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Int J Mol Sci ; 22(17)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34502139


SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causative agent of the COVID19 pandemic. The SARS-CoV-2 genome encodes for a small accessory protein termed Orf9b, which targets the mitochondrial outer membrane protein TOM70 in infected cells. TOM70 is involved in a signaling cascade that ultimately leads to the induction of type I interferons (IFN-I). This cascade depends on the recruitment of Hsp90-bound proteins to the N-terminal domain of TOM70. Binding of Orf9b to TOM70 decreases the expression of IFN-I; however, the underlying mechanism remains elusive. We show that the binding of Orf9b to TOM70 inhibits the recruitment of Hsp90 and chaperone-associated proteins. We characterized the binding site of Orf9b within the C-terminal domain of TOM70 and found that a serine in position 53 of Orf9b and a glutamate in position 477 of TOM70 are crucial for the association of both proteins. A phosphomimetic variant Orf9bS53E showed drastically reduced binding to TOM70 and did not inhibit Hsp90 recruitment, suggesting that Orf9b-TOM70 complex formation is regulated by phosphorylation. Eventually, we identified the N-terminal TPR domain of TOM70 as a second binding site for Orf9b, which indicates a so far unobserved contribution of chaperones in the mitochondrial targeting of the viral protein.

COVID-19/transmissão , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , SARS-CoV-2/patogenicidade , Animais , Sítios de Ligação/genética , COVID-19/imunologia , COVID-19/virologia , Chlorocebus aethiops , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/isolamento & purificação , Humanos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/isolamento & purificação , Mutação , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica/genética , Ligação Proteica/imunologia , Domínios Proteicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Células Vero
Sci Rep ; 10(1): 21029, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33273525


Sepsis is characterized by a dysregulated immune response, metabolic derangements and bioenergetic failure. These alterations are closely associated with a profound and persisting mitochondrial dysfunction. This however occurs despite increased expression of the nuclear-encoded transcription factor A (TFAM) that normally supports mitochondrial biogenesis and functional recovery. Since this paradox may relate to an altered intracellular distribution of TFAM in sepsis, we tested the hypothesis that enhanced extramitochondrial TFAM expression does not translate into increased intramitochondrial TFAM abundance. Accordingly, we prospectively analyzed PBMCs both from septic patients (n = 10) and lipopolysaccharide stimulated PBMCs from healthy volunteers (n = 20). Extramitochondrial TFAM protein expression in sepsis patients was 1.8-fold greater compared to controls (p = 0.001), whereas intramitochondrial TFAM abundance was approximate 80% less (p < 0.001). This was accompanied by lower mitochondrial DNA copy numbers (p < 0.001), mtND1 expression (p < 0.001) and cellular ATP content (p < 0.001) in sepsis patients. These findings were mirrored in lipopolysaccharide stimulated PBMCs taken from healthy volunteers. Furthermore, TFAM-TFB2M protein interaction within the human mitochondrial core transcription initiation complex, was 74% lower in septic patients (p < 0.001). In conclusion, our findings, which demonstrate a diminished mitochondrial TFAM abundance in sepsis and endotoxemia, may help to explain the paradox of lacking bioenergetic recovery despite enhanced TFAM expression.

Proteínas de Ligação a DNA/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Sepse/metabolismo , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Idoso , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/genética , Feminino , Dosagem de Genes , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Metiltransferases/metabolismo , Pessoa de Meia-Idade , Proteínas Mitocondriais/genética , Fatores de Transcrição/genética
Int J Mol Sci ; 21(19)2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33019591


Tom70 is a versatile adaptor protein of 70 kDa anchored in the outer membrane of mitochondria in metazoa, fungi and amoeba. The tertiary structure was resolved for the Tom70 of yeast, showing 26 α-helices, most of them participating in the formation of 11 tetratricopeptide repeat (TPR) motifs. Tom70 serves as a docking site for cytosolic chaperone proteins and co-chaperones and is thereby involved in the uptake of newly synthesized chaperone-bound proteins in mitochondrial biogenesis. In yeast, Tom70 additionally mediates ER-mitochondria contacts via binding to sterol transporter Lam6/Ltc1. In mammalian cells, TOM70 promotes endoplasmic reticulum (ER) to mitochondria Ca2+ transfer by association with the inositol-1,4,5-triphosphate receptor type 3 (IP3R3). TOM70 is specifically targeted by the Bcl-2-related protein MCL-1 that acts as an anti-apoptotic protein in macrophages infected by intracellular pathogens, but also in many cancer cells. By participating in the recruitment of PINK1 and the E3 ubiquitin ligase Parkin, TOM70 can be implicated in the development of Parkinson's disease. TOM70 acts as receptor of the mitochondrial antiviral-signaling protein (MAVS) and thereby participates in the corresponding system of innate immunity against viral infections. The protein encoded by Orf9b in the genome of SARS-CoV-2 binds to TOM70, probably compromising the synthesis of type I interferons.

Imunidade Inata , Proteínas de Transporte da Membrana Mitocondrial/química , Animais , Betacoronavirus/genética , Sítios de Ligação , Humanos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Fases de Leitura Aberta , Ligação Proteica , Transporte Proteico , SARS-CoV-2
BMC Biol ; 18(1): 156, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-33121519


BACKGROUND: The uptake of newly synthesized nuclear-encoded mitochondrial proteins from the cytosol is mediated by a complex of mitochondrial outer membrane proteins comprising a central pore-forming component and associated receptor proteins. Distinct fractions of proteins initially bind to the receptor proteins and are subsequently transferred to the pore-forming component for import. The aim of this study was the identification of the decisive elements of this machinery that determine the specific selection of the proteins that should be imported. RESULTS: We identified the essential internal targeting signal of the members of the mitochondrial metabolite carrier proteins, the largest protein family of the mitochondria, and we investigated the specific recognition of this signal by the protein import machinery at the mitochondrial outer surface. We found that the outer membrane import receptors facilitated the uptake of these proteins, and we identified the corresponding binding site, marked by cysteine C141 in the receptor protein Tom70. However, in tests both in vivo and in vitro, the import receptors were neither necessary nor sufficient for specific recognition of the targeting signals. Although these signals are unrelated to the amino-terminal presequences that mediate the targeting of other mitochondrial preproteins, they were found to resemble presequences in their strict dependence on a content of positively charged residues as a prerequisite of interactions with the import pore. CONCLUSIONS: The general import pore of the mitochondrial outer membrane appears to represent not only the central channel of protein translocation but also to form the decisive general selectivity filter in the uptake of the newly synthesized mitochondrial proteins.

J Mol Biol ; 430(24): 5233-5245, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30261167


Mimivirus (Acanthamoeba polyphaga mimivirus) was the first giant DNA virus identified in an amoeba species. Its genome contains at least 979 genes. One of these, L276, encodes a nucleotide translocator with similarities to mitochondrial metabolite carriers, provisionally named viral mitochondrial carrier 1 (VMC1). In this study, we investigated the intracellular distribution of VMC1 upon expression in HeLa cells and in the yeast Saccharomyces cerevisiae. We found that VMC1 is specifically targeted to mitochondria and to the inner mitochondrial membrane. Newly synthesized VMC1 binds to the mitochondrial outer-membrane protein Tom70 and translocates through the import channel formed by the ß-barrel protein Tom40. Derivatization of the four cysteine residues inside Tom40 by N-ethylmaleimide caused a delay in translocation but not a complete occlusion. Cell viability was not reduced by VMC1. Neither the mitochondrial membrane potential nor the intracellular production of reactive oxygen species was affected. Similar to endogenous metabolite carriers, mimivirus-encoded VMC1 appears to act as a specific translocator in the mitochondrial inner membrane. Due to its permeability for deoxyribonucleotides, VMC1 confers to the mitochondria an opportunity to contribute nucleotides for the replication of the large DNA genome of the virus.

Mimiviridae/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Saccharomyces cerevisiae/genética , Células HeLa , Humanos , Potencial da Membrana Mitocondrial , Mimiviridae/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/química , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo