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1.
PLoS Biol ; 18(8): e3000808, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32817651

RESUMO

Although dysregulation of mitochondrial dynamics has been linked to cellular senescence, which contributes to advanced age-related disorders, it is unclear how Krüppel-like factor 5 (Klf5), an essential transcriptional factor of cardiovascular remodeling, mediates the link between mitochondrial dynamics and vascular smooth muscle cell (VSMC) senescence. Here, we show that Klf5 down-regulation in VSMCs is correlated with rupture of abdominal aortic aneurysm (AAA), an age-related vascular disease. Mice lacking Klf5 in VSMCs exacerbate vascular senescence and progression of angiotensin II (Ang II)-induced AAA by facilitating reactive oxygen species (ROS) formation. Klf5 knockdown enhances, while Klf5 overexpression suppresses mitochondrial fission. Mechanistically, Klf5 activates eukaryotic translation initiation factor 5a (eIF5a) transcription through binding to the promoter of eIF5a, which in turn preserves mitochondrial integrity by interacting with mitofusin 1 (Mfn1). Accordingly, decreased expression of eIF5a elicited by Klf5 down-regulation leads to mitochondrial fission and excessive ROS production. Inhibition of mitochondrial fission decreases ROS production and VSMC senescence. Our studies provide a potential therapeutic target for age-related vascular disorders.


Assuntos
Aneurisma da Aorta Abdominal/genética , Células Endoteliais/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Mitocôndrias/metabolismo , Fatores de Iniciação de Peptídeos/genética , Proteínas de Ligação a RNA/genética , Idoso , Angiotensina II/genética , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Aorta/diagnóstico por imagem , Aorta/metabolismo , Aorta/patologia , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Senescência Celular/efeitos dos fármacos , Ecocardiografia , Células Endoteliais/patologia , Feminino , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/deficiência , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/patologia , Dinâmica Mitocondrial/efeitos dos fármacos , Fatores de Iniciação de Peptídeos/deficiência , Cultura Primária de Células , Regiões Promotoras Genéticas , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo
2.
Nat Commun ; 11(1): 3409, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641778

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is associated with high mortality and therapy resistance. Here, we show that low expression of κB-Ras GTPases is frequently detected in PDAC and correlates with higher histologic grade. In a model of KRasG12D-driven PDAC, loss of κB-Ras accelerates tumour development and shortens median survival. κB-Ras deficiency promotes acinar-to-ductal metaplasia (ADM) during tumour initiation as well as tumour progression through intrinsic effects on proliferation and invasion. κB-Ras proteins are also required for acinar regeneration after pancreatitis, demonstrating a general role in control of plasticity. Molecularly, upregulation of Ral GTPase activity and Sox9 expression underlies the observed phenotypes, identifying a previously unrecognized function of Ral signalling in ADM. Our results provide evidence for a tumour suppressive role of κB-Ras proteins and highlight low κB-Ras levels and consequent loss of Ral control as risk factors, thus emphasizing the necessity for therapeutic options that allow interference with Ral-driven signalling.


Assuntos
Células Acinares/metabolismo , Carcinoma Ductal Pancreático/genética , GTP Fosfo-Hidrolases/genética , Neoplasias Pancreáticas/genética , Pancreatite/genética , Proteínas/genética , Células Acinares/patologia , Idoso , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Feminino , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Estimativa de Kaplan-Meier , Masculino , Metaplasia/genética , Metaplasia/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Pancreatite/metabolismo , Proteínas/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Proteínas ral de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo
3.
Nature ; 584(7822): 640-645, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32612237

RESUMO

Ribosomes accurately decode mRNA by proofreading each aminoacyl-tRNA that is delivered by the elongation factor EF-Tu1. To understand the molecular mechanism of this proofreading step it is necessary to visualize GTP-catalysed elongation, which has remained a challenge2-4. Here we use time-resolved cryogenic electron microscopy to reveal 33 ribosomal states after the delivery of aminoacyl-tRNA by EF-Tu•GTP. Instead of locking cognate tRNA upon initial recognition, the ribosomal decoding centre dynamically monitors codon-anticodon interactions before and after GTP hydrolysis. GTP hydrolysis enables the GTPase domain of EF-Tu to extend away, releasing EF-Tu from tRNA. The 30S subunit then locks cognate tRNA in the decoding centre and rotates, enabling the tRNA to bypass 50S protrusions during accommodation into the peptidyl transferase centre. By contrast, the decoding centre fails to lock near-cognate tRNA, enabling the dissociation of near-cognate tRNA both during initial selection (before GTP hydrolysis) and proofreading (after GTP hydrolysis). These findings reveal structural similarity between ribosomes in initial selection states5,6 and in proofreading states, which together govern the efficient rejection of incorrect tRNA.


Assuntos
Microscopia Crioeletrônica , Guanosina Trifosfato/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Escherichia coli , GTP Fosfo-Hidrolases/metabolismo , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Hidrólise , Modelos Moleculares , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/ultraestrutura , Ribossomos/química , Rotação
4.
Proc Natl Acad Sci U S A ; 117(29): 17296-17307, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32631998

RESUMO

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of late-onset, autosomal-dominant familial Parkinson's disease (PD). LRRK2 functions as both a kinase and GTPase, and PD-linked mutations are known to influence both enzymatic activities. While PD-linked LRRK2 mutations can commonly induce neuronal damage in culture models, the mechanisms underlying these pathogenic effects remain uncertain. Rodent models containing familial LRRK2 mutations often lack robust PD-like neurodegenerative phenotypes. Here, we develop a robust preclinical model of PD in adult rats induced by the brain delivery of recombinant adenoviral vectors with neuronal-specific expression of human LRRK2 harboring the most common G2019S mutation. In this model, G2019S LRRK2 induces the robust degeneration of substantia nigra dopaminergic neurons, a pathological hallmark of PD. Introduction of a stable kinase-inactive mutation or administration of the selective kinase inhibitor, PF-360, attenuates neurodegeneration induced by G2019S LRRK2. Neuroprotection provided by pharmacological kinase inhibition is mediated by an unusual mechanism involving the robust destabilization of human LRRK2 protein in the brain relative to endogenous LRRK2. Our study further demonstrates that G2019S LRRK2-induced dopaminergic neurodegeneration critically requires normal GTPase activity, as hypothesis-testing mutations that increase GTP hydrolysis or impair GTP-binding activity provide neuroprotection although via distinct mechanisms. Taken together, our data demonstrate that G2019S LRRK2 induces neurodegeneration in vivo via a mechanism that is dependent on kinase and GTPase activity. Our study provides a robust rodent preclinical model of LRRK2-linked PD and nominates kinase inhibition and modulation of GTPase activity as promising disease-modifying therapeutic targets.


Assuntos
Neurônios Dopaminérgicos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Doença de Parkinson/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Modelos Animais de Doenças , Dopamina/metabolismo , Feminino , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Camundongos , Camundongos Knockout , Mutação , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Doença de Parkinson/patologia , Fenótipo , Projetos Piloto , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar , Substância Negra
5.
Nucleic Acids Res ; 48(14): 7924-7943, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32652011

RESUMO

Biogenesis of mammalian mitochondrial ribosomes (mitoribosomes) involves several conserved small GTPases. Here, we report that the Obg family protein GTPBP5 or MTG2 is a mitochondrial protein whose absence in a TALEN-induced HEK293T knockout (KO) cell line leads to severely decreased levels of the 55S monosome and attenuated mitochondrial protein synthesis. We show that a fraction of GTPBP5 co-sediments with the large mitoribosome subunit (mtLSU), and crosslinks specifically with the 16S rRNA, and several mtLSU proteins and assembly factors. Notably, the latter group includes MTERF4, involved in monosome assembly, and MRM2, the methyltransferase that catalyzes the modification of the 16S mt-rRNA A-loop U1369 residue. The GTPBP5 interaction with MRM2 was also detected using the proximity-dependent biotinylation (BioID) assay. In GTPBP5-KO mitochondria, the mtLSU lacks bL36m, accumulates an excess of the assembly factors MTG1, GTPBP10, MALSU1 and MTERF4, and contains hypomethylated 16S rRNA. We propose that GTPBP5 primarily fuels proper mtLSU maturation by securing efficient methylation of two 16S rRNA residues, and ultimately serves to coordinate subunit joining through the release of late-stage mtLSU assembly factors. In this way, GTPBP5 provides an ultimate quality control checkpoint function during mtLSU assembly that minimizes premature subunit joining to ensure the assembly of the mature 55S monosome.


Assuntos
Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/enzimologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , RNA Ribossômico 16S/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/enzimologia , Linhagem Celular , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Humanos , Metilação , Metiltransferases/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/fisiologia , Ribossomos Mitocondriais/metabolismo , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Fosforilação Oxidativa , Biossíntese de Proteínas , RNA Ribossômico 16S/química , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Fatores de Transcrição/metabolismo
6.
Mol Cell ; 79(4): 629-644.e4, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32679035

RESUMO

In contrast to the bacterial translation machinery, mitoribosomes and mitochondrial translation factors are highly divergent in terms of composition and architecture. There is increasing evidence that the biogenesis of mitoribosomes is an intricate pathway, involving many assembly factors. To better understand this process, we investigated native assembly intermediates of the mitoribosomal large subunit from the human parasite Trypanosoma brucei using cryo-electron microscopy. We identify 28 assembly factors, 6 of which are homologous to bacterial and eukaryotic ribosome assembly factors. They interact with the partially folded rRNA by specifically recognizing functionally important regions such as the peptidyltransferase center. The architectural and compositional comparison of the assembly intermediates indicates a stepwise modular assembly process, during which the rRNA folds toward its mature state. During the process, several conserved GTPases and a helicase form highly intertwined interaction networks that stabilize distinct assembly intermediates. The presented structures provide general insights into mitoribosomal maturation.


Assuntos
Ribossomos Mitocondriais/química , RNA Ribossômico/metabolismo , Subunidades Ribossômicas Maiores/química , Trypanosoma brucei brucei/metabolismo , Microscopia Crioeletrônica , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Ribossomos Mitocondriais/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Ribossômico/química , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores/metabolismo , Trypanosoma brucei brucei/genética
7.
Gene ; 754: 144854, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32525045

RESUMO

Alzheimer's disease (AD) is one of the most common forms of neurodegenerative diseases. Aggregation of Aß42 and hyperphosphorylated tau are two major hallmarks of AD. Whether different forms of tau (soluble or hyperphosphorylated) or Aß are the main culprit in the events observed in AD is still under investigation. Here, we examined the effect of wild-type, prone to hyperphosphorylation and hyperphosphorylated tau, and also Aß42 peptide on the brain antioxidant defense system and two mitochondrial genes, Marf (homologous to human MFN2) and Drp1 involved in mitochondrial dynamics in transgenic Drosophila melanogaster. AD is an age associated disease. Therefore, the activity of antioxidant agents, CAT, SOD, and GSH levels and the mRNA levels of Marf and Drp1 were assessed in different time points of the flies lifespan. Reduction in cognitive function and antioxidant activity was observed in all transgenic flies at any time point. The most and the least effect on the eye phenotype was exerted by hyperphosphorylated tau and Aß42, respectively. In addition, the most remarkable alteration in Marf and Drp1 mRNA levels was observed in transgenic flies expressing hyperphosphorylated tau when pan neuronal expression of transgenes was applied. However, when the disease causing gene expression was confined to the mushroom body, Marf and Drp1 mRNA levels alteration was more prominent in tauWT and tauE14 transgenic flies, respectively. In conclusion, in spite of antioxidant deficiency caused by different types of tau and Aß42, it seems that tau exerts more toxic effect on the eye phenotype and mitochondrial genes regulation (Marf and Drp1). Moreover, different mechanisms seem to be involved in mitochondrial genes dysregulation when Aß or various forms of tau are expressed.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Animais Geneticamente Modificados/metabolismo , Drosophila melanogaster/metabolismo , Dinaminas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , RNA Mensageiro/metabolismo , Proteínas tau/metabolismo , Animais , Animais Geneticamente Modificados/genética , Encéfalo/metabolismo , Drosophila melanogaster/genética , Dinaminas/genética , GTP Fosfo-Hidrolases/genética , Regulação da Expressão Gênica , Transtornos da Memória/metabolismo , Transtornos da Memória/patologia , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Fosforilação , RNA Mensageiro/genética , Proteínas tau/genética
8.
Life Sci ; 254: 117762, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32437795

RESUMO

AIMS: Patients with nonalcoholic fatty liver disease (NAFLD) have less tolerance to ischemia-reperfusion injury (IRI) of the liver than those with the healthy liver; hence have a higher incidence of severe complications after surgery. This study aimed to investigate the dynamics of the liver and mitochondrial damage and the impact of TLR4 knockout (TLR4KO) on Mfn2 expression in the composite model of NAFLD and IRI. MAIN METHODS: We performed high-fat diet (HFD) feeding and ischemia reperfusion (IR) on wild type (WT) and TLR4 knockout TLR4KO mice. KEY FINDINGS: The degree of structural and functional injuries to the liver and mitochondria (NAFLD and IRI) is greater than that caused by a single factor (NAFLD or IRI) or a simple superposition of both. The IL-6 and TNF-α expressions were significantly suppressed (P < .05), while PGC-1α and Mfn2 expressions were up-regulated considerably (P < .05) after TLR4KO. Furthermore, mitochondrial fusion increased, while ATP consumption and ROS production decreased significantly after TLR4KO (P < .05). The degree of reduction of compound injury by TLR4KO is more significant than the reduction degree of single factor injury. Also, TNF-α and IL-6 levels can be used predictive markers for mitochondrial damage and liver tolerance to NAFLD and IRI. SIGNIFICANCE: TLR4KO upregulates the expression of Mfn2 and PGC-1α in the composite model of NAFLD and IRI. This pathway may be related to IL-6 and TNF-α. This evidence provides theoretical and experimental basis for the subsequent Toll-like receptor 4 (TLR4) receptor targeted therapy.


Assuntos
GTP Fosfo-Hidrolases/biossíntese , Fígado/irrigação sanguínea , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/biossíntese , Traumatismo por Reperfusão/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Dieta Hiperlipídica , Modelos Animais de Doenças , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica , Fígado/metabolismo , Fígado/patologia , Transplante de Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Reperfusão , Traumatismo por Reperfusão/patologia , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Ativação Transcricional , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima
9.
Cardiovasc Drugs Ther ; 34(3): 345-356, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32236861

RESUMO

PURPOSE: Mitochondrial dysfunction plays a vital role in the pathophysiologic process of heart failure (HF). As a quality control system, mitochondrial fusion and fission are under control of mitochondrial fusion and fission-related proteins. The objective of this study was to investigate the effects of common variants in mitochondrial fusion and fission-related genes on the prognosis of HF. METHODS: We performed whole exome sequencing (WES) with 1000 HF patients; the statistically significant variant was further genotyped in the replicated population with 2324 HF patients. A series of function analysis including western blot, cell proliferation assay, and in vitro OMA1 activity assay were conducted to illuminate the underlying mechanism. RESULTS: We identified a missense variant rs17117699 associated with the prognosis of HF in group without ß-blocker use rather than with ß-blocker use in two-stage population: adjusted P = 0.79, HR = 0.88 (0.36-2.13) in group with ß-blocker use and adjusted P = 0.016, HR = 1.43 (1.07-1.91) in group without ß-blocker in first-stage population; adjusted P = 0.42, HR = 0.85 (0.56-1.28) in group with ß-blocker use and adjusted P = 0.015, HR = 1.39 (1.06-1.82) in group without ß-blocker in replicated stage. Functional analysis indicated that rs17117699-G allele increased the activity of OMA1 assessed by the ratio of S-OPA1 to L-OPA1 and suppressed cells proliferation under ISO treatment when compared with rs17117699-T allele. Furthermore, OMA1 functioned downstream of ß-adrenergic receptor signaling and ISO-induced OPA1 cleavage is dependent on OMA1. CONCLUSIONS: Our findings demonstrate that rs17117699T>G in OMA1 increases the risk of HF mortality via enhancing its OPA1 cleavage activity. It is a promising potential treatment target for HF. CLINICAL TRIAL REGISTRATION: NCT03461107. https://www.clinicaltrials.gov/ct2/show/NCT03461107?term=03461107&cond=Heart+Failure&cntry=CN&rank=1.


Assuntos
Insuficiência Cardíaca/genética , Metaloendopeptidases/genética , Mitocôndrias Cardíacas/genética , Dinâmica Mitocondrial/genética , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Antagonistas Adrenérgicos beta/uso terapêutico , Adulto , Idoso , Proliferação de Células , Feminino , GTP Fosfo-Hidrolases/metabolismo , Estudos de Associação Genética , Células HEK293 , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/mortalidade , Humanos , Masculino , Metaloendopeptidases/metabolismo , Pessoa de Meia-Idade , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Prognóstico , Sequenciamento Completo do Exoma
10.
J Dairy Sci ; 103(6): 5561-5574, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32278565

RESUMO

Inflammation is critical in the progression from benign hepatic lipidosis to pathological hepatic steatosis. The objective of this study was to examine the potential role of the outer mitochondrial membrane protein mitofusin 2 (MFN2) in the etiology of hepatic steatosis in dairy cows during early lactation. Using a nested case-control design, we compared blood and liver samples from 10 healthy cows and 10 age-matched cows with moderate fatty liver. Cows with moderate fatty liver had high liver triacylglycerols, elevated plasma concentrations of free fatty acids (FFA) and ß-hydroxybutyrate, and low concentrations of glucose. Cows with moderate fatty liver had overactivated inflammatory pathways in the liver, as indicated by increased abundance of phosphorylated nuclear factor κB (NF-κB) p65, NLR family pyrin domain containing 3 (NLRP3) and caspase-1 inflammasome protein, and elevated plasma concentrations and hepatic mRNA abundance of their molecular targets IL-1ß, IL-6, and tumor necrosis factor α (TNF-α). In the cell culture model, we were able to replicate our findings in cows with moderate fatty liver: 1.2 mM exogenous FFA decreased the abundance of MFN2 and upregulated phosphorylation levels of the inhibitor of NF-κB (IκB) α and NF-κB p65, the IκB kinase ß activity, and the abundance of NLRP3, caspase-1, IL-1ß, IL-6, and TNF-α. Whereas MFN2 knockdown potentiated the FFA-induced activation of these inflammatory pathways, overexpression of MFN2 attenuated the detrimental effect of excess exogenous FFA by improving mitochondrial function and decreasing the release of reactive oxygen species, suggesting that MFN2 may be a potential therapeutic target for FFA-induced hepatic inflammation in dairy cows during early lactation.


Assuntos
Doenças dos Bovinos/prevenção & controle , Ácidos Graxos não Esterificados/efeitos adversos , Fígado Gorduroso/veterinária , GTP Fosfo-Hidrolases/antagonistas & inibidores , Inflamação/veterinária , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Animais , Estudos de Casos e Controles , Bovinos , Ácidos Graxos não Esterificados/sangue , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/prevenção & controle , Feminino , GTP Fosfo-Hidrolases/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Lactação/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Espécies Reativas de Oxigênio/metabolismo
11.
PLoS Genet ; 16(3): e1008638, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32191694

RESUMO

Compromising mitochondrial fusion or fission disrupts cellular homeostasis; however, the underlying mechanism(s) are not fully understood. The loss of C. elegans fzo-1MFN results in mitochondrial fragmentation, decreased mitochondrial membrane potential and the induction of the mitochondrial unfolded protein response (UPRmt). We performed a genome-wide RNAi screen for genes that when knocked-down suppress fzo-1MFN(lf)-induced UPRmt. Of the 299 genes identified, 143 encode negative regulators of autophagy, many of which have previously not been implicated in this cellular quality control mechanism. We present evidence that increased autophagic flux suppresses fzo-1MFN(lf)-induced UPRmt by increasing mitochondrial membrane potential rather than restoring mitochondrial morphology. Furthermore, we demonstrate that increased autophagic flux also suppresses UPRmt induction in response to a block in mitochondrial fission, but not in response to the loss of spg-7AFG3L2, which encodes a mitochondrial metalloprotease. Finally, we found that blocking mitochondrial fusion or fission leads to increased levels of certain types of triacylglycerols and that this is at least partially reverted by the induction of autophagy. We propose that the breakdown of these triacylglycerols through autophagy leads to elevated metabolic activity, thereby increasing mitochondrial membrane potential and restoring mitochondrial and cellular homeostasis.


Assuntos
Autofagia/genética , Mitocôndrias/genética , Resposta a Proteínas não Dobradas/genética , Animais , Autofagia/fisiologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica/genética , Homeostase/genética , Potencial da Membrana Mitocondrial/genética , Potencial da Membrana Mitocondrial/fisiologia , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/genética , Interferência de RNA , Resposta a Proteínas não Dobradas/fisiologia
12.
Arch Biochem Biophys ; 685: 108350, 2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32220566

RESUMO

Iron is an essential requirement for the survival and virulence of most bacteria. The bacterial ferrous iron transporter protein FeoB functions as a major reduced iron transporter in prokaryotes, but its biochemical mechanism has not been fully elucidated. In the present study, we compared enzymatic properties of the cytosolic portions of pathogenic bacterial FeoBs to elucidate each bacterial strain-specific characteristic of the Feo system. We show that bacterial FeoBs are classified into two distinct groups that possess either a sole GTPase or an NTPase with a substrate promiscuity. This difference in nucleotide preference alters cellular requirements for monovalent and divalent cations. While the hydrolytic activity of the GTP-dependent FeoBs was stimulated by potassium, the action of the NTP-dependent FeoBs was not significantly affected by the presence of monovalent cations. Mutation of Asn11, having a role in potassium-dependent GTP hydrolysis, changed nucleotide specificity of the NTP-dependent FeoB, resulting in loss of ATPase activity. Sequence analysis suggested a possible association of alanine in the G5 motif for the NTP-dependent activity in FeoBs. This demonstration of the distinct enzymatic properties of bacterial FeoBs provides important insights into mechanistic details of Feo iron transport processes, as well as offers a promising species-specific anti-virulence target.


Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte de Cátions/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Bactérias/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/química , Hidrólise , Mutagênese Sítio-Dirigida , Mutação , Nucleosídeo-Trifosfatase/química , Nucleosídeo-Trifosfatase/metabolismo , Potássio/metabolismo , Ligação Proteica , Alinhamento de Sequência , Especificidade por Substrato
13.
Nucleic Acids Res ; 48(8): 4572-4584, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32196113

RESUMO

The single G protein of the spliceosome, Snu114, has been proposed to facilitate splicing as a molecular motor or as a regulatory G protein. However, available structures of spliceosomal complexes show Snu114 in the same GTP-bound state, and presently no Snu114 GTPase-regulatory protein is known. We determined a crystal structure of Snu114 with a Snu114-binding region of the Prp8 protein, in which Snu114 again adopts the same GTP-bound conformation seen in spliceosomes. Snu114 and the Snu114-Prp8 complex co-purified with endogenous GTP. Snu114 exhibited weak, intrinsic GTPase activity that was abolished by the Prp8 Snu114-binding region. Exchange of GTP-contacting residues in Snu114, or of Prp8 residues lining the Snu114 GTP-binding pocket, led to temperature-sensitive yeast growth and affected the same set of splicing events in vivo. Consistent with dynamic Snu114-mediated protein interactions during splicing, our results suggest that the Snu114-GTP-Prp8 module serves as a relay station during spliceosome activation and disassembly, but that GTPase activity may be dispensable for splicing.


Assuntos
Guanosina Trifosfato/química , Processamento de RNA , Ribonucleoproteína Nuclear Pequena U4-U6/química , Ribonucleoproteína Nuclear Pequena U5/química , Proteínas de Saccharomyces cerevisiae/química , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Modelos Moleculares , Conformação Proteica , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
14.
Med Sci Monit ; 26: e918216, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32129321

RESUMO

BACKGROUND Chemoresistance is a primary hindrance for current cancer treatments. The influence of abnormal mitochondria in chemotherapy resistance is not well known. To explore the correlation between mitochondria and acquired chemoresistance, this work studied alterations in mitochondrial dynamics, biogenesis, and functions for paclitaxel-resistant cancer cell line A549/Taxol and its parental line A549. MATERIAL AND METHODS Mitochondrial morphology was observed by transmission electron microscopy and confocal microscopy. We measured the mitochondrial mass and mitochondrial membrane potential using fluorescent dyes. The glucose metabolic profile and ATP (adenosine triphosphate) content were determined by bioluminescent cell assays. Seahorse bio-energy analyzer XF24 was used to detect the mitochondrial respiratory function. The expressions of mitochondrial dynamics and biogenesis related genes were quantified using real-time polymerase chain reaction. RESULTS We observed fusion morphology of the mitochondrial network in A549/Taxol cells, with upregulation of fusion genes (Mfn1 and Mfn2) and downregulation of fission gene Fis1. In A549/Taxol cells, mitochondrial mass showed a significant decrease, while the mitochondrial biogenesis pathway was strongly activated. Despite the decreased mitochondrial membrane potential, the capability for mitochondrial respiration was not impaired in A549/Taxol cells. CONCLUSIONS Our study revealed a series changes of mitochondrial characteristics in paclitaxel-resistant cells. Mfn1 and Mfn2 and PGC-1alpha increased, while Fis1 expression and mitochondrial oxidative phosphorylation decreased in A549/Taxol cell lines. These changes to mitochondrial fusion, fission, and biological function contributed to the occurrence of paclitaxel resistance in tumor cells which induced paclitaxel resistance.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Dinâmica Mitocondrial , Biogênese de Organelas , Paclitaxel/farmacologia , Células A549 , GTP Fosfo-Hidrolases/metabolismo , Humanos , Potencial da Membrana Mitocondrial , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo
15.
Am J Physiol Renal Physiol ; 318(4): F878-F887, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32003595

RESUMO

Disruption of mitochondrial dynamics is an important pathogenic event in both acute and chronic kidney diseases, but the underlying mechanism remains poorly understood. Here, we report the regulation of mitofusin-2 (Mfn2; a key mitochondrial fusion protein) by microRNA-214 (miR-214) in renal ischemia-reperfusion that contributes to mitochondrial fragmentation, renal tubular cell death, and ischemic acute kidney injury (AKI). miR-214 was induced, whereas Mfn2 expression was decreased, in mouse ischemic AKI and cultured rat kidney proximal tubular cells (RPTCs) following ATP depletion treatment. Overexpression of miR-214 decreased Mfn2. Conversely, inhibition of miR-214 with anti-miR-214 prevented Mfn2 downregulation in RPTCs following ATP depletion. Anti-miR-214 further ameliorated mitochondrial fragmentation and apoptosis, whereas overexpression of miR-214 increased apoptosis, in ATP-depleted RPTCs. To test regulation in vivo, we established a mouse model with miR-214 specifically deleted from kidney proximal tubular cells (PT-miR-214-/-). Compared with wild-type mice, PT-miR-214-/- mice had less severe tissue damage, fewer apoptotic cells, and better renal function after ischemic AKI. miR-214 induction in ischemic AKI was suppressed in PT-miR-214-/- mice, accompanied by partial preservation of Mfn2 in kidneys. These results unveil the miR-214/Mfn2 axis that contributes to the disruption of mitochondrial dynamics and tubular cell death in ischemic AKI, offering new therapeutic targets.


Assuntos
Lesão Renal Aguda/metabolismo , Apoptose , GTP Fosfo-Hidrolases/metabolismo , Túbulos Renais Proximais/metabolismo , MicroRNAs/metabolismo , Proteínas Mitocondriais/metabolismo , Traumatismo por Reperfusão/metabolismo , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Trifosfato de Adenosina/deficiência , Animais , Linhagem Celular , Modelos Animais de Doenças , Regulação para Baixo , GTP Fosfo-Hidrolases/genética , Túbulos Renais Proximais/patologia , Camundongos Knockout , MicroRNAs/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Dinâmica Mitocondrial , Proteínas Mitocondriais/genética , Ratos , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais
16.
Int J Nanomedicine ; 15: 633-645, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32099356

RESUMO

Background: With the increased application of Silver nanoparticles (AgNP), its potential concerns to the health of human beings remain to be defined. This study aims to explore the harmful effects of AgNP on lung tissue in animals and to examine the mechanisms of protection achieved by sodium selenite. Methods: Sprague-Dawley(SD) rats were exposed to AgNP (200 µL,1mg/mL) through a single intratracheal instillation. Sodium selenite (0.2mg/kg) was i.p. injected. Malondialdehyde (MDA) and glutathione (GSH) were measured using a spectrophotometer. Histological outcomes and ultrastructural changes were assessed by hematoxylin and eosin (HE) staining and electronic microscopy. Caspases and mitochondrial fission and fusion markers were measured by Western blotting. Results: The histopathologic findings showed that AgNP significantly increased the thickness of alveolar septa, accumulation of macrophage, and the formation of pulmonary bullae and pulmonary consolidation. Ultrastructural studies showed localization of AgNP inside the mitochondria, hyperplasia and vacuolation of type I and type II alveolar cells, lysis of osmiophilic lamellar bodies, and swollen of the mitochondria. AgNP elevated MDA and reduced GSH levels. AgNP activated caspases-3, increased mitochondrial fission markers Dynamin-related protein 1 (Drp1) and phospho-Drp1(p-Drp1), and decreased fusion proteins optic atrophy 1 (Opa1) and mitofusins 2 (Mfn2). Treatment with sodium selenite for 7 days corrected the AgNP-caused alterations in morphological, ultrastructural, oxidative stress, caspase-3 activation and mitochondrial dynamic imbalance. Conclusion: We conclude that the exposure of AgNP causes lung tissue damage by enhances oxidative stress, activates caspases-3, and triggers mitochondrial dynamic imbalance towards fission. Sodium selenite effectively detoxifies the AgNP-induced damage to the lung tissue by preventing the above alterations.


Assuntos
Pulmão/efeitos dos fármacos , Nanopartículas Metálicas/efeitos adversos , Dinâmica Mitocondrial/efeitos dos fármacos , Prata/efeitos adversos , Selenito de Sódio/farmacologia , Animais , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Dinaminas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Glutationa/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Masculino , Malondialdeído/metabolismo , Nanopartículas Metálicas/química , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Ratos Sprague-Dawley , Prata/química
17.
Nat Commun ; 11(1): 605, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001718

RESUMO

Techniques of protein regulation, such as conditional gene expression, RNA interference, knock-in and knock-out, lack sufficient spatiotemporal accuracy, while optogenetic tools suffer from non-physiological response due to overexpression artifacts. Here we present a near-infrared light-activatable optogenetic system, which combines the specificity and orthogonality of intrabodies with the spatiotemporal precision of optogenetics. We engineer optically-controlled intrabodies to regulate genomically expressed protein targets and validate the possibility to further multiplex protein regulation via dual-wavelength optogenetic control. We apply this system to regulate cytoskeletal and enzymatic functions of two non-tagged endogenous proteins, actin and RAS GTPase, involved in complex functional networks sensitive to perturbations. The optogenetically-enhanced intrabodies allow fast and reversible regulation of both proteins, as well as simultaneous monitoring of RAS signaling with visible-light biosensors, enabling all-optical approach. Growing number of intrabodies should make their incorporation into optogenetic tools the versatile technology to regulate endogenous targets.


Assuntos
Optogenética , Proteínas/metabolismo , Actinas/metabolismo , Movimento Celular/efeitos da radiação , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Luz , Engenharia de Proteínas
18.
FASEB J ; 34(2): 3165-3178, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31908049

RESUMO

Dense granule protein 12 (GRA12) is implicated in a range of processes related to the establishment of Toxoplasma gondii infection, such as the formation of the intravacuolar network (IVN) within the parasitophorous vacuole (PV). This protein is also thought to be important for T. gondii-host interaction, pathogenesis, and immune evasion, but their exact roles remain unknown. In this study, the contributions of GRA12 to the molecular pathogenesis of T. gondii infection were examined in vitro and in vivo. Deletion of GRA12 in type I RH and type II Pru T. gondii strains did not affect the parasite growth and replication in vitro, however, it caused a significant reduction in the parasite virulence and tissue cyst burden in vivo. T. gondii Δgra12 mutants were more vulnerable to be eliminated by host immunity, without the accumulation of immunity-related GTPase a6 (Irga6) onto the PV membrane. The ultrastructure of IVN in Δgra12 mutants appeared normal, suggesting that GRA12 is not required for biogenesis of the IVN. Combined deletion of GRA12 and ROP18 induced more severe attenuation of virulence compared to single Δgra12 or Δrop18 mutant strains. These data suggest a functional association between GRA12 and ROP18 that is revealed by the severe attenuation of virulence in a double mutant relative to the single individual mutations. Future studies are needed to define the molecular basis of this putative association. Collectively these findings indicate that although GRA12 is not essential for the parasite growth and replication in vitro, it contributes to the virulence and growth of T. gondii in mice.


Assuntos
Antígenos de Protozoários/metabolismo , Toxoplasma/patogenicidade , Toxoplasmose/parasitologia , Animais , Antígenos de Protozoários/genética , Células Cultivadas , GTP Fosfo-Hidrolases/metabolismo , Interações Hospedeiro-Parasita , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Toxoplasma/genética , Toxoplasma/imunologia , Toxoplasmose/metabolismo , Vacúolos/metabolismo , Virulência/genética
19.
Cancer Cell ; 37(1): 71-84.e7, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31935373

RESUMO

Cancer cells rely on altered metabolism to support abnormal proliferation. We performed a CRISPR/Cas9 functional genomic screen targeting metabolic enzymes and identified PDXK-an enzyme that produces pyridoxal phosphate (PLP) from vitamin B6-as an acute myeloid leukemia (AML)-selective dependency. PDXK kinase activity is required for PLP production and AML cell proliferation, and pharmacological blockade of the vitamin B6 pathway at both PDXK and PLP levels recapitulated PDXK disruption effects. PDXK disruption reduced intracellular concentrations of key metabolites needed for cell division. Furthermore, disruption of PLP-dependent enzymes ODC1 or GOT2 selectively inhibited AML cell proliferation and their downstream products partially rescued PDXK disruption induced proliferation blockage. Our work identifies the vitamin B6 pathway as a pharmacologically actionable dependency in AML.


Assuntos
Leucemia Mieloide Aguda/enzimologia , Fosfotransferases/metabolismo , Fosfato de Piridoxal/metabolismo , Vitamina B 6/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células , GTP Fosfo-Hidrolases/metabolismo , Regulação Leucêmica da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosfotransferases/genética , Poliaminas/metabolismo , RNA Interferente Pequeno/metabolismo
20.
J Nutr ; 150(5): 1004-1011, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31965176

RESUMO

Mechanistic target of rapamycin complex 1 (mTORC1) is a highly evolutionarily conserved serine/threonine kinase that regulates cell growth and metabolism in response to multiple environmental cues, such as nutrients, hormones, energy, and stress. Deregulation of mTORC1 can lead to diseases such as diabetes, obesity, and cancer. A series of small GTPases, including Rag, Ras homolog enriched in brain (Rheb), adenosine diphosphate ribosylation factor 1 (Arf1), Ras-related protein Ral-A, Ras homolog (Rho), and Rab, are involved in regulating mTORC1 in response to nutrients, and mTORC1 is differentially regulated via these small GTPases according to specific conditions. Leucine and arginine sensing are considered to be well-confirmed amino acid-sensing signals, activating mTORC1 via a Rag GTPase-dependent mechanism as well as the Ragulator complex and vacuolar H+-adenosine triphosphatase (v-ATPase). Glutamine promotes mTORC1 activation via Arf1 independently of the Rag GTPase. In this review, we summarize current knowledge regarding the regulation of mTORC1 activity by small GTPases in response to nutrients, focusing on the function of small GTPases in mTORC1 activation and how small GTPases are regulated by nutrients.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Nutrientes/farmacologia , GTP Fosfo-Hidrolases/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética
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