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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.
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
4.
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
5.
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
6.
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
7.
EMBO Rep ; 22(9): e51954, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34296790

RESUMO

Mfn2 is a mitochondrial fusion protein with bioenergetic functions implicated in the pathophysiology of neuronal and metabolic disorders. Understanding the bioenergetic mechanism of Mfn2 may aid in designing therapeutic approaches for these disorders. Here we show using endoplasmic reticulum (ER) or mitochondria-targeted Mfn2 that Mfn2 stimulation of the mitochondrial metabolism requires its localization in the ER, which is independent of its fusion function. ER-located Mfn2 interacts with mitochondrial Mfn1/2 to tether the ER and mitochondria together, allowing Ca2+ transfer from the ER to mitochondria to enhance mitochondrial bioenergetics. The physiological relevance of these findings is shown during neurite outgrowth, when there is an increase in Mfn2-dependent ER-mitochondria contact that is necessary for correct neuronal arbor growth. Reduced neuritic growth in Mfn2 KO neurons is recovered by the expression of ER-targeted Mfn2 or an artificial ER-mitochondria tether, indicating that manipulation of ER-mitochondria contacts could be used to treat pathologic conditions involving Mfn2.


Assuntos
Retículo Endoplasmático , GTP Fosfo-Hidrolases , Retículo Endoplasmático/metabolismo , Metabolismo Energético , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo
8.
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
9.
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
10.
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
11.
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
12.
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
13.
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
14.
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
15.
Mol Med Rep ; 24(2)2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34080026

RESUMO

The dynamic regulation of mitochondrial morphology is key for eukaryotic cells to manage physiological challenges. Therefore, it is important to understand the molecular basis of mitochondrial dynamic regulation. The aim of the present study was to explore the role of HIG1 hypoxia inducible domain family member 1B (HIGD­1B) in hypoxia­induced mitochondrial fragmentation. Protein expression was determined via western blotting. Immunofluorescence assays were performed to detect the subcellular location of HIGD­1B. Cell Counting Kit­8 assays and flow cytometry were carried out to measure cell viability and apoptosis, respectively. Protein interactions were evaluated by co­immunoprecipitation. In the present study, it was found that HIGD­1B serves a role in cell survival by maintaining the integrity of the mitochondria under hypoxic conditions. Knockdown of HIGD­1B promoted mitochondrial fragmentation, while overexpression of HIGD­1B increased survival by preventing activation of caspase­3 and ­9. HIGD­1B expression was associated with cell viability and apoptosis in cardiomyocytes. Furthermore, HIGD­1B delayed the cleavage process of optic atrophy 1 (OPA1) and stabilized mitochondrial morphology by interacting with OPA1. Collectively, the results from the present study identified a role for HIGD­1B as an inhibitor of the mitochondrial fission in cardiomyocytes.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Dinâmica Mitocondrial/fisiologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , Apoptose/genética , Morte Celular/genética , Hipóxia Celular/fisiologia , Linhagem Celular , Sobrevivência Celular/genética , Técnicas de Silenciamento de Genes , Humanos , Miócitos Cardíacos/citologia
16.
J Cell Sci ; 134(13)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34110411

RESUMO

Mitochondria-endoplasmic reticulum contacts (MERCs) play an essential role in multiple cell physiological processes. Although Mfn2 was the first protein implicated in the formation of MERCs, there is debate as to whether it acts as a tether or antagonizer, largely based on in vitro studies. To understand the role of Mfn2 in MERCs in vivo, we characterized ultrastructural and biochemical changes of MERCs in pyramidal neurons of hippocampus in Mfn2 conditional knockout mice and in Mfn2 overexpressing mice, and found that Mfn2 ablation caused reduced close contacts, whereas Mfn2 overexpression caused increased close contacts between the endoplasmic reticulum (ER) and mitochondria in vivo. Functional studies on SH-SY5Y cells with Mfn2 knockout or overexpression demonstrating similar biochemical changes found that mitochondrial calcium uptake along with IP3R3-Grp75 interaction was decreased in Mfn2 knockout cells but increased in Mfn2 overexpressing cells. Lastly, we found Mfn2 knockout decreased and Mfn2 overexpression increased the interaction between the ER-mitochondria tethering pair of VAPB-PTPIP51. In conclusion, our study supports the notion that Mfn2 plays a critical role in ER-mitochondrial tethering and the formation of close contacts in neuronal cells in vivo.


Assuntos
Retículo Endoplasmático , Proteínas Mitocondriais , Animais , Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Hipocampo/metabolismo , Camundongos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Neurônios/metabolismo , Proteínas Tirosina Fosfatases/metabolismo
17.
Nucleic Acids Res ; 49(12): 7053-7074, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34125911

RESUMO

Eukaryotic ribosome biogenesis is an elaborate process during which ribosomal proteins assemble with the pre-rRNA while it is being processed and folded. Hundreds of assembly factors (AF) are required and transiently recruited to assist the sequential remodeling events. One of the most intricate ones is the stepwise removal of the internal transcribed spacer 2 (ITS2), between the 5.8S and 25S rRNAs, that constitutes together with five AFs the pre-60S 'foot'. In the transition from nucleolus to nucleoplasm, Nop53 replaces Erb1 at the basis of the foot and recruits the RNA exosome for the ITS2 cleavage and foot disassembly. Here we comprehensively analyze the impact of Nop53 recruitment on the pre-60S compositional changes. We show that depletion of Nop53, different from nop53 mutants lacking the exosome-interacting motif, not only causes retention of the unprocessed foot in late pre-60S intermediates but also affects the transition from nucleolar state E particle to subsequent nuclear stages. Additionally, we reveal that Nop53 depletion causes the impairment of late maturation events such as Yvh1 recruitment. In light of recently described pre-60S cryo-EM structures, our results provide biochemical evidence for the structural role of Nop53 rearranging and stabilizing the foot interface to assist the Nog2 particle formation.


Assuntos
Proteínas Nucleares/fisiologia , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Fosfatases de Especificidade Dupla/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Biogênese de Organelas , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
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.
J Cell Biol ; 220(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34019080

RESUMO

Neuronal extracellular vesicles (EVs) play important roles in intercellular communication and pathogenic protein propagation in neurological disease. However, it remains unclear how cargoes are selectively packaged into neuronal EVs. Here, we show that loss of the endosomal retromer complex leads to accumulation of EV cargoes including amyloid precursor protein (APP), synaptotagmin-4 (Syt4), and neuroglian (Nrg) at Drosophila motor neuron presynaptic terminals, resulting in increased release of these cargoes in EVs. By systematically exploring known retromer-dependent trafficking mechanisms, we show that EV regulation is separable from several previously identified roles of neuronal retromer. Conversely, mutations in rab11 and rab4, regulators of endosome-plasma membrane recycling, cause reduced EV cargo levels, and rab11 suppresses cargo accumulation in retromer mutants. Thus, EV traffic reflects a balance between Rab4/Rab11 recycling and retromer-dependent removal from EV precursor compartments. Our data shed light on previous studies implicating Rab11 and retromer in competing pathways in Alzheimer's disease, and suggest that misregulated EV traffic may be an underlying defect.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Vesículas Extracelulares/metabolismo , Terminações Pré-Sinápticas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/metabolismo , Animais , Animais Geneticamente Modificados , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestrutura , Vesículas Extracelulares/genética , Vesículas Extracelulares/ultraestrutura , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Terminações Pré-Sinápticas/ultraestrutura , Transporte Proteico , Sinaptotagminas/genética , Sinaptotagminas/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/genética
20.
Nat Commun ; 12(1): 2736, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980844

RESUMO

Endothelial barrier integrity is ensured by the stability of the adherens junction (AJ) complexes comprised of vascular endothelial (VE)-cadherin as well as accessory proteins such as ß-catenin and p120-catenin. Disruption of the endothelial barrier due to disassembly of AJs results in tissue edema and the influx of inflammatory cells. Using three-dimensional structured illumination microscopy, we observe that the mitochondrial protein Mitofusin-2 (Mfn2) co-localizes at the plasma membrane with VE-cadherin and ß-catenin in endothelial cells during homeostasis. Upon inflammatory stimulation, Mfn2 is sulfenylated, the Mfn2/ß-catenin complex disassociates from the AJs and Mfn2 accumulates in the nucleus where Mfn2 negatively regulates the transcriptional activity of ß-catenin. Endothelial-specific deletion of Mfn2 results in inflammatory activation, indicating an anti-inflammatory role of Mfn2 in vivo. Our results suggest that Mfn2 acts in a non-canonical manner to suppress the inflammatory response by stabilizing cell-cell adherens junctions and by binding to the transcriptional activator ß-catenin.


Assuntos
Junções Aderentes/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas Mitocondriais/metabolismo , beta Catenina/metabolismo , Animais , Antígenos CD/metabolismo , Western Blotting , Caderinas/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Feminino , Humanos , Imunoprecipitação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia
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