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1.
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
2.
Int J Mol Sci ; 22(11)2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34198873

RESUMO

Nicotinamide nucleotide transhydrogenase (NNT) is a proton pump in the inner mitochondrial membrane that generates reducing equivalents in the form of NAPDH, which can be used for anabolic pathways or to remove reactive oxygen species (ROS). A number of studies have linked NNT dysfunction to cardiomyopathies and increased risk of atherosclerosis; however, biallelic mutations in humans commonly cause a phenotype of adrenal insufficiency, with rare occurrences of cardiac dysfunction and testicular tumours. Here, we compare the transcriptomes of the hearts, adrenals and testes from three mouse models: the C57BL/6N, which expresses NNT; the C57BL/6J, which lacks NNT; and a third mouse, expressing the wild-type NNT sequence on the C57BL/6J background. We saw enrichment of oxidative phosphorylation genes in the C57BL/B6J in the heart and adrenal, possibly indicative of an evolved response in this substrain to loss of Nnt. However, differential gene expression was mainly driven by mouse background with some changes seen in all three tissues, perhaps reflecting underlying genetic differences between the C57BL/B6J and -6N substrains.


Assuntos
Aterosclerose/genética , Cardiomiopatias/genética , Miocárdio/metabolismo , NADP Trans-Hidrogenase Específica para A ou B/genética , Fosforilação Oxidativa , Glândulas Suprarrenais/metabolismo , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Cardiomiopatias/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Testículo/metabolismo
3.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208592

RESUMO

Mitochondrial DNA depletion and multiple deletions syndromes (MDDS) constitute a group of mitochondrial diseases defined by dysfunctional mitochondrial DNA (mtDNA) replication and maintenance. As is the case for many other mitochondrial diseases, the options for the treatment of these disorders are rather limited today. Some aggressive treatments such as liver transplantation or allogeneic stem cell transplantation are among the few available options for patients with some forms of MDDS. However, in recent years, significant advances in our knowledge of the biochemical pathomechanisms accounting for dysfunctional mtDNA replication have been achieved, which has opened new prospects for the treatment of these often fatal diseases. Current strategies under investigation to treat MDDS range from small molecule substrate enhancement approaches to more complex treatments, such as lentiviral or adenoassociated vector-mediated gene therapy. Some of these experimental therapies have already reached the clinical phase with very promising results, however, they are hampered by the fact that these are all rare disorders and so the patient recruitment potential for clinical trials is very limited.


Assuntos
DNA Mitocondrial , Mitocôndrias/genética , Doenças Mitocondriais/etiologia , Doenças Mitocondriais/terapia , Animais , Terapia Combinada , Replicação do DNA , Gerenciamento Clínico , Suscetibilidade a Doenças , Regulação da Expressão Gênica , Humanos , Mitocôndrias/metabolismo , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação
4.
Nat Commun ; 12(1): 4203, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244519

RESUMO

Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we establish an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generate a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms ( http://steinmetzlab.embl.de/iBrowser/ ). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identify 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by the discovery of IMMT isoforms mis-spliced by RBM20 mutations. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms, providing more immediate biological interpretation and higher resolution transcriptome comparisons.


Assuntos
Processamento Alternativo , Cardiomiopatia Dilatada/genética , Miócitos Cardíacos/patologia , Proteínas de Ligação a RNA/genética , RNA-Seq/métodos , Sistemas CRISPR-Cas/genética , Cardiomiopatia Dilatada/patologia , Diferenciação Celular/genética , Linhagem Celular , Estudos de Viabilidade , Edição de Genes , Humanos , Células-Tronco Pluripotentes Induzidas , Proteínas Mitocondriais/genética , Anotação de Sequência Molecular , Proteínas Musculares/genética , Mutação , Isoformas de RNA/genética , RNA Guia/genética
5.
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
6.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073512

RESUMO

Gene clusters are becoming promising tools for gene identification. The study reveals the purposive genomic distribution of genes toward higher inheritance rates of intact metabolic pathways/phenotypes and, thereby, higher fitness. The co-localization of co-expressed, co-interacting, and functionally related genes was found as genome-wide trends in humans, mouse, golden eagle, rice fish, Drosophila, peanut, and Arabidopsis. As anticipated, the analyses verified the co-segregation of co-localized events. A negative correlation was notable between the likelihood of co-localization events and the inter-loci distances. The evolution of genomic blocks was also found convergent and uniform along the chromosomal arms. Calling a genomic block responsible for adjacent metabolic reactions is therefore recommended for identification of candidate genes and interpretation of cellular functions. As a case story, a function in the metabolism of energy and secondary metabolites was proposed for Slc25A44, based on its genomic local information. Slc25A44 was further characterized as an essential housekeeping gene which has been under evolutionary purifying pressure and belongs to the phylogenetic ETC-clade of SLC25s. Pathway enrichment mapped the Slc25A44s to the energy metabolism. The expression of peanut and human Slc25A44s in oocytes and Saccharomyces cerevisiae strains confirmed the transport of common precursors for secondary metabolites and ubiquinone. These results suggest that SLC25A44 is a mitochondrion-ER-nucleus zone transporter with biotechnological applications. Finally, a conserved three-amino acid signature on the cytosolic face of transport cavity was found important for rational engineering of SLC25s.


Assuntos
Sistemas de Transporte de Aminoácidos , Evolução Molecular , Genoma Humano , Proteínas Mitocondriais , Proteínas Carreadoras de Solutos , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Animais , Transporte Biológico Ativo , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Carreadoras de Solutos/genética , Proteínas Carreadoras de Solutos/metabolismo , Xenopus
7.
Front Immunol ; 12: 665773, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34108966

RESUMO

The COVID-19 pandemic has caused more than three million deaths globally. The severity of the disease is characterized, in part, by a dysregulated immune response. CD16+ monocytes are innate immune cells involved in inflammatory responses to viral infections, and tissue repair, among other functions. We characterized the transcriptional changes in CD16+ monocytes from PBMC of people with COVID-19, and from healthy individuals using publicly available single cell RNA sequencing data. CD16+ monocytes from people with COVID-19 compared to those from healthy individuals expressed transcriptional changes indicative of increased cell activation, and induction of a migratory phenotype. We also analyzed COVID-19 cases based on severity of the disease and found that mild cases were characterized by upregulation of interferon response and MHC class II related genes, whereas the severe cases had dysregulated expression of mitochondrial and antigen presentation genes, and upregulated inflammatory, cell movement, and apoptotic gene signatures. These results suggest that CD16+ monocytes in people with COVID-19 contribute to a dysregulated host response characterized by decreased antigen presentation, and an elevated inflammatory response with increased monocytic infiltration into tissues. Our results show that there are transcriptomic changes in CD16+ monocytes that may impact the functions of these cells, contributing to the pathogenesis and severity of COVID-19.


Assuntos
COVID-19/virologia , Monócitos/virologia , Receptores de IgG/metabolismo , SARS-CoV-2/patogenicidade , Transcrição Genética , Transcriptoma , Adulto , Idoso , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , COVID-19/genética , COVID-19/imunologia , COVID-19/metabolismo , Estudos de Casos e Controles , Citocinas/genética , Citocinas/metabolismo , Feminino , Proteínas Ligadas por GPI/metabolismo , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Mediadores da Inflamação/metabolismo , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , RNA-Seq , SARS-CoV-2/imunologia , Índice de Gravidade de Doença , Análise de Célula Única , Adulto Jovem
8.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072612

RESUMO

The BAG proteins are a family of multi-functional co-chaperones. In plants, BAG proteins were found to play roles both in abiotic and biotic stress tolerance. However, the function of Arabidopsis BAG2 remains largely unknown, whereas BAG6 is required for plants' defense to pathogens, although it remains unknown whether BAG6 is involved in plants' tolerance to abiotic stresses. Here, we show that both BAG2 and BAG6 are expressed in various tissues and are upregulated by salt, mannitol, and heat treatments and by stress-related hormones including ABA, ethylene, and SA. Germination of bag2, bag6 and bag2 bag6 seeds is less sensitive to ABA compared to the wild type (WT), whereas BAG2 and BAG6 overexpression lines are hypersensitive to ABA. bag2, bag6, and bag2 bag6 plants show higher survival rates than WT in drought treatment but display lower survival rates in heat-stress treatment. Consistently, these mutants showed differential expression of several stress- and ABA-related genes such as RD29A, RD29B, NCED3 and ABI4 compared to the WT. Furthermore, these mutants exhibit lower levels of ROS after drought and ABA treatment but higher ROS accumulation after heat treatment than the WT. These results suggest that BAG2 and BAG6 are negatively involved in drought stress but play a positive role in heat stress in Arabidopsis.


Assuntos
Adaptação Fisiológica , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Mitocondriais/genética , Chaperonas Moleculares/genética , Proteínas Nucleares/genética , Estresse Fisiológico , Proteínas de Arabidopsis/metabolismo , Secas , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Desenvolvimento Vegetal/genética , Reguladores de Crescimento de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo
9.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071169

RESUMO

Polygodial is a "hot" peppery-tasting sesquiterpenoid that was first described for its anti-feedant activity against African armyworms. Using the haploid deletion mutant library of Saccharomyces cerevisiae, a genome-wide mutant screen was performed to shed more light on polygodial's antifungal mechanism of action. We identified 66 deletion strains that were hypersensitive and 47 that were highly resistant to polygodial treatment. Among the hypersensitive strains, an enrichment was found for genes required for vacuolar acidification, amino acid biosynthesis, nucleosome mobilization, the transcription mediator complex, autophagy and vesicular trafficking, while the resistant strains were enriched for genes encoding cytoskeleton-binding proteins, ribosomal proteins, mitochondrial matrix proteins, components of the heme activator protein (HAP) complex, and known regulators of the target of rapamycin complex 1 (TORC1) signaling. WE confirm that polygodial triggers a dose-dependent vacuolar alkalinization and that it increases Ca2+ influx and inhibits glucose-induced Ca2+ signaling. Moreover, we provide evidence suggesting that TORC1 signaling and its protective agent ubiquitin play a central role in polygodial resistance, suggesting that they can be targeted by polygodial either directly or via altered Ca2+ homeostasis.


Assuntos
Antifúngicos/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Antifúngicos/química , Cálcio , Farmacorresistência Fúngica/genética , Homeostase/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Testes de Sensibilidade Microbiana , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Nucleossomos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Transdução de Sinais , ATPases Vacuolares Próton-Translocadoras
10.
Methods Mol Biol ; 2277: 157-173, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080151

RESUMO

Mitochondria have complex ultrastructure which includes continuous subcompartments, such as matrix, intermembrane space, and two membranes, as well as focal structures, such as nucleoids, RNA granules, and mitoribosomes. Comprehensive studies of the spatial distribution of proteins and RNAs inside the mitochondria are necessary to understand organellar gene expression processes and macromolecule targeting pathways. Here we give examples of distribution analysis of mitochondrial proteins and transcripts by conventional microscopy and the super-resolution technique 3D STORM. We provide detailed protocols and discuss limitations of immunolabeling of mitochondrial proteins and newly synthesized mitochondrial RNAs by bromouridine incorporation and single-molecule RNA FISH in hepatocarcinoma cells.


Assuntos
Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Microscopia Confocal/métodos , Proteínas Mitocondriais/metabolismo , Bromouracila/análogos & derivados , Bromouracila/química , Células Hep G2 , Humanos , Processamento de Imagem Assistida por Computador/métodos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , RNA Mitocondrial/química , Imagem Individual de Molécula/métodos , Uridina/análogos & derivados , Uridina/química
11.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 37(7): 616-622, 2021 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-34140073

RESUMO

Objective To investigate the effect of inner mitochondrial membrane peptidase 2-like (IMMP2L) gene mutation on cerebral ischemic injury and its mechanism. Methods The cerebral ischemia/reperfusion (I/R) model was established in wild-type (WT) mice and mice with IMMP2L gene mutation (IMMP2L+/-) by middle cerebral artery occlusion (MCAO), and cortical tissues were collected at 0, 1, 5 and 24 hours after reperfusion. Laser speckle contrast imaging (LSCI) was used to monitor the change in cerebral blood flow (CBF). Longa behavioral score was used to evaluate neurological function. triphenyltetrazolium chloride (TTC), HE staining was used to evaluate cerebral infarction and neuron injury, TUNEL was used to evaluate the neuronal apoptosis. The protein expression of cleaved caspase-3 and apoptosis-inducing factor (AIF)was analyzed by Western blotting. The changes of cerebral blood flow (CBF) were monitored by laser speckle contrast imaging (LSCI), neurological function was evaluated by longa behavioral score, and cerebral infarction area and neuronal injury were observed by TTC staining and HE staining respectively; the changes of neuronal apoptosis were analyzed by TUNEL, and the protein expressions of cleaved caspase-3 (c-caspase-3) and apoptosis inducing factor (AIF) were detected by Western blotting. Results The neurobehavioral score was significantly higher in the IMMP2L+/- versus WT mice. The volume of the infarcted region, the number of degenerated neurons, and the degree of cerebral edema all increased at 5 and 24 hours after reperfusion. The apoptotic neurons increased at 0, 1, 5 and 24 hours after reperfusion and the protein levels of c-caspase-3 and AIF were up-regulated at 5 and 24 hours after I/R. Conclusion IMMP2L mutation aggravates cerebral ischemic injury by activating the mitochondrial apoptosis pathway.


Assuntos
Isquemia Encefálica , Endopeptidases/genética , Proteínas Mitocondriais/genética , Traumatismo por Reperfusão , Animais , Apoptose/genética , Isquemia Encefálica/genética , Infarto da Artéria Cerebral Média/genética , Camundongos , Mutação , Traumatismo por Reperfusão/genética
12.
FASEB J ; 35(7): e21688, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34143516

RESUMO

The mitochondria-associated membrane (MAM) is a functional subdomain of the endoplasmic reticulum membrane that tethers to the mitochondrial outer membrane and is essential for cellular homeostasis. A defect in MAM is involved in various neurological diseases, including amyotrophic lateral sclerosis (ALS). Recently, we and others reported that MAM was disrupted in the models expressing several ALS-linked genes, including SOD1, SIGMAR1, VAPB, TARDBP, and FUS, suggesting that MAM disruption is deeply involved in the pathomechanism of ALS. However, it is still uncertain whether MAM disruption is a common pathology in ALS, mainly due to the absence of a simple, quantitative tool for monitoring the status of MAM. In this study, to examine the effects of various ALS-causative genes on MAM, we created the following two novel MAM reporters: MAMtracker-Luc and MAMtracker-Green. The MAMtrackers could detect MAM disruption caused by suppression of SIGMAR1 or the overexpression of ALS-linked mutant SOD1 in living cells. Moreover, the MAMtrackers have an advantage in their ability to monitor reversible changes in the MAM status induced by nutritional conditions. We used the MAMtrackers with an expression plasmid library of ALS-causative genes and noted that 76% (16/21) of the genes altered MAM integrity. Our results suggest that MAM disruption is a common pathological feature in ALS. Furthermore, we anticipate our MAMtrackers, which are suitable for high-throughput assays, to be valuable tools to understand MAM dynamics.


Assuntos
Esclerose Amiotrófica Lateral/patologia , Mitocôndrias/patologia , Membranas Mitocondriais/patologia , Proteínas Mitocondriais/metabolismo , Mutação , Neuroblastoma/patologia , Esclerose Amiotrófica Lateral/etiologia , Esclerose Amiotrófica Lateral/metabolismo , Animais , Humanos , Camundongos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo
13.
Int J Mol Sci ; 22(9)2021 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-34063696

RESUMO

Multiple mitochondrial dysfunctions syndrome (MMDS) is a rare neurodegenerative disorder associated with mutations in genes with a vital role in the biogenesis of mitochondrial [4Fe-4S] proteins. Mutations in one of these genes encoding for BOLA3 protein lead to MMDS type 2 (MMDS2). Recently, a novel phenotype for MMDS2 with complete clinical recovery was observed in a patient containing a novel variant (c.176G > A, p.Cys59Tyr) in compound heterozygosity. In this work, we aimed to rationalize this unique phenotype observed in MMDS2. To do so, we first investigated the structural impact of the Cys59Tyr mutation on BOLA3 by NMR, and then we analyzed how the mutation affects both the formation of a hetero-complex between BOLA3 and its protein partner GLRX5 and the iron-sulfur cluster-binding properties of the hetero-complex by various spectroscopic techniques and by experimentally driven molecular docking. We show that (1) the mutation structurally perturbed the iron-sulfur cluster-binding region of BOLA3, but without abolishing [2Fe-2S]2+ cluster-binding on the hetero-complex; (2) tyrosine 59 did not replace cysteine 59 as iron-sulfur cluster ligand; and (3) the mutation promoted the formation of an aberrant apo C59Y BOLA3-GLRX5 complex. All these aspects allowed us to rationalize the unique phenotype observed in MMDS2 caused by Cys59Tyr mutation.


Assuntos
Glutarredoxinas/genética , Mitocôndrias/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Cisteína/genética , Glutarredoxinas/ultraestrutura , Humanos , Proteínas Ferro-Enxofre/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/ultraestrutura , Simulação de Acoplamento Molecular , Complexos Multiproteicos , Mutação , Ressonância Magnética Nuclear Biomolecular , Fenótipo
14.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070437

RESUMO

Although sequences of mitogenomes have been widely used for investigating phylogenetic relationship, population genetics, and biogeography in many members of Fulgoroidea, only one complete mitogenome of a member of Flatidae has been sequenced. Here, the complete mitogenomes of Cerynia lineola, Cromna sinensis, and Zecheuna tonkinensis are sequenced. The gene arrangements of the three new mitogenomes are consistent with ancestral insect mitogenomes. The strategy of using mitogenomes in phylogenetics remains in dispute due to the heterogeneity in base composition and the possible variation in evolutionary rates. In this study, we found compositional heterogeneity and variable evolutionary rates among planthopper mitogenomes. Phylogenetic analysis based on site-homogeneous models showed that the families (Delphacidae and Derbidae) with high values of Ka/Ks and A + T content tended to fall together at a basal position on the trees. Using a site-heterogeneous mixture CAT + GTR model implemented in PhyloBayes yielded almost the same topology. Our results recovered the monophyly of Fulgoroidea. In this study, we apply the heterogeneous mixture model to the planthoppers' phylogenetic analysis for the first time. Our study is based on a large sample and provides a methodological reference for future phylogenetic studies of Fulgoroidea.


Assuntos
Genoma Mitocondrial , Hemípteros/genética , Mitocôndrias/genética , Animais , Composição de Bases , Uso do Códon , Ordem dos Genes , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas Mitocondriais/genética , Filogenia , RNA Ribossômico/genética , RNA de Transferência/genética , Alinhamento de Sequência , Sequências de Repetição em Tandem/genética
15.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34063907

RESUMO

Plant mitochondria have large genomes to house a small number of key genes. Most mitochondria do not contain a whole genome. Despite these latter characteristics, the mitochondrial genome is faithfully maternally inherited. To maintain the mitochondrial genes-so important for energy production-the fusion and fission of mitochondria are critical. Fission in plants is better understood than fusion, with the dynamin-related proteins (DRP 3A and 3B) driving the constriction of the mitochondrion. How the endoplasmic reticulum and the cytoskeleton are linked to the fission process is not yet fully understood. The fusion mechanism is less well understood, as obvious orthologues are not present. However, there is a recently described gene, MIRO2, that appears to have a significant role, as does the ER and cytoskeleton. Massive mitochondrial fusion (MMF or hyperfusion) plays a significant role in plants. MMF occurs at critical times of the life cycle, prior to flowering, in the enlarging zygote and at germination, mixing the cells' mitochondrial population-the so-called "discontinuous whole". MMF in particular aids genome repair, the conservation of critical genes and possibly gives an energy boost to important stages of the life cycle. MMF is also important in plant regeneration, an important component of plant biotechnology.


Assuntos
Genoma Mitocondrial/genética , Estágios do Ciclo de Vida/fisiologia , Mitocôndrias/genética , Mitocôndrias/fisiologia , Dinâmica Mitocondrial/genética , Dinâmica Mitocondrial/fisiologia , Plantas/genética , Animais , Citoesqueleto/genética , Humanos , Estágios do Ciclo de Vida/genética , Proteínas Mitocondriais/genética
16.
Gene ; 790: 145697, 2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-33964376

RESUMO

Human Mitoribosomal Small Subunit unit (MRPS) family of genes appears to have role in cancer. Gene expression analysis of select MRPS genes (n = 9) in 15 cancer cell lines showed altered expression in cancer cells. Protein levels of MRPS6, MRPS23 showed significant overexpression in breast cancer cells and tissues. Interestingly, their overexpression did not correlate with mitochondrial ribosome translated COX2 protein levels in breast cancer. Subcellular fractionation analysis showed a distinct presence of MRPS23 in the nuclear fraction. GST/MRP6 and GST/MRPS23 pulldown assays identified 32 novel protein-protein interactions (PPIs) and MRPS23-RIPK3 interaction was validated. Co-expression module identification tool (CEMi) analysis of breast cancer gene expression and MRPS6 and MRPS23 interactions revealed hub interactions in gene expression modules having functional roles in cancer-associated cellular processes. Based on PPI network analysis a novel interaction MRPS23-p53 was validated. Knockdown of MRPS6 and MRPS23 decreased proliferation, expression of select mesenchymal markers, oncogenes, and increased expression of tumor suppressor genes. Taken together present study has revealed that MRPS6 and MRPS23 genes have pro-tumorigenic functions in breast cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Carcinogênese , Regulação Neoplásica da Expressão Gênica , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , Apoptose , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proliferação de Células , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Proteínas Mitocondriais/genética , Prognóstico , Células Tumorais Cultivadas
17.
Nat Commun ; 12(1): 2947, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011950

RESUMO

The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion.


Assuntos
Evolução Molecular , Mitocôndrias/genética , Mitocôndrias/metabolismo , Sistemas de Secreção Tipo II/genética , Sistemas de Secreção Tipo II/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Eucariotos/classificação , Eucariotos/genética , Eucariotos/metabolismo , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Proteínas Mitocondriais/classificação , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Modelos Moleculares , Naegleria/classificação , Naegleria/genética , Naegleria/metabolismo , Peroxissomos/metabolismo , Filogenia , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Homologia de Sequência de Aminoácidos , Sistemas de Secreção Tipo II/classificação
18.
Nat Commun ; 12(1): 2616, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972548

RESUMO

FUN14 domain-containing protein 1 (FUNDC1) is an integral mitochondrial outer-membrane protein, and mediates the formation of mitochondria-associated endoplasmic reticulum membranes (MAMs). This study aims to determine the contributions of FUNDC1-mediated MAMs to angiogenesis in vitro and in vivo. In cultured endothelial cells, VEGF significantly increases the formation of MAMs and MAM-related proteins, including FUNDC1. Endothelial cell-specific deletion of FUNDC1, which disrupts MAM formation in endothelial cells, lowers VEGFR2 expression and reduces tube formation, spheroid-sprouting, and functional blood vessel formation in vitro and in vivo. Conversely, increased MAM formation using MAM linkers mimics the effects of VEGF and promotes endothelial angiogenesis. Mechanistically, increased MAMs formation led to increased levels of Ca2+ in cytosol, promoted the phosphorylation of serum response factor (SRF) and enhanced the binding of SRF to VEGFR2 promoter, resulting in increased VEGFR2 production, with consequent angiogenesis. Moreover, blocking FUNDC1-related MAM formation with a cell-penetrating inhibitory peptide significantly suppresses the expressions of downstream angiogenic genes and inhibits tumor angiogenesis. We conclude that decreased MAMs formation by silencing FUNDC1 can inhibit angiogenesis by decreasing VEGFR2 expression, and targeting FUNDC1-dependent MAMs might be a promising approach for treating human disorders characterized by defective angiogenesis.


Assuntos
Retículo Endoplasmático/metabolismo , Células Endoteliais/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neovascularização Patológica/genética , Neovascularização Fisiológica/genética , Animais , Cálcio/metabolismo , Citoplasma/metabolismo , Retículo Endoplasmático/ultraestrutura , Inativação Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Membranas Intracelulares/efeitos dos fármacos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Neovascularização Fisiológica/efeitos dos fármacos , Fosforilação , RNA Interferente Pequeno , Retina/metabolismo , Fator de Resposta Sérica/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Mol Cell ; 81(11): 2460-2476.e11, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33974913

RESUMO

Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation.


Assuntos
Proteínas Mitocondriais/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Mitocondriais/classificação , Proteínas Mitocondriais/metabolismo , Transporte Proteico , Proteólise , Proteômica/métodos , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Treonina/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/classificação , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
20.
Methods Mol Biol ; 2228: 253-270, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33950496

RESUMO

Stable isotope labeling by amino acids in cell culture (SILAC) combined with high-resolution mass spectrometry is a quantitative strategy for the comparative analysis of (sub)proteomes. It is based on the metabolic incorporation of stable isotope-coded amino acids during growth of cells or organisms. Here, complete labeling of proteins with the amino acid(s) selected for incorporation needs to be guaranteed to enable accurate quantification on a proteomic scale. Wild-type strains of baker's yeast (Saccharomyces cerevisiae ), which is a widely accepted and well-studied eukaryotic model organism, are generally able to synthesize all amino acids on their own (i.e., prototrophic). To render them amenable to SILAC, auxotrophies are introduced by genetic manipulations. We addressed this limitation by developing a generic strategy for complete "native" labeling of prototrophic S. cerevisiae with isotope-coded arginine and lysine, referred to as "2nSILAC". It allows for directly using and screening several genome-wide yeast mutant collections that are easily accessible to the scientific community for functional proteomic studies but are based on prototrophic variants of S. cerevisiae.


Assuntos
Proteínas Mitocondriais/análise , Proteoma , Proteômica , Proteínas de Saccharomyces cerevisiae/análise , Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas em Tandem , Cromatografia Líquida de Alta Pressão , Regulação Fúngica da Expressão Gênica , Marcação por Isótopo , Proteínas Mitocondriais/genética , Mutação , Projetos de Pesquisa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
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