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1.
Proc Natl Acad Sci U S A ; 117(28): 16383-16390, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601238

RESUMO

Calcium uptake by the mitochondrial calcium uniporter coordinates cytosolic signaling events with mitochondrial bioenergetics. During the past decade all protein components of the mitochondrial calcium uniporter have been identified, including MCU, the pore-forming subunit. However, the specific lipid requirements, if any, for the function and formation of this channel complex are currently not known. Here we utilize yeast, which lacks the mitochondrial calcium uniporter, as a model system to address this problem. We use heterologous expression to functionally reconstitute human uniporter machinery both in wild-type yeast as well as in mutants defective in the biosynthesis of phosphatidylethanolamine, phosphatidylcholine, or cardiolipin (CL). We uncover a specific requirement of CL for in vivo reconstituted MCU stability and activity. The CL requirement of MCU is evolutionarily conserved with loss of CL triggering rapid turnover of MCU homologs and impaired calcium transport. Furthermore, we observe reduced abundance and activity of endogenous MCU in mammalian cellular models of Barth syndrome, which is characterized by a partial loss of CL. MCU abundance is also decreased in the cardiac tissue of Barth syndrome patients. Our work raises the hypothesis that impaired mitochondrial calcium transport contributes to the pathogenesis of Barth syndrome, and more generally, showcases the utility of yeast phospholipid mutants in dissecting the phospholipid requirements of ion channel complexes.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Síndrome de Barth/genética , Síndrome de Barth/metabolismo , Transporte Biológico , Canais de Cálcio/química , Canais de Cálcio/genética , Cardiolipinas/genética , Cardiolipinas/metabolismo , Humanos , Camundongos , Mitocôndrias/química , Mitocôndrias/genética , Mioblastos/metabolismo , Fosfolipídeos , Estabilidade Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
2.
J Biol Chem ; 293(28): 10870-10883, 2018 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-29866881

RESUMO

Cardiolipin (CL) is a signature phospholipid of the mitochondria required for the formation of mitochondrial respiratory chain (MRC) supercomplexes. The destabilization of MRC supercomplexes is the proximal cause of the pathology associated with the depletion of CL in patients with Barth syndrome. Thus, promoting supercomplex formation could ameliorate mitochondrial dysfunction associated with CL depletion. However, to date, physiologically relevant small-molecule regulators of supercomplex formation have not been identified. Here, we report that ethanolamine (Etn) supplementation rescues the MRC defects by promoting supercomplex assembly in a yeast model of Barth syndrome. We discovered this novel role of Etn while testing the hypothesis that elevating mitochondrial phosphatidylethanolamine (PE), a phospholipid suggested to overlap in function with CL, could compensate for CL deficiency. We found that the Etn supplementation rescues the respiratory growth of CL-deficient Saccharomyces cerevisiae cells in a dose-dependent manner but independently of its incorporation into PE. The rescue was specifically dependent on Etn but not choline or serine, the other phospholipid precursors. Etn improved mitochondrial function by restoring the expression of MRC proteins and promoting supercomplex assembly in CL-deficient cells. Consistent with this mechanism, overexpression of Cox4, the MRC complex IV subunit, was sufficient to promote supercomplex formation in CL-deficient cells. Taken together, our work identifies a novel role of a ubiquitous metabolite, Etn, in attenuating mitochondrial dysfunction caused by CL deficiency.


Assuntos
Cardiolipinas/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Etanolaminas/farmacologia , Mitocôndrias/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte de Elétrons , Mitocôndrias/patologia , Saccharomyces cerevisiae/efeitos dos fármacos
3.
J Biol Chem ; 289(2): 1092-105, 2014 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-24275663

RESUMO

One of the mechanisms for establishment of infection employed by intra-macrophage pathogen-like Leishmania is inhibition of oxidative burst-mediated macrophage apoptosis to protect their niche for survival and replication. We tried to elucidate the underlying mechanism for this by using H2O2 for induction of apoptosis. Leishmania donovani-infected macrophages were much more resistant to H2O2-mediated apoptosis compared with control. Although infected cells were capable of comparable reactive oxygen species production, there was less activation of the downstream cascade consisting of caspase-3 and -7 and cleaved poly(ADP)-ribose polymerase. Suppressors of cytokine signaling (SOCS) 1 and 3 proteins and reactive oxygen species scavenging enzyme thioredoxin, known to be involved in stabilization of protein-tyrosine phosphatases, were found to be induced during infection. Induction of SOCS proteins may be mediated by Egr1, and silencing of Socs1 and -3 either alone or in combination resulted in reduced thioredoxin levels, enhanced activation of caspases, and increased apoptosis of infected macrophages. The induction of protein-tyrosine phosphatases, thioredoxin, SOCS, and Egr1 in L. donovani-infected macrophages was found to be unaffected by H2O2 treatment. SOCS knocked down cells also displayed decreased parasite survival thus marking reduction in disease progression. Taken together, these results suggest that L. donovani may exploit SOCS for subverting macrophage apoptotic machinery toward establishing its replicative niche inside the host.


Assuntos
Apoptose/fisiologia , Leishmania donovani/crescimento & desenvolvimento , Macrófagos/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Expressão Gênica , Interações Hospedeiro-Patógeno , Peróxido de Hidrogênio/farmacologia , Immunoblotting , Leishmania donovani/fisiologia , Macrófagos/microbiologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oxidantes/farmacologia , Fagocitose/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Interferência de RNA , Explosão Respiratória/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Tiorredoxinas/metabolismo
4.
J Immunol ; 187(3): 1322-32, 2011 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-21705615

RESUMO

To reside and multiply successfully within the host macrophages, Leishmania parasites impair the generation of reactive oxygen species (ROS), which are a major host defense mechanism against any invading pathogen. Mitochondrial uncoupling proteins are associated with mitochondrial ROS generation, which is the major contributor of total cellular ROS generation. In the present study we have demonstrated that Leishmania donovani infection is associated with strong upregulation of uncoupling protein 2 (UCP2), a negative regulator of mitochondrial ROS generation located at the inner membrane of mitochondria. Functional knockdown of macrophage UCP2 by small interfering RNA-mediated silencing was associated with increased mitochondrial ROS generation, lower parasite survival, and induction of marked proinflammatory cytokine response. Induction of proinflammatory cytokine response in UCP2 knocked-down cells was a direct consequence of p38 and ERK1/2 MAPK activation, which resulted from ROS-mediated inhibition of protein tyrosine phosphatases (PTPs). Administration of ROS quencher, N-acetyl-l-cysteine, abrogated PTP inhibition in UCP2 knocked-down infected cells, implying a role of ROS in inactivating PTP. Short hairpin RNA-mediated in vivo silencing of UCP2 resulted in decreased Src homology 2 domain-containing tyrosine phosphatase 1 and PTP-1B activity and host-protective proinflammatory cytokine response resulting in effective parasite clearance. To our knowledge, this study, for the first time, reveals the induction of host UCP2 expression during Leishmania infection to downregulate mitochondrial ROS generation, thereby possibly preventing ROS-mediated PTP inactivation to suppress macrophage defense mechanisms.


Assuntos
Regulação para Baixo/imunologia , Mediadores da Inflamação/fisiologia , Canais Iônicos/fisiologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/prevenção & controle , Mitocôndrias/metabolismo , Proteínas Mitocondriais/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Espécies Reativas de Oxigênio/antagonistas & inibidores , Animais , Linhagem Celular , Regulação para Baixo/genética , Ativação Enzimática/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Mediadores da Inflamação/metabolismo , Canais Iônicos/deficiência , Canais Iônicos/genética , Leishmania donovani/imunologia , Leishmaniose Visceral/patologia , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/genética , Mitocôndrias/imunologia , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Proteínas Tirosina Fosfatases/metabolismo , RNA Interferente Pequeno/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteína Desacopladora 2
5.
Nanomaterials (Basel) ; 12(3)2022 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-35159648

RESUMO

Nanoclusters possess an ultrasmall size, amongst other favorable attributes, such as a high fluorescence and long-term colloidal stability, and consequently, they carry several advantages when applied in biological systems for use in diagnosis and therapy. Particularly, the early diagnosis of diseases may be facilitated by the right combination of bioimaging modalities and suitable probes. Amongst several metallic nanoclusters, copper nanoclusters (Cu NCs) present advantages over gold or silver NCs, owing to their several advantages, such as high yield, raw abundance, low cost, and presence as an important trace element in biological systems. Additionally, their usage in diagnostics and therapeutic modalities is emerging. As a result, the fluorescent properties of Cu NCs are exploited for use in optical imaging technology, which is the most commonly used research tool in the field of biomedicine. Optical imaging technology presents a myriad of advantages over other bioimaging technologies, which are discussed in this review, and has a promising future, particularly in early cancer diagnosis and imaging-guided treatment. Furthermore, we have consolidated, to the best of our knowledge, the recent trends and applications of copper nanoclusters (Cu NCs), a class of metal nanoclusters that have been gaining much traction as ideal bioimaging probes, in this review. The potential modes in which the Cu NCs are used for bioimaging purposes (e.g., as a fluorescence, magnetic resonance imaging (MRI), two-photon imaging probe) are firstly delineated, followed by their applications as biosensors and bioimaging probes, with a focus on disease detection.

6.
Artigo em Inglês | MEDLINE | ID: mdl-32058032

RESUMO

Mitochondrial membrane biogenesis requires the import of phospholipids; however, the molecular mechanisms underlying this process remain elusive. Recent work has implicated membrane contact sites between the mitochondria, endoplasmic reticulum (ER), and vacuole in phospholipid transport. Utilizing a genetic approach focused on these membrane contact site proteins, we have discovered a 'moonlighting' role of the membrane contact site and vesicular fusion protein, Vps39, in phosphatidylethanolamine (PE) transport to the mitochondria. We show that the deletion of Vps39 prevents ethanolamine-stimulated elevation of mitochondrial PE levels without affecting PE biosynthesis in the ER or its transport to other sub-cellular organelles. The loss of Vps39 did not alter the levels of other mitochondrial phospholipids that are biosynthesized ex situ, implying a PE-specific role of Vps39. The abundance of Vps39 and its recruitment to the mitochondria and the ER is dependent on PE levels in each of these organelles, directly implicating Vps39 in the PE transport process. Deletion of essential subunits of Vps39-containing complexes, vCLAMP and HOPS, did not abrogate ethanolamine-stimulated PE elevation in the mitochondria, suggesting an independent role of Vps39 in intracellular PE trafficking. Our work thus identifies Vps39 as a novel player in ethanolamine-stimulated PE transport to the mitochondria.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Fosfatidiletanolaminas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Retículo Endoplasmático/metabolismo , Etanolamina/metabolismo , Técnicas de Silenciamento de Genes , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
7.
FEBS Lett ; 592(8): 1273-1290, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29067684

RESUMO

Mitochondrial structure and function are influenced by the unique phospholipid composition of its membranes. While mitochondria contain all the major classes of phospholipids, recent studies have highlighted specific roles of the nonbilayer-forming phospholipids phosphatidylethanolamine (PE) and cardiolipin (CL) in the assembly and activity of mitochondrial respiratory chain (MRC) complexes. The nonbilayer phospholipids are cone-shaped molecules that introduce curvature stress in the bilayer membrane and have been shown to impact mitochondrial fusion and fission. In addition to their overlapping roles in these mitochondrial processes, each nonbilayer phospholipid also plays a unique role in mitochondrial function; for example, CL is specifically required for MRC supercomplex formation. Recent discoveries of mitochondrial PE- and CL-trafficking proteins and prior knowledge of their biosynthetic pathways have provided targets for precisely manipulating nonbilayer phospholipid levels in the mitochondrial membranes in vivo. Thus, the genetic mutants of these pathways could be valuable tools in illuminating molecular functions and biophysical properties of nonbilayer phospholipids in driving mitochondrial bioenergetics and dynamics.


Assuntos
Cardiolipinas/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Fosfatidiletanolaminas/metabolismo , Animais , Cardiolipinas/genética , Transporte de Elétrons/fisiologia , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Fosfatidiletanolaminas/genética , Transporte Proteico/fisiologia
8.
Mol Biol Cell ; 27(14): 2161-71, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27226479

RESUMO

Mitochondrial membrane phospholipid composition affects mitochondrial function by influencing the assembly of the mitochondrial respiratory chain (MRC) complexes into supercomplexes. For example, the loss of cardiolipin (CL), a signature non-bilayer-forming phospholipid of mitochondria, results in disruption of MRC supercomplexes. However, the functions of the most abundant mitochondrial phospholipids, bilayer-forming phosphatidylcholine (PC) and non-bilayer-forming phosphatidylethanolamine (PE), are not clearly defined. Using yeast mutants of PE and PC biosynthetic pathways, we show a specific requirement for mitochondrial PE in MRC complex III and IV activities but not for their formation, whereas loss of PC does not affect MRC function or formation. Unlike CL, mitochondrial PE or PC is not required for MRC supercomplex formation, emphasizing the specific requirement of CL in supercomplex assembly. Of interest, PE biosynthesized in the endoplasmic reticulum (ER) can functionally substitute for the lack of mitochondrial PE biosynthesis, suggesting the existence of PE transport pathway from ER to mitochondria. To understand the mechanism of PE transport, we disrupted ER-mitochondrial contact sites formed by the ERMES complex and found that, although not essential for PE transport, ERMES facilitates the efficient rescue of mitochondrial PE deficiency. Our work highlights specific roles of non-bilayer-forming phospholipids in MRC function and formation.


Assuntos
Transporte de Elétrons/fisiologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Fosfolipídeos/metabolismo , Cardiolipinas/metabolismo , Retículo Endoplasmático/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Saccharomyces cerevisiae/metabolismo
9.
Int J Biochem Cell Biol ; 55: 196-208, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25218172

RESUMO

Establishment of infection by an intracellular pathogen depends on successful internalization with a concomitant neutralization of host defense machinery. Leishmania donovani, an intramacrophage pathogen, targets host SREBP2, a critical transcription factor, to regulate macrophage plasma membrane cholesterol and mitochondrial reactive oxygen species generation, favoring parasite invasion and persistence. Leishmania infection triggered membrane-raft reorientation-dependent Lyn-PI3K/Akt pathway activation which in turn deactivated GSK3ß to stabilize nuclear SREBP2. Moreover, cells perceiving less available intracellular cholesterol due to its sequestration at the plasma membrane resulted in the deregulation of the ER-residing SCAP-SREBP2-Insig circuit thereby assisting increased nuclear translocation of SREBP2. Both increased nuclear transport and stabilization of SREBP2 caused HMGCR-catalyzed cholesterol biosynthesis-mediated plasma membrane cholesterol enrichment leading to decreased membrane-fluidity and plausibly assisting delay in phagosomal acidification. Parasite survival ensuing entry was further ensured by SREBP2-dependent transcriptional up-regulation of UCP2, which suppressed mitochondrial ROS generation, one of the primary microbicidal molecules in macrophages recognized for its efficacy against Leishmania. Functional knock-down of SREBP2 both in vitro and in vivo was associated with reduction in macrophage plasma membrane cholesterol, increased ROS production and lower parasite survival. To our knowledge, this study, for the first time, reveals that Leishmania exploits macrophage cholesterol-dependent SREBP2 circuit to facilitate its entry and survival within the host.


Assuntos
Colesterol/imunologia , Leishmania donovani/imunologia , Macrófagos/imunologia , Mitocôndrias/imunologia , Oxidantes/imunologia , Proteína de Ligação a Elemento Regulador de Esterol 2/imunologia , Animais , Western Blotting , Membrana Celular/imunologia , Membrana Celular/metabolismo , Células Cultivadas , Colesterol/metabolismo , Feminino , Interações Hospedeiro-Parasita/imunologia , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/imunologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismo , Leishmania donovani/fisiologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/parasitologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Mitocôndrias/parasitologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Oxidantes/metabolismo , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/imunologia , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Proteína Desacopladora 2 , Quinases da Família src/imunologia , Quinases da Família src/metabolismo
10.
Cell Mol Immunol ; 11(3): 263-74, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24561457

RESUMO

Fucoidan can cure both antimony-sensitive and antimony-resistant visceral leishmaniasis through immune activation. However, the signaling events underlying this cellular response remain uncharacterized. The present study reveals that fucoidan induces activation of p38 and ERK1/2 and NF-κB DNA binding in both normal and Leishmania donovani-infected macrophages, as revealed by western blotting and electrophoretic mobility shift assay (EMSA), respectively. Pharmacological inhibition of p38, ERK1/2 or the NF-κB pathway markedly attenuated fucoidan-induced pro-inflammatory cytokine synthesis and inducible nitric oxide synthase (iNOS) gene transcription, resulting in a reduction of parasite clearance. To decipher the underlying mechanism of fucoidan-mediated parasite suppression, the expression and functionality of various protein kinase C (PKC) isoforms were evaluated by immunoblotting and enzyme activity assay. Fucoidan elicited an increase in expression and activity of PKC-α, -ßI and -ßII isoforms in infected macrophages. Functional knockdown of PKC-α and -ß resulted in downregulation of p38 and ERK1/2, along with a marked reduction of IL-12 and TNF-α production in fucoidan-treated infected macrophages. Collectively, these results suggest that the curative effect of fucoidan is mediated by PKC-dependent activation of the mitogen-activated protein kinase (MAPK)/NF-κB pathway, which ultimately results in the production of nitric oxide (NO) and disease-resolving pro-inflammatory cytokines.


Assuntos
Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Polissacarídeos/uso terapêutico , Proteína Quinase C/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Imunidade/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Isoenzimas/metabolismo , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Polissacarídeos/farmacologia , Tiazinas/farmacologia , Fator de Transcrição AP-1/metabolismo
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