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2.
J Hematol Oncol ; 14(1): 171, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34663417

RESUMO

GTPase-activating protein (GAP) is a negative regulator of GTPase protein that is thought to promote the conversion of the active GTPase-GTP form to the GTPase-GDP form. Based on its ability to regulate GTPase proteins and other domains, GAPs are directly or indirectly involved in various cell requirement processes. We reviewed the existing evidence of GAPs regulating regulated cell death (RCD), mainly apoptosis and autophagy, as well as some novel RCDs, with particular attention to their association in diseases, especially cancer. We also considered that GAPs could affect tumor immunity and attempted to link GAPs, RCD and tumor immunity. A deeper understanding of the GAPs for regulating these processes could lead to the discovery of new therapeutic targets to avoid pathologic cell loss or to mediate cancer cell death.


Assuntos
Proteínas Ativadoras de GTPase/imunologia , Neoplasias/imunologia , Morte Celular Regulada , Animais , GTP Fosfo-Hidrolases/imunologia , Guanosina Trifosfato/imunologia , Humanos , Neoplasias/patologia
4.
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
5.
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
6.
Int J Mol Sci ; 22(18)2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34575944

RESUMO

Mitigation of calcium-dependent destruction of skeletal muscle mitochondria is considered as a promising adjunctive therapy in Duchenne muscular dystrophy (DMD). In this work, we study the effect of intraperitoneal administration of a non-immunosuppressive inhibitor of calcium-dependent mitochondrial permeability transition (MPT) pore alisporivir on the state of skeletal muscles and the functioning of mitochondria in dystrophin-deficient mdx mice. We show that treatment with alisporivir reduces inflammation and improves muscle function in mdx mice. These effects of alisporivir were associated with an improvement in the ultrastructure of mitochondria, normalization of respiration and oxidative phosphorylation, and a decrease in lipid peroxidation, due to suppression of MPT pore opening and an improvement in calcium homeostasis. The action of alisporivir was associated with suppression of the activity of cyclophilin D and a decrease in its expression in skeletal muscles. This was observed in both mdx mice and wild-type animals. At the same time, alisporivir suppressed mitochondrial biogenesis, assessed by the expression of Ppargc1a, and altered the dynamics of organelles, inhibiting both DRP1-mediated fission and MFN2-associated fusion of mitochondria. The article discusses the effects of alisporivir administration and cyclophilin D inhibition on mitochondrial reprogramming and networking in DMD and the consequences of this therapy on skeletal muscle health.


Assuntos
Dinaminas/genética , Distrofina/genética , GTP Fosfo-Hidrolases/genética , Distrofia Muscular de Duchenne/tratamento farmacológico , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Animais , Ciclofilinas/genética , Ciclosporina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos mdx , Mitocôndrias/efeitos dos fármacos , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/genética , Dinâmica Mitocondrial/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial/farmacologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia
7.
Int J Mol Sci ; 22(18)2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34575980

RESUMO

Mitochondrial impairments in dynamic behavior (fusion/fission balance) associated with mitochondrial dysfunction play a key role in cell lipotoxicity and lipid-induced metabolic diseases. The present work aimed to evaluate dose- and time-dependent effects of the monounsaturated fatty acid oleate on mitochondrial fusion/fission proteins in comparison with the saturated fatty acid palmitate in hepatic cells. To this end, HepG-2 cells were treated with 0, 10 µM, 50 µM, 100 µM, 250 µM or 500 µM of either oleate or palmitate for 8 or 24 h. Cell viability and lipid accumulation were evaluated to assess lipotoxicity. Mitochondrial markers of fusion (mitofusin 2, MFN2) and fission (dynamin-related protein 1, DRP1) processes were evaluated by Western blot analysis. After 8 h, the highest dose of oleate induced a decrease in DRP1 content without changes in MFN2 content in association with cell viability maintenance, whereas palmitate induced a decrease in cell viability associated with a decrease mainly in MFN2 content. After 24 h, oleate induced MFN2 increase, whereas palmitate induced DRP1 increase associated with a higher decrease in cell viability with high doses compared to oleate. This finding could be useful to understand the role of mitochondria in the protective effects of oleate as a bioactive compound.


Assuntos
Dinaminas/genética , GTP Fosfo-Hidrolases/genética , Doenças Metabólicas/genética , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/genética , Ácido Oleico/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ácidos Graxos Monoinsaturados/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/toxicidade , Doenças Metabólicas/etiologia , Doenças Metabólicas/metabolismo , Doenças Metabólicas/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/patologia , Dinâmica Mitocondrial/genética , Ácido Oleico/farmacologia , Palmitatos/metabolismo , Palmitatos/farmacologia
8.
Infect Immun ; 89(11): e0030621, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34370506

RESUMO

A mitochondrion, as a highly dynamic organelle, continuously changes morphology and position during its life cycle. Mitochondrial dynamics, including fission and fusion, play a critical role in maintaining functional mitochondria for ATP production, which is directly linked to host defense against Mycobacterium tuberculosis infection. However, how macrophages regulate mitochondrial dynamics during M. tuberculosis infection remains elusive. In this study, we found that M. tuberculosis infection induced mitochondrial fusion by enhancing the expression of mitofusin 1 (MFN1), which resulted in increased ATP production. Silencing of MFN1 inhibited mitochondrial fusion and subsequently reduced ATP production, which, in turn, severely impaired macrophages' mycobactericidal activity by inhibiting autophagy. Impairment of mycobactericidal activity and autophagy was replicated using oligomycin, an inhibitor of ATP synthase. In summary, our study revealed that MFN1-mediated mitochondrial fusion is essential for macrophages' mycobactericidal activity through the regulation of ATP-dependent autophagy. The MFN1-mediated metabolism pathway might be a target for the development of a host direct therapy (HDT) strategy against tuberculosis.


Assuntos
Autofagia/fisiologia , GTP Fosfo-Hidrolases/fisiologia , Macrófagos/imunologia , Dinâmica Mitocondrial/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Tuberculose/imunologia , Trifosfato de Adenosina/biossíntese , Humanos , Células THP-1 , Tuberculose/tratamento farmacológico
9.
Infect Immun ; 89(11): e0020221, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34338548

RESUMO

Gamma interferon (IFN-γ)-induced immunity-related GTPases (IRGs) confer cell-autonomous immunity to the intracellular protozoan pathogen Toxoplasma gondii. Effector IRGs are loaded onto the Toxoplasma-containing parasitophorous vacuole (PV), where they recruit ubiquitin ligases, ubiquitin-binding proteins, and IFN-γ-inducible guanylate-binding proteins (Gbps), prompting PV lysis and parasite destruction. Host cells lacking the regulatory IRGs Irgm1 and Irgm3 fail to load effector IRGs, ubiquitin, and Gbps onto the PV and are consequently defective for cell-autonomous immunity to Toxoplasma. However, the role of the third regulatory IRG, Irgm2, in cell-autonomous immunity to Toxoplasma has remained unexplored. Here, we report that Irgm2 unexpectedly plays a limited role in the targeting of effector IRGs, ubiquitin, and Gbps to the Toxoplasma PV. Instead, Irgm2 is instrumental in the decoration of PVs with γ-aminobutyric acid receptor-associated protein-like 2 (GabarapL2). Cells lacking Irgm2 are as defective for cell-autonomous host defense to Toxoplasma as pan-Irgm-/- cells lacking all three Irgm proteins, and Irgm2-/- mice succumb to Toxoplasma infections as readily as pan-Irgm-/- mice. These findings demonstrate that, relative to Irgm1 and Irgm3, Irgm2 plays a distinct but critically important role in host resistance to Toxoplasma.


Assuntos
GTP Fosfo-Hidrolases/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Toxoplasmose/imunologia , Animais , Proteínas Reguladoras de Apoptose/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/fisiologia , Ubiquitina/fisiologia , Vacúolos/fisiologia
10.
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
11.
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
12.
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
13.
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
14.
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
15.
Clin Immunol ; 231: 108837, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34455097

RESUMO

RAS-associated autoimmune leukoproliferative disease (RALD) is a rare immune dysregulation syndrome caused by somatic gain-of-function mutations of either NRAS or KRAS gene in hematopoietic cells. We describe a 27-year-old patient presenting at 5 months of age with recurrent infections and generalized lymphadenopathy who developed a complex multi-organ autoimmune syndrome with hypogammaglobulinemia, partially controlled with oral steroids, hydroxichloroquine, mofetil mycophenolate and IVIG prophylaxis. Activation of type I interferon pathway was observed in peripheral blood. Since 18 years of age, the patient developed regenerative nodular hyperplasia of the liver evolving into hepatopulmonary syndrome. Whole exome sequencing analysis of the peripheral blood DNA showed the NRAS p.Gly13Asp mutation validated as somatic. Our report highlights the possibility of detecting somatic NRAS gene mutations in patients with inflammatory immune dysregulation and type I interferon activation.


Assuntos
Síndrome Linfoproliferativa Autoimune/genética , Síndrome Linfoproliferativa Autoimune/imunologia , GTP Fosfo-Hidrolases/genética , Interferon Tipo I/imunologia , Hepatopatias/genética , Proteínas de Membrana/genética , Adulto , Síndrome Linfoproliferativa Autoimune/complicações , Humanos , Hepatopatias/imunologia , Mutação
16.
Redox Biol ; 46: 102098, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34418601

RESUMO

Diabetes disrupts mitochondrial function and often results in diabetic cardiomyopathy (DCM). Paeonol is a bioactive compound that has been reported to have pharmacological potential for cardiac and mitochondrial protection. This study aims to explore the effects of paeonol on mitochondrial disorderes in DCM and the underlying mechanisms. We showed that paeonol promoted Opa1-mediated mitochondrial fusion, inhibited mitochondrial oxidative stress, and preserved mitochondrial respiratory capacity and cardiac performance in DCM in vivo and in vitro. Knockdown of Opa1 blunted the above protective effects of paeonol in both diabetic hearts and high glucose-treated cardiomyocytes. Mechanistically, inhibitor screening, siRNA knockdown and chromatin immunoprecipitation experiments showed that paeonol-promoted Opa1-mediated mitochondrial fusion required the activation of Stat3, which directly bound to the promoter of Opa1 to upregulate its transcriptional expression. Moreover, pharmmapper screening and molecular docking studies revealed that CK2α served as a direct target of paeonol that interacted with Jak2 and induced the phosphorylation and activation of Jak2-Stat3. Knockdown of CK2α blunted the promoting effect of paeonol on Jak2-Stat3 phosphorylation and Opa1-mediated mitochondrial fusion. Collectively, we have demonstrated for the first time that paeonol is a novel mitochondrial fusion promoter in protecting against hyperglycemia-induced mitochondrial oxidative injury and DCM at least partially via an Opa1-mediated mechanism, a process in which paeonol interacts with CK2α and restores its kinase activity that subsequently increasing Jak2-Stat3 phosphorylation and enhancing the transcriptional level of Opa1. These findings suggest that paeonol or the promotion of mitochondrial fusion might be a promising strategy for the treatment of DCM.


Assuntos
Diabetes Mellitus , Cardiomiopatias Diabéticas , Acetofenonas/farmacologia , Cardiomiopatias Diabéticas/tratamento farmacológico , Cardiomiopatias Diabéticas/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Dinâmica Mitocondrial , Simulação de Acoplamento Molecular , Fator de Transcrição STAT3/genética
17.
Biomolecules ; 11(8)2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34439767

RESUMO

Parkinson's Disease (PD) affects millions of people worldwide with no cure to halt the progress of the disease. Leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause of PD and, as such, LRRK2 inhibitors are promising therapeutic agents. In the last decade, great progress in the LRRK2 field has been made. This review provides a comprehensive overview of the current state of the art, presenting recent developments and challenges in developing LRRK2 inhibitors, and discussing extensively the potential targeting strategies from the protein perspective. As currently there are three LRRK2-targeting agents in clinical trials, more developments are predicted in the upcoming years.


Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Doença de Parkinson/tratamento farmacológico , Sítio Alostérico , Animais , Biomarcadores/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Doenças Neurodegenerativas/metabolismo , Doença de Parkinson/metabolismo , Fosforilação , Conformação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Resultado do Tratamento
18.
PLoS One ; 16(7): e0253987, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34242285

RESUMO

Autosomal dominant optic atrophy is one of the most common inherited optic neuropathies. This disease is genetically heterogeneous, but most cases are due to pathogenic variants in the OPA1 gene: depending on the population studied, 32-90% of cases harbor pathogenic variants in this gene. The aim of this study was to provide a comprehensive overview of the entire spectrum of likely pathogenic variants in the OPA1 gene in a large cohort of patients. Over a period of 20 years, 755 unrelated probands with a diagnosis of bilateral optic atrophy were referred to our laboratory for molecular genetic investigation. Genetic testing of the OPA1 gene was initially performed by a combined analysis using either single-strand conformation polymorphism or denaturing high performance liquid chromatography followed by Sanger sequencing to validate aberrant bands or melting profiles. The presence of copy number variations was assessed using multiplex ligation-dependent probe amplification. Since 2012, genetic testing was based on next-generation sequencing platforms. Genetic screening of the OPA1 gene revealed putatively pathogenic variants in 278 unrelated probands which represent 36.8% of the entire cohort. A total of 156 unique variants were identified, 78% of which can be considered null alleles. Variant c.2708_2711del/p.(V903Gfs*3) was found to constitute 14% of all disease-causing alleles. Special emphasis was placed on the validation of splice variants either by analyzing cDNA derived from patients´ blood samples or by heterologous splice assays using minigenes. Splicing analysis revealed different aberrant splicing events, including exon skipping, activation of exonic or intronic cryptic splice sites, and the inclusion of pseudoexons. Forty-eight variants that we identified were novel. Nine of them were classified as pathogenic, 34 as likely pathogenic and five as variant of uncertain significance. Our study adds a significant number of novel variants to the mutation spectrum of the OPA1 gene and will thereby facilitate genetic diagnostics of patients with suspected dominant optic atrophy.


Assuntos
GTP Fosfo-Hidrolases/genética , Predisposição Genética para Doença , Mutação/genética , Atrofia Óptica Autossômica Dominante/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Criança , Estudos de Coortes , Feminino , GTP Fosfo-Hidrolases/sangue , GTP Fosfo-Hidrolases/química , Humanos , Masculino , Pessoa de Meia-Idade , Atrofia Óptica Autossômica Dominante/sangue , Adulto Jovem
19.
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
20.
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
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