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1.
J Hazard Mater ; 416: 126177, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34492951

RESUMO

Previous study showed that lead (Pb) could induce ATM-dependent mitophagy. However, whether Pb has any impact on mitochondrial fusion and fission, the upstream events of mitophagy, and how ATM connects to these processes remain unclear. In this study, we found that Pb can disrupt mitochondrial network morphology as indicated by increased percentage of shortened mitochondria and by decreased mitochondrial footprints. Correspondingly, the expression of fission protein Drp1 and its association with mitochondrial marker Hsp60 were significantly increased, while those of fusion proteins Mfn2 and Opa1 and their co-localization with Hsp60 were drastically attenuated. Notably, the expression of p-Drp1 (Ser616) and its translocation to mitochondria were dramatically elevated. Moreover, a small amount of ATM could be detected in the cytoplasm around mitochondria in response to Pb, and the co-localization of p-ATM (Ser1981) with Drp1 and p-Drp1 (Ser616) was obviously increased while its co-localization with Mfn2 and Opa1 was dramatically decreased. Furthermore, siRNA silencing of ATM evidently promoted greater fission in response to Pb stress, indicating that ATM is involved in mitochondrial fragmentation. Our results suggest that cytoplasmic ATM is an important regulator of Pb-induced mitochondrial fission.


Assuntos
Chumbo , Dinâmica Mitocondrial , Dinaminas , Fibroblastos , GTP Fosfo-Hidrolases/genética , Proteínas Associadas aos Microtúbulos , Proteínas Mitocondriais/genética
2.
Nat Commun ; 12(1): 5393, 2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34518553

RESUMO

Dynamin belongs to the large GTPase superfamily, and mediates the fission of vesicles during endocytosis. Dynamin molecules are recruited to the neck of budding vesicles to assemble into a helical collar and to constrict the underlying membrane. Two helical forms were observed: the one-start helix in the constricted state and the two-start helix in the super-constricted state. Here we report the cryoEM structure of a super-constricted two-start dynamin 1 filament at 3.74 Å resolution. The two strands are joined by the conserved GTPase dimeric interface. In comparison with the one-start structure, a rotation around Hinge 1 is observed, essential for communicating the chemical power of the GTPase domain and the mechanical force of the Stalk and PH domain onto the underlying membrane. The Stalk interfaces are well conserved and serve as fulcrums for adapting to changing curvatures. Relative to one-start, small rotations per interface accumulate to bring a drastic change in the helical pitch. Elasticity theory rationalizes the diversity of dynamin helical symmetries and suggests corresponding functional significance.


Assuntos
Microscopia Crioeletrônica/métodos , Dinamina I/química , Dinamina I/ultraestrutura , Simulação de Dinâmica Molecular , Domínios de Homologia à Plecstrina , Conformação Proteica , Multimerização Proteica , Algoritmos , Dinamina I/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Mutação , Termodinâmica
3.
Structure ; 29(8): 779-781, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34358462

RESUMO

COPII vesicle biogenesis at the endoplasmic reticulum requires activation of the Sar1 GTPase, which recruits the COP II coat protein complex to drive membrane budding. In this issue of Structure, Joiner and Fromme (2021) investigate the enigmatic structural basis for Sar1 activation by the Sec12 guanine nucleotide exchange factor.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório , Proteínas de Transporte Vesicular , Retículo Endoplasmático , GTP Fosfo-Hidrolases , Fatores de Troca do Nucleotídeo Guanina , Humanos , Proteínas de Transporte Vesicular/genética
4.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360957

RESUMO

In recent years, the "non-autonomous motor neuron death" hypothesis has become more consolidated behind amyotrophic lateral sclerosis (ALS). It postulates that cells other than motor neurons participate in the pathology. In fact, the involvement of the autonomic nervous system is fundamental since patients die of sudden death when they become unable to compensate for cardiorespiratory arrest. Mitochondria are thought to play a fundamental role in the physiopathology of ALS, as they are compromised in multiple ALS models in different cell types, and it also occurs in other neurodegenerative diseases. Our study aimed to uncover mitochondrial alterations in the sympathoadrenal system of a mouse model of ALS, from a structural, bioenergetic and functional perspective during disease instauration. We studied the adrenal chromaffin cell from mutant SOD1G93A mouse at pre-symptomatic and symptomatic stages. The mitochondrial accumulation of the mutated SOD1G93A protein and the down-regulation of optic atrophy protein-1 (OPA1) provoke mitochondrial ultrastructure alterations prior to the onset of clinical symptoms. These changes affect mitochondrial fusion dynamics, triggering mitochondrial maturation impairment and cristae swelling, with increased size of cristae junctions. The functional consequences are a loss of mitochondrial membrane potential and changes in the bioenergetics profile, with reduced maximal respiration and spare respiratory capacity of mitochondria, as well as enhanced production of reactive oxygen species. This study identifies mitochondrial dynamics regulator OPA1 as an interesting therapeutic target in ALS. Additionally, our findings in the adrenal medulla gland from presymptomatic stages highlight the relevance of sympathetic impairment in this disease. Specifically, we show new SOD1G93A toxicity pathways affecting cellular energy metabolism in non-motor neurons, which offer a possible link between cell specific metabolic phenotype and the progression of ALS.


Assuntos
Esclerose Amiotrófica Lateral/genética , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/metabolismo , Superóxido Dismutase-1/genética , Glândulas Suprarrenais/citologia , Esclerose Amiotrófica Lateral/metabolismo , Animais , Células Cultivadas , Células Cromafins/metabolismo , Regulação para Baixo , GTP Fosfo-Hidrolases/genética , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/ultraestrutura , Mutação de Sentido Incorreto , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase-1/metabolismo
5.
Nat Commun ; 12(1): 4696, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349113

RESUMO

Productive ribosomal RNA (rRNA) compaction during ribosome assembly necessitates establishing correct tertiary contacts between distant secondary structure elements. Here, we quantify the response of the yeast proteome to low temperature (LT), a condition where aberrant mis-paired RNA folding intermediates accumulate. We show that, at LT, yeast cells globally boost production of their ribosome assembly machinery. We find that the LT-induced assembly factor, Puf6, binds to the nascent catalytic RNA-rich subunit interface within the 60S pre-ribosome, at a site that eventually loads the nuclear export apparatus. Ensemble Förster resonance energy transfer studies show that Puf6 mimics the role of Mg2+ to usher a unique long-range tertiary contact to compact rRNA. At LT, puf6 mutants accumulate 60S pre-ribosomes in the nucleus, thus unveiling Puf6-mediated rRNA compaction as a critical temperature-regulated rescue mechanism that counters rRNA misfolding to prime export competence.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a RNA/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Temperatura Baixa , GTP Fosfo-Hidrolases/metabolismo , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteoma/metabolismo , Dobramento de RNA , Precursores de RNA/química , Precursores de RNA/metabolismo , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Subunidades Ribossômicas Maiores de Eucariotos/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
6.
Biomolecules ; 11(8)2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34439794

RESUMO

Recent breakthroughs have reignited interest in RAS GEFs as direct therapeutic targets. To search for new inhibitors of SOS GEF activity, a repository of known/approved compounds (NIH-NACTS) and a library of new marine compounds (Biomar Microbial Technologies) were screened by means of in vitro RAS-GEF assays using purified, bacterially expressed SOS and RAS constructs. Interestingly, all inhibitors identified in our screenings (two per library) shared related chemical structures belonging to the anthraquinone family of compounds. All our anthraquinone SOS inhibitors were active against the three canonical RAS isoforms when tested in our SOS GEF assays, inhibited RAS activation in mouse embryonic fibroblasts, and were also able to inhibit the growth of different cancer cell lines harboring WT or mutant RAS genes. In contrast to the commercially available anthraquinone inhibitors, our new marine anthraquinone inhibitors did not show in vivo cardiotoxicity, thus providing a lead for future discovery of stronger, clinically useful anthraquinone SOS GEF blockers.


Assuntos
Antraquinonas/farmacologia , Antineoplásicos/farmacologia , GTP Fosfo-Hidrolases/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Cardiotoxicidade/prevenção & controle , Linhagem Celular Transformada , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Idarubicina/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína SOS1/genética , Proteína SOS1/metabolismo , Proteínas Son Of Sevenless/deficiência , Proteínas Son Of Sevenless/genética
7.
Nutr Metab Cardiovasc Dis ; 31(10): 2979-2986, 2021 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-34362635

RESUMO

BACKGROUND AND AIMS: Coronary heart disease is a major global health concern. Further, severity of this condition is greatly influenced by myocardial ischemia/reperfusion (I/R) injury. Branched-chain amino acids (BCAAs) have cardioprotective effects against I/R via mammalian target of rapamycin (mTOR) activity, wherein Leu is considered to particularly regulate mTOR activation. However, the mechanism underlying cardioprotective effects of Leu via mTOR activity is not fully elucidated. Here, we aimed to study the signaling pathway of cardioprotection and mitochondrial function induced by Leu treatment. METHODS AND RESULTS: Cardiac myocytes isolated from adult male Wistar rats were incubated and exposed to simulated I/R (SI/R) injury by replacing the air content. Cardiac myocytes were treated with Leu and subsequently, their survival rate was calculated. To elucidate the signaling pathway and mitochondrial function, immunoblots and mitochondrial permeability transition pore were examined. Cell survival rate was decreased with SI/R but improved by 160 µM Leu (38.5 ± 3.6% vs. 64.5 ± 4.2%, respectively, p < 0.001). Although rapamycin (mTOR inhibitor) prevented this cardioprotective effect induced by Leu, wortmannin (PI3K inhibitor) did not interfere with this effect. In addition, we indicated that overexpression of Opa-1 and mitochondrial function are ameliorated via Leu-induced mitochondrial biogenesis. In contrast, knockdown of Opa-1 suppressed Leu-induced cardioprotection. CONCLUSION: Leu treatment is critical in rendering a cardioprotective effect exhibited by BCAAs via mTOR signaling. Furthermore, Leu improved mitochondrial function.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Leucina/farmacologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , GTP Fosfo-Hidrolases/genética , Masculino , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/patologia , Dinâmica Mitocondrial/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Biogênese de Organelas , Ratos Wistar , Transdução de Sinais
8.
Nat Commun ; 12(1): 4371, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272364

RESUMO

Metabolic programming and mitochondrial dynamics along with T cell differentiation affect T cell fate and memory development; however, how to control metabolic reprogramming and mitochondrial dynamics in T cell memory development is unclear. Here, we provide evidence that the SUMO protease SENP1 promotes T cell memory development via Sirt3 deSUMOylation. SENP1-Sirt3 signalling augments the deacetylase activity of Sirt3, promoting both OXPHOS and mitochondrial fusion. Mechanistically, SENP1 activates Sirt3 deacetylase activity in T cell mitochondria, leading to reduction of the acetylation of mitochondrial metalloprotease YME1L1. Consequently, deacetylation of YME1L1 suppresses its activity on OPA1 cleavage to facilitate mitochondrial fusion, which results in T cell survival and promotes T cell memory development. We also show that the glycolytic intermediate fructose-1,6-bisphosphate (FBP) as a negative regulator suppresses AMPK-mediated activation of the SENP1-Sirt3 axis and reduces memory development. Moreover, glucose limitation reduces FBP production and activates AMPK during T cell memory development. These data show that glucose limitation activates AMPK and the subsequent SENP1-Sirt3 signalling for T cell memory development.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Linfócitos T CD8-Positivos/imunologia , Cisteína Endopeptidases/metabolismo , Memória Imunológica , Mitocôndrias/metabolismo , Sirtuína 3/metabolismo , Linfócitos T/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Acetilação , Aloenxertos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Neoplasias do Colo/imunologia , Frutosedifosfatos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Glucose/deficiência , Memória Imunológica/genética , Metabolômica , Metaloendopeptidases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Sirtuína 3/antagonistas & inibidores , Sirtuína 3/genética , Sumoilação , Linfócitos T/imunologia
9.
Redox Biol ; 45: 102055, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34214709

RESUMO

BACKGROUND: Chronic lung diseases, such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are linked to several mitochondrial alterations. Cigarette smoke (CS) alters the structure and function of mitochondria. OPA1 is the main inner mitochondrial GTPase responsible for the fusion events. OPA1 undergoes proteolytic cleavage from long to short forms during acute stress and mitophagy. However, the exact role of OPA1 isoforms and related proteins during CS-induced mitophagy and COPD is not clear. METHODS: Lung tissues from non-smokers, smokers, COPD and IPF were used to determine the relative expression of OPA1 and related proteins. Additionally, we used mouse lungs from chronic (6 months) CS exposure to evaluate the status of OPA1. Primary lung fibroblasts from normal and COPD patients and naked mole rat (NMR) lung fibroblasts, human fetal lung fibroblast (HFL1), mouse embryonic fibroblast from wild type (WT), OPA1-/-, MFN1 and MFN2-/- were used to determine the effect of CS on OPA1 isoforms. Various mitochondrial fusion promoters/activators (BGP-15, leflunomide, M1) and fission inhibitor (DRP1) were used to determine their effect on OPA1 status and cigarette smoke extract (CSE)-induced lung epithelial (BEAS2B) cell damage, respectively. Seahorse flux analyzer was used to determine the effect of these compounds in BEAS2B cells with and without CSE exposure. FINDINGS: Short OPA1 isoforms were predominantly detected and significantly increased in COPD subjects. Acute CSE treatment in various cell lines except NMR was found to increase the conversion of long to short OPA1 isoforms. CSE treatment significantly increased mitochondrial stress-related protein SLP2 in all the cells used. OPA1 interacting partners like prohibitins (PHB1 and 2) were also altered depending on the CS exposure. Finally, BGP-15 and leflunomide treatment were able to preserve the long OPA1 isoform in cells treated with CSE. INTERPRETATION/CONCLUSION: The long OPA1 isoform along with SLP2 and prohibitins play a crucial role in CS-induced lung damage, causing mitophagy/mitochondrial dysfunction in COPD, which may be used as a novel therapeutic target in COPD.


Assuntos
Proteínas Mitocondriais , Doença Pulmonar Obstrutiva Crônica , Animais , Fibroblastos , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Pulmão/metabolismo , Camundongos , Mitocôndrias , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fumaça/efeitos adversos , Tabaco , Fumar Tabaco
10.
Nat Commun ; 12(1): 4164, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230493

RESUMO

Spi-1 Proto-Oncogene (SPI1) fusion genes are recurrently found in T-cell acute lymphoblastic leukemia (T-ALL) cases but are insufficient to drive leukemogenesis. Here we show that SPI1 fusions in combination with activating NRAS mutations drive an immature T-ALL in vivo using a conditional bone marrow transplant mouse model. Addition of the oncogenic fusion to the NRAS mutation also results in a higher leukemic stem cell frequency. Mechanistically, genetic deletion of the ß-catenin binding domain within Transcription factor 7 (TCF7)-SPI1 or use of a TCF/ß-catenin interaction antagonist abolishes the oncogenic activity of the fusion. Targeting the TCF7-SPI1 fusion in vivo with a doxycycline-inducible knockdown results in increased differentiation. Moreover, both pharmacological and genetic inhibition lead to down-regulation of SPI1 targets. Together, our results reveal an example where TCF7-SPI1 leukemia is vulnerable to pharmacological targeting of the TCF/ß-catenin interaction.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator 1 de Transcrição de Linfócitos T/metabolismo , Transativadores/metabolismo , beta Catenina/metabolismo , Animais , Transplante de Medula Óssea , Carcinogênese/genética , Modelos Animais de Doenças , Feminino , GTP Fosfo-Hidrolases/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Oncogenes , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogênicas/genética , Fator 1 de Transcrição de Linfócitos T/genética , Linfócitos T/metabolismo , Transativadores/genética , Transcriptoma , beta Catenina/genética
11.
Science ; 373(6550): 77-81, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34210880

RESUMO

Brain postnatal development is characterized by critical periods of experience-dependent remodeling of neuronal circuits. Failure to end these periods results in neurodevelopmental disorders. The cellular processes defining critical-period timing remain unclear. Here, we show that in the mouse visual cortex, astrocytes control critical-period closure. We uncover the underlying pathway, which involves astrocytic regulation of the extracellular matrix, allowing interneuron maturation. Unconventional astrocyte connexin signaling hinders expression of extracellular matrix-degrading enzyme matrix metalloproteinase 9 (MMP9) through RhoA-guanosine triphosphatase activation. Thus, astrocytes not only influence the activity of single synapses but also are key elements in the experience-dependent wiring of brain circuits.


Assuntos
Astrócitos/fisiologia , Período Crítico Psicológico , Plasticidade Neuronal , Córtex Visual/crescimento & desenvolvimento , Animais , Astrócitos/metabolismo , Conexina 30/metabolismo , Ativação Enzimática , GTP Fosfo-Hidrolases/metabolismo , Interneurônios/metabolismo , Interneurônios/fisiologia , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sinapses/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
12.
Nucleic Acids Res ; 49(14): 8384-8395, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34255843

RESUMO

Bacteria have evolved sophisticated mechanisms to deliver potent toxins into bacterial competitors or into eukaryotic cells in order to destroy rivals and gain access to a specific niche or to hijack essential metabolic or signaling pathways in the host. Delivered effectors carry various activities such as nucleases, phospholipases, peptidoglycan hydrolases, enzymes that deplete the pools of NADH or ATP, compromise the cell division machinery, or the host cell cytoskeleton. Effectors categorized in the family of polymorphic toxins have a modular structure, in which the toxin domain is fused to additional elements acting as cargo to adapt the effector to a specific secretion machinery. Here we show that Photorhabdus laumondii, an entomopathogen species, delivers a polymorphic antibacterial toxin via a type VI secretion system. This toxin inhibits protein synthesis in a NAD+-dependent manner. Using a biotinylated derivative of NAD, we demonstrate that translation is inhibited through ADP-ribosylation of the ribosomal 23S RNA. Mapping of the modification further showed that the adduct locates on helix 44 of the thiostrepton loop located in the GTPase-associated center and decreases the GTPase activity of the EF-G elongation factor.


Assuntos
Toxinas Bacterianas/farmacologia , GTP Fosfo-Hidrolases/genética , RNA Ribossômico 23S/genética , Sistemas de Secreção Tipo VI/efeitos dos fármacos , ADP-Ribosilação/efeitos dos fármacos , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , NAD/genética , Fator G para Elongação de Peptídeos/genética , Photorhabdus/química , Photorhabdus/genética , Biossíntese de Proteínas/efeitos dos fármacos , RNA Ribossômico 23S/efeitos dos fármacos , Tioestreptona/química , Tioestreptona/farmacologia
13.
Nature ; 596(7871): 281-284, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34290409

RESUMO

The mTOR complex 1 (mTORC1) controls cell growth in response to amino acid levels1. Here we report SAR1B as a leucine sensor that regulates mTORC1 signalling in response to intracellular levels of leucine. Under conditions of leucine deficiency, SAR1B inhibits mTORC1 by physically targeting its activator GATOR2. In conditions of leucine sufficiency, SAR1B binds to leucine, undergoes a conformational change and dissociates from GATOR2, which results in mTORC1 activation. SAR1B-GATOR2-mTORC1 signalling is conserved in nematodes and has a role in the regulation of lifespan. Bioinformatic analysis reveals that SAR1B deficiency correlates with the development of lung cancer. The silencing of SAR1B and its paralogue SAR1A promotes mTORC1-dependent growth of lung tumours in mice. Our results reveal that SAR1B is a conserved leucine sensor that has a potential role in the development of lung cancer.


Assuntos
Leucina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Transdução de Sinais , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sequência Conservada , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Humanos , Leucina/deficiência , Longevidade/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/agonistas , Camundongos , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/deficiência , Proteínas Monoméricas de Ligação ao GTP/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Proteins ; 89(11): 1541-1556, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34245187

RESUMO

The expansion of three-dimensional protein structures and enhanced computing power have significantly facilitated our understanding of protein sequence/structure/function relationships. A challenge in structural genomics is to predict the function of uncharacterized proteins. Protein function deconvolution based on global sequence or structural homology is impracticable when a protein relates to no other proteins with known function, and in such cases, functional relationships can be established by detecting their local ligand binding site similarity. Here, we introduce a sequence order-independent comparison algorithm, PocketShape, for structural proteome-wide exploration of protein functional site by fully considering the geometry of the backbones, orientation of the sidechains, and physiochemical properties of the pocket-lining residues. PocketShape is efficient in distinguishing similar from dissimilar ligand binding site pairs by retrieving 99.3% of the similar pairs while rejecting 100% of the dissimilar pairs on a dataset containing 1538 binding site pairs. This method successfully classifies 83 enzyme structures with diverse functions into 12 clusters, which is highly in accordance with the actual structural classification of proteins classification. PocketShape also achieves superior performances than other methods in protein profiling based on experimental data. Potential new applications for representative SARS-CoV-2 drugs Remdesivir and 11a are predicted. The high accuracy and time-efficient characteristics of PocketShape will undoubtedly make it a promising complementary tool for proteome-wide protein function inference and drug repurposing study.


Assuntos
Algoritmos , Antivirais/farmacologia , Reposicionamento de Medicamentos/métodos , Proteínas/metabolismo , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Alanina/farmacologia , Antivirais/química , Sítios de Ligação , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Bases de Dados de Proteínas , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Fosfoglicerato Mutase/química , Fosfoglicerato Mutase/metabolismo , Proteínas/química , Proteínas/classificação , Curva ROC , SARS-CoV-2/efeitos dos fármacos
15.
FASEB J ; 35(8): e21771, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34275172

RESUMO

Impaired mitochondrial fusion, due in part to decreased mitofusin 2 (Mfn2) expression, contributes to unrestricted cell proliferation and apoptosis-resistance in hyperproliferative diseases like pulmonary arterial hypertension (PAH) and non-small cell lung cancer (NSCLC). We hypothesized that Mfn2 levels are reduced due to increased proteasomal degradation of Mfn2 triggered by its phosphorylation at serine 442 (S442) and investigated the potential kinase mediators. Mfn2 expression was decreased and Mfn2 S442 phosphorylation was increased in pulmonary artery smooth muscle cells from PAH patients and in NSCLC cells. Mfn2 phosphorylation was mediated by PINK1 and protein kinase A (PKA), although only PINK1 expression was increased in these diseases. We designed a S442 phosphorylation deficient Mfn2 construct (PD-Mfn2) and a S442 constitutively phosphorylated Mfn2 construct (CP-Mfn2). The effects of these modified Mfn2 constructs on Mfn2 expression and biological function were compared with those of the wildtype Mfn2 construct (WT-Mfn2). WT-Mfn2 increased Mfn2 expression and mitochondrial fusion in both PAH and NSCLC cells resulting in increased apoptosis and decreased cell proliferation. Compared to WT-Mfn2, PD-Mfn2 caused greater Mfn2 expression, suppression of proliferation, apoptosis induction, and cell cycle arrest. Conversely, CP-Mfn2 caused only a small increase in Mfn2 expression and did not restore mitochondrial fusion, inhibit cell proliferation, or induce apoptosis. Silencing PINK1 or PKA, or proteasome blockade using MG132, increased Mfn2 expression, enhanced mitochondrial fusion and induced apoptosis. In a xenotransplantation NSCLC model, PD-Mfn2 gene therapy caused greater tumor regression than did therapy with WT-Mfn2. Mfn2 deficiency in PAH and NSCLC reflects proteasomal degradation triggered by Mfn2-S442 phosphorylation by PINK1 and/or PKA. Inhibiting Mfn2 phosphorylation has potential therapeutic benefit in PAH and lung cancer.


Assuntos
Proliferação de Células , GTP Fosfo-Hidrolases/metabolismo , Hipertensão Pulmonar/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Quinases/metabolismo , Proteólise , Células A549 , Animais , GTP Fosfo-Hidrolases/genética , Humanos , Hipertensão Pulmonar/genética , Neoplasias Pulmonares/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Mitocondriais/genética , Proteínas de Neoplasias/genética , Fosforilação/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Quinases/genética
16.
J Biol Chem ; 297(1): 100861, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34116056

RESUMO

Cellular growth and proliferation are primarily dictated by the mechanistic target of rapamycin complex 1 (mTORC1), which balances nutrient availability against the cell's anabolic needs. Central to the activity of mTORC1 is the RagA-RagC GTPase heterodimer, which under favorable conditions recruits the complex to the lysosomal surface to promote its activity. The RagA-RagC heterodimer has a unique architecture in that both subunits are active GTPases. To promote mTORC1 activity, the RagA subunit is loaded with GTP and the RagC subunit is loaded with GDP, while the opposite nucleotide-loading configuration inhibits this signaling pathway. Despite its unique molecular architecture, how the Rag GTPase heterodimer maintains the oppositely loaded nucleotide state remains elusive. Here, we applied structure-function analysis approach to the crystal structures of the Rag GTPase heterodimer and identified a key hydrogen bond that stabilizes the GDP-loaded state of the Rag GTPases. This hydrogen bond is mediated by the backbone carbonyl of Asn30 in the nucleotide-binding domain of RagA or Lys84 of RagC and the hydroxyl group on the side chain of Thr210 in the C-terminal roadblock domain of RagA or Ser266 of RagC, respectively. Eliminating this interdomain hydrogen bond abolishes the ability of the Rag GTPase to maintain its functional state, resulting in a distorted response to amino acid signals. Our results reveal that this long-distance interdomain interaction within the Rag GTPase is required for the maintenance and regulation of the mTORC1 nutrient-sensing pathway.


Assuntos
Aminoácidos/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteínas Monoméricas de Ligação ao GTP/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/ultraestrutura , Guanosina Trifosfato/química , Humanos , Ligação de Hidrogênio , Hidrólise , Alvo Mecanístico do Complexo 1 de Rapamicina/ultraestrutura , Proteínas Monoméricas de Ligação ao GTP/ultraestrutura , Conformação Proteica , Domínios Proteicos/genética , Multimerização Proteica/genética , Transdução de Sinais/genética
17.
Cancer Treat Rev ; 99: 102238, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34098219

RESUMO

Genetic alterations affecting RAS proteins are commonly found in human cancers. Roughly a fourth of melanoma patients carry activating NRAS mutations, rendering this malignancy particularly challenging to treat. Although the development of targeted as well as immunotherapies led to a substantial improvement in the overall survival of non-NRASmut melanoma patients (e.g. BRAFmut), patients with NRASmut melanomas have an overall poorer prognosis due to the high aggressiveness of RASmut tumors, lack of efficient targeted therapies or rapidly emerging resistance to existing treatments. Understanding how NRAS-driven melanomas develop therapy resistance by maintaining cell cycle progression and survival is crucial to develop more effective and specific treatments for this group of melanoma patients. In this review, we provide an updated summary of currently available therapeutic options for NRASmut melanoma patients with a focus on combined inhibition of MAPK signaling and CDK4/6-driven cell cycle progression and mechanisms of the inevitably developing resistance to these treatments. We conclude with an outlook on the most promising novel therapeutic approaches for melanoma patients with constitutively active NRAS. STATEMENT OF SIGNIFICANCE: An estimated 75000 patients are affected by NRASmut melanoma each year and these patients still have a shorter progression-free survival than BRAFmut melanomas. Both intrinsic and acquired resistance occur in NRAS-driven melanomas once treated with single or combined targeted therapies involving MAPK and CDK4/6 inhibitors and/or checkpoint inhibiting immunotherapy. Oncolytic viruses, mRNA-based vaccinations, as well as targeted triple-agent therapy are promising alternatives, which could soon contribute to improved progression-free survival of the NRASmut melanoma patient group.


Assuntos
GTP Fosfo-Hidrolases/genética , Melanoma/genética , Melanoma/terapia , Proteínas de Membrana/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/terapia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Terapia Combinada , Humanos , Melanoma/enzimologia , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , Ensaios Clínicos Controlados Aleatórios como Assunto , Neoplasias Cutâneas/enzimologia
18.
Methods Mol Biol ; 2276: 325-332, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060052

RESUMO

Mitochondrial fusion depends on proteolytic processing of the dynamin-related GTPase protein, OPA1, which is regulated by the mitochondrial zinc metalloproteinase, OMA1. Last year we published a report describing a novel approach to directly measure the enzymatic activity of OMA1 in whole cell lysates. This fluorescence-based reporter assay utilizes an eight amino acid peptide sequence referred to as the S1 cleavage site where OMA1 cleaves within OPA1 and is flanked by a fluorophore and quencher. In this chapter, we provide additional insight into the OMA1 activity assay.


Assuntos
Ensaios Enzimáticos/métodos , Corantes Fluorescentes/química , GTP Fosfo-Hidrolases/metabolismo , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Peptídeos/química , Células Cultivadas , Humanos , Dinâmica Mitocondrial
19.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072974

RESUMO

This study investigates whether reduced optic atrophy 1 (Opa1) level promotes apoptosis and retinal vascular lesions associated with diabetic retinopathy (DR). Four groups of mice: wild type (WT) control mice, streptozotocin (STZ)-induced diabetic mice, Opa1+/- mice, and diabetic Opa1+/- mice were used in this study. 16 weeks after diabetes onset, retinas were assessed for Opa1 and Bax levels by Western blot analysis, and retinal networks were examined for acellular capillaries (AC) and pericyte loss (PL). Apoptotic cells were detected in retinal capillaries using TUNEL assay, and caspase-3 activity was assessed using fluorometric analysis. Opa1 expression was significantly downregulated in retinas of diabetic and Opa1+/- mice compared with those of WT mice. Inducing diabetes further decreased Opa1 expression in retinas of Opa1+/- mice. Increased cytochrome c release concomitant with increased level of pro-apoptotic Bax and elevated caspase-3 activity were observed in retinas of diabetic and Opa1+/- mice; the number of TUNEL-positive cells and AC/PL was also significantly increased. An additional decrease in the Opa1 level in retinas of diabetic Opa1+/- mice exacerbated the development of apoptotic cells and AC/PL compared with those of diabetic mice. Diabetes-induced Opa1 downregulation contributes, at least in part, to the development of retinal vascular lesions characteristic of DR.


Assuntos
Capilares , Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/metabolismo , GTP Fosfo-Hidrolases/fisiologia , Vasos Retinianos , Animais , Apoptose , Capilares/metabolismo , Capilares/patologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/etiologia , Retinopatia Diabética/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia
20.
FASEB J ; 35(7): e21678, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34133045

RESUMO

Hypertension is associated with excessive reactive oxygen species (ROS) production in vascular cells. Mitochondria undergo fusion and fission, a process playing a role in mitochondrial function. OPA1 is essential for mitochondrial fusion. Loss of OPA1 is associated with ROS production and cell dysfunction. We hypothesized that mitochondria fusion could reduce oxidative stress that defect in fusion would exacerbate hypertension. Using (a) Opa1 haploinsufficiency in isolated resistance arteries from Opa1+/- mice, (b) primary vascular cells from Opa1+/- mice, and (c) RNA interference experiments with siRNA against Opa1 in vascular cells, we investigated the role of mitochondria fusion in hypertension. In hypertension, Opa1 haploinsufficiency induced altered mitochondrial cristae structure both in vascular smooth muscle and endothelial cells but did not modify protein level of long and short forms of OPA1. In addition, we demonstrated an increase of mitochondrial ROS production, associated with a decrease of superoxide dismutase 1 protein expression. We also observed an increase of apoptosis in vascular cells and a decreased VSMCs proliferation. Blood pressure, vascular contractility, as well as endothelium-dependent and -independent relaxation were similar in Opa1+/- , WT, L-NAME-treated Opa1+/- and WT mice. Nevertheless, chronic NO-synthase inhibition with L-NAME induced a greater hypertension in Opa1+/- than in WT mice without compensatory arterial wall hypertrophy. This was associated with a stronger reduction in endothelium-dependent relaxation due to excessive ROS production. Our results highlight the protective role of mitochondria fusion in the vasculature during hypertension by limiting mitochondria ROS production.


Assuntos
GTP Fosfo-Hidrolases/fisiologia , Hipertensão/prevenção & controle , Dinâmica Mitocondrial , Substâncias Protetoras/administração & dosagem , Animais , Apoptose , Inibidores Enzimáticos/toxicidade , Hipertensão/induzido quimicamente , Hipertensão/metabolismo , Hipertensão/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , NG-Nitroarginina Metil Éster/toxicidade , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo
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