondria remain poorly understood. In this study, we show that rotenone-induced ROS triggers the Ser727 phosphorylation of STAT3 and its increased mitochondrial localisation. Furthermore, we show that STAT3-depleted cells display increased ROS levels during rotenone treatment. Targeted expression in mitochondria of wild-type STAT3 - but not S727A mutant - lowers ROS levels, indicating the importance of Ser727 phosphorylation, both in rotenone-induced mitochondrial targeting and quenching of ROS levels. Together, our results demonstrate a novel STAT3-mediated feedback mechanism to maintain redox homeostasis during stress.
Assuntos
Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rotenona/farmacologia , Fator de Transcrição STAT3/metabolismo , Células HeLa , Humanos , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Fosforilação/efeitos dos fármacos , Fator de Transcrição STAT3/genética , Serina/metabolismoRESUMO
Human MIA40, an intermembrane space (IMS) import receptor of mitochondria harbors twin CX9C motifs for stability while its CPC motif is known to facilitate the import of IMS bound proteins. Site-directed mutagenesis complemented by MALDI on in vivo hMIA40 protein shows that a portion of MIA40 undergoes reversible S-glutathionylation at three cysteines in the twin CX9C motifs and the lone cysteine 4 residue. We find that HEK293T cells expressing hMIA40 mutant defective for glutathionylation are compromised in the activities of complexes III and IV of the Electron Transport Chain (ETC) and enhance Reactive Oxygen Species (ROS) levels. Immunocapture studies show MIA40 interacting with complex III. Interestingly, glutathionylated MIA40 can transfer electrons to cytochrome C directly. However, Fe-S clusters associated with the CPC motif are essential to facilitate the two-electron to one-electron transfer for reducing cytochrome C. These results suggest that hMIA40 undergoes glutathionylation to maintain ROS levels and for optimum function of complexes III and IV of ETC. Our studies shed light on a novel post-translational modification of hMIA40 and its ability to act as a redox switch to regulate the ETC and cellular redox homeostasis.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons , Proteínas de Transporte da Membrana Mitocondrial , Transporte de Elétrons , Células HEK293 , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Oxirredução , Transporte Proteico , Espécies Reativas de OxigênioRESUMO
Perturbations in mitochondrial redox levels oxidize nucleotide exchanger Mge1, compromising its ability to bind to the Hsp70, while the Mxr2 enzyme reduces the oxidized Mge1. However, the effects of persistent oxidative stress on Mge1 structure and function are not known. In this study, we show that oxidation-induced selective and local structural adaptations cause the detachment of Mge1 from Hsp70. Notably, persistent oxidative stress causes monomeric Mge1 to aggregate and to generate amyloid-type particles. Mxr2 appears to protect Mge1 from oxidative stress induced aggregation. We conclude that the Mxr2-Mge1-Hsp70 protein triad is finely regulated through structural alterations of Mge1 mediated by redox levels.
Assuntos
Adaptação Biológica , Proteínas de Choque Térmico HSP70/metabolismo , Metionina Sulfóxido Redutases/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Chaperonas Moleculares/metabolismo , Estresse Oxidativo , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Choque Térmico HSP70/genética , Metionina Sulfóxido Redutases/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Chaperonas Moleculares/genética , Oxirredução , Ligação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Cells across evolution employ reversible oxidative modification of methionine and cysteine amino acids within proteins to regulate responses to redox stress. Previously we have shown that mitochondrial localized methionine sulfoxide reductase (Mxr2) reversibly regulates oxidized yeast Mge1 (yMge1), a co-chaperone of Hsp70/Ssc1 to maintain protein homeostasis during oxidative stress. However, the specificity and the conservation of the reversible methionine oxidation mechanism in higher eukaryotes is debatable as human GrpEL1 (hGrpEL1) unlike its homolog yMge1 harbors two methionine residues and multiple cysteines besides the mammalian mitochondria hosting R and S types of Mxrs/Msrs. In this study, using yeast as a surrogate system, we show that hGRPEL1 and R type MSRs but not the S type MSRs complement the deletion of yeast MGE1 or MXR2 respectively. Our investigations show that R type Msrs interact selectively with oxidized hGrpEL1/yMge1 in an oxidative stress dependent manner, reduce the conserved hGrpEL1-Met146-SO and rescue the Hsp70 ATPase activity. In addition, a single point mutation in hGrpEL1-M146L rescues the slow growth phenotype of yeast MXR2 deletion under oxidative duress. Our study illustrates the evolutionarily conserved formation of specific Met-R-SO in hGrpEL1/yMge1 and the essential and canonical role of R type Msrs/Mxrs in mitochondrial redox mechanism.
Assuntos
Ferredoxina-NADP Redutase/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Metionina Sulfóxido Redutases/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Cisteína/metabolismo , Ferredoxina-NADP Redutase/genética , Teste de Complementação Genética , Proteínas de Choque Térmico HSP70/genética , Humanos , Metionina/metabolismo , Metionina Sulfóxido Redutases/genética , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oxirredução , Estresse Oxidativo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Peptide methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in protein(s). Although these reductases have been implicated in several human diseases, there is a dearth of information on the identity of their physiological substrates. By using Saccharomyces cerevisiae as a model, we show that of the two methionine sulfoxide reductases (MXR1, MXR2), deletion of mitochondrial MXR2 renders yeast cells more sensitive to oxidative stress than the cytosolic MXR1. Our earlier studies showed that Mge1, an evolutionarily conserved nucleotide exchange factor of Hsp70, acts as an oxidative sensor to regulate mitochondrial Hsp70. In the present study, we show that Mxr2 regulates Mge1 by selectively reducing MetO at position 155 and restores the activity of Mge1 both in vitro and in vivo. Mge1 M155L mutant rescues the slow-growth phenotype and aggregation of proteins of mxr2Δ strain during oxidative stress. By identifying the first mitochondrial substrate for Mxrs, we add a new paradigm to the regulation of the oxidative stress response pathway.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Metionina Sulfóxido Redutases/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Chaperonas Moleculares/metabolismo , Estresse Oxidativo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Metionina/metabolismo , Proteínas Mutantes , Oxirredução , OxirredutasesRESUMO
Adaptation to changes in extracellular tonicity is essential for cell survival. However, severe or chronic hyperosmotic stress induces apoptosis, which involves cytochrome c (Cyt c) release from mitochondria and subsequent apoptosome formation. Here, we show that angiogenin-induced accumulation of tRNA halves (or tiRNAs) is accompanied by increased survival in hyperosmotically stressed mouse embryonic fibroblasts. Treatment of cells with angiogenin inhibits stress-induced formation of the apoptosome and increases the interaction of small RNAs with released Cyt c in a ribonucleoprotein (Cyt c-RNP) complex. Next-generation sequencing of RNA isolated from the Cyt c-RNP complex reveals that 20 tiRNAs are highly enriched in the Cyt c-RNP complex. Preferred components of this complex are 5' and 3' tiRNAs of specific isodecoders within a family of isoacceptors. We also demonstrate that Cyt c binds tiRNAs in vitro, and the pool of Cyt c-interacting RNAs binds tighter than individual tiRNAs. Finally, we show that angiogenin treatment of primary cortical neurons exposed to hyperosmotic stress also decreases apoptosis. Our findings reveal a connection between angiogenin-generated tiRNAs and cell survival in response to hyperosmotic stress and suggest a novel cellular complex involving Cyt c and tiRNAs that inhibits apoptosome formation and activity.
Assuntos
Apoptose/genética , Apoptossomas/biossíntese , Citocromos c/metabolismo , Clivagem do RNA , RNA de Transferência/metabolismo , Ribonuclease Pancreático/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptossomas/antagonistas & inibidores , Fator Apoptótico 1 Ativador de Proteases/genética , Sequência de Bases , Caspase 3/metabolismo , Caspase 9/metabolismo , Sobrevivência Celular , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Fibroblastos , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Pressão Osmótica , Ribonuclease Pancreático/farmacologia , Ribonucleoproteínas/genética , Análise de Sequência de RNARESUMO
Despite the growing evidence of the role of oxidative stress in disease, its molecular mechanism of action remains poorly understood. The yeast Saccharomyces cerevisiae provides a valuable model system in which to elucidate the effects of oxidative stress on mitochondria in higher eukaryotes. Dimeric yeast Mge1, the cochaperone of heat shock protein 70 (Hsp70), is essential for exchanging ATP for ADP on Hsp70 and thus for recycling of Hsp70 for mitochondrial protein import and folding. Here we show an oxidative stress-dependent decrease in Mge1 dimer formation accompanied by a concomitant decrease in Mge1-Hsp70 complex formation in vitro. The Mge1-M155L substitution mutant stabilizes both Mge1 dimer and Mge1-Hsp70 complex formation. Most important, the Mge1-M155L mutant rescues the slow-growth phenomenon associated with the wild-type Mge1 strain in the presence of H2O2 in vivo, stimulation of the ATPase activity of Hsp70, and the protein import defect during oxidative stress in vitro. Furthermore, cross-linking studies reveal that Mge1-Hsp70 complex formation in mitochondria isolated from wild-type Mge1 cells is more susceptible to reactive oxygen species compared with mitochondria from Mge1-M155L cells. This novel oxidative sensor capability of yeast Mge1 might represent an evolutionarily conserved function, given that human recombinant dimeric Mge1 is also sensitive to H2O2.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Peróxido de Hidrogênio/farmacologia , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutação , Estresse Oxidativo , Multimerização Proteica , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de SequênciaRESUMO
Aminoacyl tRNA synthetases play a central role in protein synthesis by charging tRNAs with amino acids. Yeast mitochondrial lysyl tRNA synthetase (Msk1), in addition to the aminoacylation of mitochondrial tRNA, also functions as a chaperone to facilitate the import of cytosolic lysyl tRNA. In this report, we show that human mitochondrial Kars (lysyl tRNA synthetase) can complement the growth defect associated with the loss of yeast Msk1 and can additionally facilitate the in vitro import of tRNA into mitochondria. Surprisingly, the import of lysyl tRNA can occur independent of Msk1 in vivo. This suggests that an alternative mechanism is present for the import of lysyl tRNA in yeast.