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1.
Immunity ; 56(11): 2523-2541.e8, 2023 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-37924812

RESUMO

Gasdermin D (GSDMD)-activated inflammatory cell death (pyroptosis) causes mitochondrial damage, but its underlying mechanism and functional consequences are largely unknown. Here, we show that the N-terminal pore-forming GSDMD fragment (GSDMD-NT) rapidly damaged both inner and outer mitochondrial membranes (OMMs) leading to reduced mitochondrial numbers, mitophagy, ROS, loss of transmembrane potential, attenuated oxidative phosphorylation (OXPHOS), and release of mitochondrial proteins and DNA from the matrix and intermembrane space. Mitochondrial damage occurred as soon as GSDMD was cleaved prior to plasma membrane damage. Mitochondrial damage was independent of the B-cell lymphoma 2 family and depended on GSDMD-NT binding to cardiolipin. Canonical and noncanonical inflammasome activation of mitochondrial damage, pyroptosis, and inflammatory cytokine release were suppressed by genetic ablation of cardiolipin synthase (Crls1) or the scramblase (Plscr3) that transfers cardiolipin to the OMM. Phospholipid scramblase-3 (PLSCR3) deficiency in a tumor compromised pyroptosis-triggered anti-tumor immunity. Thus, mitochondrial damage plays a critical role in pyroptosis.


Assuntos
Gasderminas , Piroptose , Proteínas de Neoplasias/metabolismo , Cardiolipinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Inflamassomos/metabolismo
2.
Arterioscler Thromb Vasc Biol ; 42(9): 1169-1185, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35924558

RESUMO

BACKGROUND: Endothelial dysfunction is a critical component in the pathogenesis of cardiovascular diseases and is closely associated with nitric oxide (NO) levels and oxidative stress. Here, we report on novel findings linking endothelial expression of CD70 (also known as CD27 ligand) with alterations in NO and reactive oxygen species. METHODS: CD70 expression was genetically manipulated in human aortic and pulmonary artery endothelial cells. Intracellular NO and hydrogen peroxide (H2O2) were measured using genetically encoded biosensors, and cellular phenotypes were assessed. RESULTS: An unbiased phenome-wide association study demonstrated that polymorphisms in CD70 associate with vascular phenotypes. Endothelial cells treated with CD70-directed short-interfering RNA demonstrated impaired wound closure, decreased agonist-stimulated NO levels, and reduced eNOS (endothelial nitric oxide synthase) protein. These changes were accompanied by reduced NO bioactivity, increased 3-nitrotyrosine levels, and a decrease in the eNOS binding partner heat shock protein 90. Following treatment with the thioredoxin inhibitor auranofin or with agonist histamine, intracellular H2O2 levels increased up to 80% in the cytosol, plasmalemmal caveolae, and mitochondria. There was increased expression of NADPH oxidase 1 complex and gp91phox; expression of copper/zinc and manganese superoxide dismutases was also elevated. CD70 knockdown reduced levels of the H2O2 scavenger catalase; by contrast, glutathione peroxidase 1 expression and activity were increased. CD70 overexpression enhanced endothelial wound closure, increased NO levels, and attenuated the reduction in eNOS mRNA induced by TNFα. CONCLUSIONS: Taken together, these data establish CD70 as a novel regulatory protein in endothelial NO and reactive oxygen species homeostasis, with implications for human vascular disease.


Assuntos
Ligante CD27 , Células Endoteliais , Óxido Nítrico , Ligante CD27/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo
3.
Am J Physiol Heart Circ Physiol ; 322(3): H451-H465, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35089810

RESUMO

The failing heart is characterized by elevated levels of reactive oxygen species. We have developed an animal model of heart failure induced by chemogenetic production of oxidative stress in the heart using a recombinant adeno-associated virus (AAV9) expressing yeast d-amino acid oxidase (DAAO) targeted to cardiac myocytes. When DAAO-infected animals are fed the DAAO substrate d-alanine, the enzyme generates hydrogen peroxide (H2O2) in the cardiac myocytes, leading to dilated cardiomyopathy. However, the underlying mechanisms of oxidative stress-induced heart failure remain incompletely understood. Therefore, we investigated the effects of chronic oxidative stress on the cardiac transcriptome and metabolome. Rats infected with recombinant cardiotropic AAV9 expressing DAAO or control AAV9 were treated for 7 wk with d-alanine to stimulate chemogenetic H2O2 production by DAAO and generate dilated cardiomyopathy. After hemodynamic assessment, left and right ventricular tissues were processed for RNA sequencing and metabolomic profiling. DAAO-induced dilated cardiomyopathy was characterized by marked changes in the cardiac transcriptome and metabolome both in the left and right ventricle. Downregulated transcripts are related to energy metabolism and mitochondrial function, accompanied by striking alterations in metabolites involved in cardiac energetics, redox homeostasis, and amino acid metabolism. Upregulated transcripts are involved in cytoskeletal organization and extracellular matrix. Finally, we noted increased metabolite levels of antioxidants glutathione and ascorbate. These findings provide evidence that chemogenetic generation of oxidative stress leads to a robust heart failure model with distinct transcriptomic and metabolomic signatures and set the basis for understanding the underlying pathophysiology of chronic oxidative stress in the heart.NEW & NOTEWORTHY We have developed a "chemogenetic" heart failure animal model that recapitulates a central feature of human heart failure: increased cardiac redox stress. We used a recombinant DAAO enzyme to generate H2O2 in cardiomyocytes, leading to cardiomyopathy. Here we report striking changes in the cardiac metabolome and transcriptome following chemogenetic heart failure, similar to changes observed in human heart failure. Our findings help validate chemogenetic approaches for the discovery of novel therapeutic targets in heart failure.


Assuntos
Cardiomiopatia Dilatada , Insuficiência Cardíaca , Alanina/farmacologia , Aminoácidos/metabolismo , Aminoácidos/farmacologia , Aminoácidos/uso terapêutico , Animais , Cardiomiopatia Dilatada/metabolismo , Dependovirus/metabolismo , Modelos Animais de Doenças , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Peróxido de Hidrogênio/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , Ratos , Transcriptoma
4.
Cell Physiol Biochem ; 52(1): 57-75, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30790505

RESUMO

BACKGROUND/AIMS: In pancreatic ß-cells, the intracellular Ca²âº homeostasis is an essential regulator of the cells major functions. The endoplasmic reticulum (ER) as interactive intracellular Ca²âº store balances cellular Ca²âº. In this study basal ER Ca²âº homeostasis was evaluated in order to reveal potential ß-cell-specificity of ER Ca²âº handling and its consequences for mitochondrial Ca²âº, ATP and respiration. METHODS: The two pancreatic cell lines INS-1 and MIN-6, freshly isolated pancreatic islets, and the two non-pancreatic cell lines HeLA and EA.hy926 were used. Cytosolic, ER and mitochondrial Ca²âº and ATP measurements were performed using single cell fluorescence microscopy and respective (genetically-encoded) sensors/dyes. Mitochondrial respiration was monitored by respirometry. GSK3ß activity was measured with ELISA. RESULTS: An atypical ER Ca²âº leak was observed exclusively in pancreatic islets and ß-cells. This continuous ER Ca²âº efflux is directed to mitochondria and increases basal respiration and organellar ATP levels, is established by GSK3ß-mediated phosphorylation of presenilin-1, and is prevented by either knockdown of presenilin-1 or an inhibition/knockdown of GSK3ß. Expression of a presenlin-1 mutant that mimics GSK3ß-mediated phosphorylation established a ß-cell-like ER Ca²âº leak in HeLa and EA.hy926 cells. The ER Ca²âº loss in ß-cells was compensated at steady state by Ca²âº entry that is linked to the activity of TRPC3. CONCLUSION: Pancreatic ß-cells establish a cell-specific ER Ca²âº leak that is under the control of GSK3ß and directed to mitochondria, thus, reflecting a cell-specific intracellular Ca²âº handling for basal mitochondrial activity.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Presenilina-1/metabolismo , Animais , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Glicogênio Sintase Quinase 3 beta/genética , Células HeLa , Humanos , Masculino , Camundongos , Mitocôndrias/genética , Fosforilação , Presenilina-1/genética , Ratos
5.
Cell Physiol Biochem ; 53(3): 573-586, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31529929

RESUMO

BACKGROUND/AIMS: In our recent work, the importance of GSK3ß-mediated phosphorylation of presenilin-1 as crucial process to establish a Ca2+ leak in the endoplasmic reticulum and, subsequently, the pre-activation of resting mitochondrial activity in ß-cells was demonstrated. The present work is a follow-up and reveals the importance of GSK3ß-phosphorylated presenilin-1 for responsiveness of pancreatic islets and ß-cells to elevated glucose in terms of cytosolic Ca2+ spiking and insulin secretion. METHODS: Freshly isolated pancreatic islets and the two pancreatic ß-cell lines INS-1 and MIN-6 were used. Cytosolic Ca2+ was fluorometrically monitored using Fura-2/AM and cellular insulin content and secretion were measured by ELISA. RESULTS: Our data strengthened our previous findings of the existence of a presenilin-1-mediated ER-Ca2+ leak in ß-cells, since a reduction of presenilin-1 expression strongly counteracted the ER Ca2+ leak. Furthermore, our data revealed that cytosolic Ca2+ spiking upon administration of high D-glucose was delayed in onset time and strongly reduced in amplitude and frequency upon siRNA-mediated knock-down of presenilin-1 or the inhibition of GSK3ß in the pancreatic ß-cells. Moreover, glucose-triggered initial insulin secretion disappeared by depletion from presenilin-1 and inhibition of GSK3ß in the pancreatic ß-cells and isolated pancreatic islets, respectively. CONCLUSION: These data complement our previous work and demonstrate that the sensitivity of pancreatic islets and ß-cells to glucose illustrated as glucose-triggered cytosolic Ca2+ spiking and initial but not long-lasting insulin secretion crucially depends on a strong ER Ca2+ leak that is due to the phosphorylation of presenilin-1 by GSK3ß, a phenomenon that might be involved in the development of type 2 diabetes.


Assuntos
Retículo Endoplasmático/metabolismo , Glucose/farmacologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Presenilina-1/metabolismo , Animais , Antracenos/farmacologia , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Humanos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , MAP Quinase Quinase 4/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo
6.
Mol Pharmacol ; 93(4): 335-343, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29358221

RESUMO

According to current views, oxidation of aldehyde dehydrogenase-2 (ALDH2) during glyceryltrinitrate (GTN) biotransformation is essentially involved in vascular nitrate tolerance and explains the dependence of this reaction on added thiols. Using a novel fluorescent intracellular nitric oxide (NO) probe expressed in vascular smooth muscle cells (VSMCs), we observed ALDH2-catalyzed formation of NO from GTN in the presence of exogenously added dithiothreitol (DTT), whereas only a short burst of NO, corresponding to a single turnover of ALDH2, occurred in the absence of DTT. This short burst of NO associated with oxidation of the reactive C302 residue in the active site was followed by formation of low-nanomolar NO, even without added DTT, indicating slow recovery of ALDH2 activity by an endogenous reductant. In addition to the thiol-reversible oxidation of ALDH2, thiol-refractive inactivation was observed, particularly under high-turnover conditions. Organ bath experiments with rat aortas showed that relaxation by GTN lasted longer than that caused by the NO donor diethylamine/NONOate, in line with the long-lasting nanomolar NO generation from GTN observed in VSMCs. Our results suggest that an endogenous reductant with low efficiency allows sustained generation of GTN-derived NO in the low-nanomolar range that is sufficient for vascular relaxation. On a longer time scale, mechanism-based, thiol-refractive irreversible inactivation of ALDH2, and possibly depletion of the endogenous reductant, will render blood vessels tolerant to GTN. Accordingly, full reactivation of oxidized ALDH2 may not occur in vivo and may not be necessary to explain GTN-induced vasodilation.


Assuntos
Aldeído-Desidrogenase Mitocondrial/metabolismo , Tolerância a Medicamentos/fisiologia , Músculo Liso Vascular/metabolismo , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Nitroglicerina/metabolismo , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Ditiotreitol/farmacologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Nitratos/farmacologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley
7.
Pflugers Arch ; 470(8): 1193-1203, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29527615

RESUMO

Mitochondria are multifunctional organelles that essentially contribute to cell signaling by sophisticated mechanisms of communications. Live cell imaging studies showed that mitochondria are dynamic and complex structures that form ramified networks by directed movements, fission, and fusion events. There is emerging evidence that the morphology of mitochondria determines cellular functions and vice versa. Several intracellular signaling pathways and messengers including Ca2+ dynamically influence the architecture of mitochondria. Because electron microscopy cannot be utilized for an assessment of dynamics of mitochondrial morphology in intact cells, most studies were performed using wide-field or laser confocal fluorescence microscopies that, due to limitations of their spatial resolution, do not allow investigating sub-mitochondrial structures. Accordingly, our understanding of the dynamics of substructures of mitochondria is quite limited. Here, we present a robust super-resolution method to quantify the dynamics of mitochondrial cristae, the main substructures of the inner mitochondrial membrane, exploiting structured illumination microscopy (SIM). We observed that knockdown of the dynamin-like 120-kDa protein, which is encoded by the OPA1 gene, specifically reduces the dynamics of the mitochondrial cristae membranes (CM), while the inner boundary membrane (IBM) remained flexible. We further used dual color SIM to quantify the dynamics of CM in the junction between mitochondria and the endoplasmic reticulum (ER; mitochondrial associated membranes, MAMs). Intracellular Ca2+ release spatially reduced CM-dynamics in MAMs. Moreover, CM-dynamics was independent from matrix Ca2+ signal. Our data suggest that local Ca2+ signals specifically control CM-dynamics and structure to facilitate a well-balanced functional (Ca2+) interplay between mitochondria and the ER.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/fisiologia , Células HeLa , Humanos , Mitocôndrias/fisiologia , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/fisiologia
8.
J Biol Chem ; 291(46): 24076-24084, 2016 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-27679490

RESUMO

Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN), resulting in activation of soluble guanylate cyclase (sGC) and cGMP-mediated vasodilation. We have previously shown that a minor reaction of ALDH2-catalyzed GTN bioconversion, accounting for about 5% of the main clearance-based turnover yielding inorganic nitrite, results in direct NO formation and concluded that this minor pathway could provide the link between vascular GTN metabolism and activation of sGC. However, lack of detectable NO at therapeutically relevant GTN concentrations (≤1 µm) in vascular tissue called into question the biological significance of NO formation by purified ALDH2. We addressed this issue and used a novel, highly sensitive genetically encoded fluorescent NO probe (geNOp) to visualize intracellular NO formation at low GTN concentrations (≤1 µm) in cultured vascular smooth muscle cells (VSMC) expressing an ALDH2 mutant that reduces GTN to NO but lacks clearance-based GTN denitration activity. NO formation was compared with GTN-induced activation of sGC. The addition of 1 µm GTN to VSMC expressing either wild-type or C301S/C303S ALDH2 resulted in pronounced intracellular NO elevation, with maximal concentrations of 7 and 17 nm, respectively. Formation of GTN-derived NO correlated well with activation of purified sGC in VSMC lysates and cGMP accumulation in intact porcine aortic endothelial cells infected with wild-type or mutant ALDH2. Formation of NO and cGMP accumulation were inhibited by ALDH inhibitors chloral hydrate and daidzin. The present study demonstrates that ALDH2-catalyzed NO formation is necessary and sufficient for GTN bioactivation in VSMC.


Assuntos
Aldeído-Desidrogenase Mitocondrial/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Óxido Nítrico/metabolismo , Nitroglicerina/farmacocinética , Aldeído-Desidrogenase Mitocondrial/antagonistas & inibidores , Aldeído-Desidrogenase Mitocondrial/genética , Substituição de Aminoácidos , Animais , Bovinos , Hidrato de Cloral/farmacologia , Humanos , Isoflavonas/farmacologia , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Nitroglicerina/farmacologia , Suínos
9.
Nitric Oxide ; 70: 59-67, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28882669

RESUMO

The members of the nitric oxide synthase (NOS) family, eNOS, nNOS and iNOS, are well-characterized enzymes. However, due to the lack of suitable direct NO sensors, little is known about the kinetic properties of cellular NO generation by the different nitric oxide synthase isoenzymes. Very recently, we developed a novel class of fluorescent protein-based NO-probes, the geNOps, which allow real-time measurement of cellular NO generation and fluctuation. By applying these genetic NO biosensors to nNOS-, eNOS- and iNOS-expressing HEK293 cells we were able to characterize the respective NO dynamics in single cells that exhibited identical Ca2+ signaling as comparable activator of nNOS and eNOS. Our data demonstrate that upon Ca2+ mobilization nNOS-derived NO signals occur instantly and strictly follow the Ca2+ elevation while NO release by eNOS occurs gradually and sustained. To detect high NO levels in cells expressing iNOS, a new ratiometric probe based on two fluorescent proteins was developed. This novel geNOp variant allows the measurement of the high NO levels in cells expressing iNOS. Moreover, we used this probe to study the L-arginine-dependency of NO generation by iNOS on the level of single cells. Our experiments highlight that the geNOps technology is suitable to detect obvious differences in the kinetics, amplitude and substrate-dependence of cellular NO signals-derived from all three nitric oxide synthase isoforms.


Assuntos
Óxido Nítrico Sintase Tipo III/análise , Óxido Nítrico Sintase Tipo II/análise , Óxido Nítrico Sintase Tipo I/análise , Óxido Nítrico/biossíntese , Arginina/metabolismo , Técnicas Biossensoriais/instrumentação , Cálcio/metabolismo , Corantes Fluorescentes/química , Células HEK293/enzimologia , Humanos , Isoenzimas , Cinética , Proteínas Luminescentes/química , Microscopia de Fluorescência , Óxido Nítrico/análise , Óxido Nítrico/química
10.
J Cell Sci ; 127(Pt 13): 2944-55, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24806964

RESUMO

Mitochondria contribute to cell signaling by controlling store-operated Ca(2+) entry (SOCE). SOCE is activated by Ca(2+) release from the endoplasmic reticulum (ER), whereupon stromal interacting molecule 1 (STIM1) forms oligomers, redistributes to ER-plasma-membrane junctions and opens plasma membrane Ca(2+) channels. The mechanisms by which mitochondria interfere with the complex process of SOCE are insufficiently clarified. In this study, we used an shRNA approach to investigate the direct involvement of mitochondrial Ca(2+) buffering in SOCE. We demonstrate that knockdown of either of two proteins that are essential for mitochondrial Ca(2+) uptake, the mitochondrial calcium uniporter (MCU) or uncoupling protein 2 (UCP2), results in decelerated STIM1 oligomerization and impaired SOCE following cell stimulation with an inositol-1,4,5-trisphosphate (IP3)-generating agonist. Upon artificially augmented cytosolic Ca(2+) buffering or ER Ca(2+) depletion by sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitors, STIM1 oligomerization did not rely on intact mitochondrial Ca(2+) uptake. However, MCU-dependent mitochondrial sequestration of Ca(2+) entering through the SOCE pathway was essential to prevent slow deactivation of SOCE. Our findings show a stimulus-specific contribution of mitochondrial Ca(2+) uptake to the SOCE machinery, likely through a role in shaping cytosolic Ca(2+) micro-domains.


Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas de Neoplasias/metabolismo , Canais de Cálcio/genética , Células HeLa , Humanos , Inositol 1,4,5-Trifosfato/genética , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Molécula 1 de Interação Estromal
11.
Cell Physiol Biochem ; 39(4): 1404-20, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27606689

RESUMO

BACKGROUND/AIMS: Resveratrol and its derivate piceatannol are known to induce cancer cell-specific cell death. While multiple mechanisms of actions have been described including the inhibition of ATP synthase, changes in mitochondrial membrane potential and ROS levels, the exact mechanisms of cancer specificity of these polyphenols remain unclear. This paper is designed to reveal the molecular basis of the cancer-specific initiation of cell death by resveratrol and piceatannol. METHODS: The two cancer cell lines EA.hy926 and HeLa, and somatic short-term cultured HUVEC were used. Cell viability and caspase 3/7 activity were tested. Mitochondrial, cytosolic and endoplasmic reticulum Ca2+ as well as cytosolic and mitochondrial ATP levels were measured using single cell fluorescence microscopy and respective genetically-encoded sensors. Mitochondria-ER junctions were analyzed applying super-resolution SIM and ImageJ-based image analysis. RESULTS: Resveratrol and piceatannol selectively trigger death in cancer but not somatic cells. Hence, these polyphenols strongly enhanced mitochondrial Ca2+ uptake in cancer exclusively. Resveratrol and piceatannol predominantly affect mitochondrial but not cytosolic ATP content that yields in a reduced SERCA activity. Decreased SERCA activity and the strongly enriched tethering of the ER and mitochondria in cancer cells result in an enhanced MCU/Letm1-dependent mitochondrial Ca2+ uptake upon intracellular Ca2+ release exclusively in cancer cells. Accordingly, resveratrol/piceatannol-induced cancer cell death could be prevented by siRNA-mediated knock-down of MCU and Letm1. CONCLUSIONS: Because their greatly enriched ER-mitochondria tethering, cancer cells are highly susceptible for resveratrol/piceatannol-induced reduction of SERCA activity to yield mitochondrial Ca2+ overload and subsequent cancer cell death.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Cálcio/agonistas , Retículo Endoplasmático/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Estilbenos/farmacologia , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Transporte de Íons/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Especificidade de Órgãos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Resveratrol , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
12.
Pflugers Arch ; 467(12): 2509-18, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26275882

RESUMO

The mitochondrial Ca(2+) uniporter is a highly Ca(2+)-selective protein complex that consists of the pore-forming mitochondrial Ca(2+) uniporter protein (MCU), the scaffolding essential MCU regulator (EMRE), and mitochondrial calcium uptake 1 and 2 (MICU1/2), which negatively regulate mitochondrial Ca(2+) uptake. We have previously reported that uncoupling proteins 2 and 3 (UCP2/3) are also engaged in the activity of mitochondrial Ca(2+) uptake under certain conditions, while the mechanism by which UCP2/3 facilitates mitochondrial Ca(2+) uniport remains elusive. This work was designed to investigate the impact of UCP2 on the three distinct mitochondrial Ca(2+) currents found in mitoplasts isolated from HeLa cells, the intermediate- (i-), burst- (b-) and extra-large (xl-) mitochondrial/mitoplast Ca(2+) currents (MCC). Using the patch clamp technique on mitoplasts from cells with reduced MCU and EMRE unveiled a very high affinity of MCU for xl-MCC that succeeds that for i-MCC, indicating the coexistence of at least two MCU/EMRE-dependent Ca(2+) currents. The manipulation of the expression level of UCP2 by either siRNA-mediated knockdown or overexpression changed exclusively the open probability (NPo) of xl-MCC by approx. 38% decrease or nearly a 3-fold increase, respectively. These findings confirm a regulatory role of UCP2 in mitochondrial Ca(2+) uptake and identify UCP2 as a selective modulator of just one distinct MCU/EMRE-dependent mitochondrial Ca(2+) inward current.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Canais Iônicos/metabolismo , Proteínas Mitocondriais/metabolismo , Células HeLa , Humanos , Canais Iônicos/genética , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Proteínas Mitocondriais/genética , Proteína Desacopladora 2
13.
Sensors (Basel) ; 15(6): 13052-68, 2015 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-26053751

RESUMO

Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs (GFP, OFP, RFP) have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store.


Assuntos
Cálcio/análise , Cálcio/química , Retículo Endoplasmático/química , Corantes Fluorescentes/química , Proteínas Luminescentes/química , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Microscopia Confocal
14.
J Biol Chem ; 288(21): 15367-79, 2013 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-23592775

RESUMO

The transfer of Ca(2+) across the inner mitochondrial membrane is an important physiological process linked to the regulation of metabolism, signal transduction, and cell death. While the definite molecular composition of mitochondrial Ca(2+) uptake sites remains unknown, several proteins of the inner mitochondrial membrane, that are likely to accomplish mitochondrial Ca(2+) fluxes, have been described: the novel uncoupling proteins 2 and 3, the leucine zipper-EF-hand containing transmembrane protein 1 and the mitochondrial calcium uniporter. It is unclear whether these proteins contribute to one unique mitochondrial Ca(2+) uptake pathway or establish distinct routes for mitochondrial Ca(2+) sequestration. In this study, we show that a modulation of Ca(2+) release from the endoplasmic reticulum by inhibition of the sarco/endoplasmatic reticulum ATPase modifies cytosolic Ca(2+) signals and consequently switches mitochondrial Ca(2+) uptake from an uncoupling protein 3- and mitochondrial calcium uniporter-dependent, but leucine zipper-EF-hand containing transmembrane protein 1-independent to a leucine zipper-EF-hand containing transmembrane protein 1- and mitochondrial calcium uniporter-mediated, but uncoupling protein 3-independent pathway. Thus, the activity of sarco/endoplasmatic reticulum ATPase is significant for the mode of mitochondrial Ca(2+) sequestration and determines which mitochondrial proteins might actually accomplish the transfer of Ca(2+) across the inner mitochondrial membrane. Moreover, our findings herein support the existence of distinct mitochondrial Ca(2+) uptake routes that might be essential to ensure an efficient ion transfer into mitochondria despite heterogeneous cytosolic Ca(2+) rises.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Canais Iônicos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Retículo Endoplasmático/genética , Células HeLa , Humanos , Canais Iônicos/genética , Mitocôndrias/genética , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Proteína Desacopladora 1
15.
Pflugers Arch ; 466(7): 1411-20, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24162235

RESUMO

A protein referred to as CCDC109A and then renamed to mitochondrial calcium uniporter (MCU) has recently been shown to accomplish mitochondrial Ca(2+) uptake in different cell types. In this study, we investigated whole-mitoplast inward cation currents and single Ca(2+) channel activities in mitoplasts prepared from stable MCU knockdown HeLa cells using the patch-clamp technique. In whole-mitoplast configuration, diminution of MCU considerably reduced inward Ca(2+) and Na(+) currents. This was accompanied by a decrease in occurrence of single channel activity of the intermediate conductance mitochondrial Ca(2+) current (i-MCC). However, ablation of MCU yielded a compensatory 2.3-fold elevation in the occurrence of the extra large conductance mitochondrial Ca(2+) current (xl-MCC), while the occurrence of bursting currents (b-MCC) remained unaltered. These data reveal i-MCC as MCU-dependent current while xl-MCC and b-MCC seem to be rather MCU-independent, thus, pointing to the engagement of at least two molecularly distinct mitochondrial Ca(2+) channels.


Assuntos
Potenciais de Ação , Canais de Cálcio/metabolismo , Membranas Mitocondriais/metabolismo , Cálcio/metabolismo , Canais de Cálcio/genética , Células HeLa , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Sódio/metabolismo
16.
Redox Biol ; 73: 103214, 2024 07.
Artigo em Inglês | MEDLINE | ID: mdl-38805973

RESUMO

The chaperone protein EROS ("Essential for Reactive Oxygen Species") was recently discovered in phagocytes. EROS was shown to regulate the abundance of the ROS-producing enzyme NADPH oxidase isoform 2 (NOX2) and to control ROS-mediated cell killing. Reactive oxygen species are important not only in immune surveillance, but also modulate physiological signaling responses in multiple tissues. The roles of EROS have not been previously explored in the context of oxidant-modulated cell signaling. Here we show that EROS plays a key role in ROS-dependent signal transduction in vascular endothelial cells. We used siRNA-mediated knockdown and developed CRISPR/Cas9 knockout of EROS in human umbilical vein endothelial cells (HUVEC), both of which cause a significant decrease in the abundance of NOX2 protein, associated with a marked decrease in RAC1, a small G protein that activates NOX2. Loss of EROS also attenuates receptor-mediated hydrogen peroxide (H2O2) and Ca2+ signaling, disrupts cytoskeleton organization, decreases cell migration, and promotes cellular senescence. EROS knockdown blocks agonist-modulated eNOS phosphorylation and nitric oxide (NO●) generation. These effects of EROS knockdown are strikingly similar to the alterations in endothelial cell responses that we previously observed following RAC1 knockdown. Proteomic analyses following EROS or RAC1 knockdown in endothelial cells showed that reduced abundance of these two distinct proteins led to largely overlapping effects on endothelial biological processes, including oxidoreductase, protein phosphorylation, and endothelial nitric oxide synthase (eNOS) pathways. These studies demonstrate that EROS plays a central role in oxidant-modulated endothelial cell signaling by modulating NOX2 and RAC1.


Assuntos
Células Endoteliais da Veia Umbilical Humana , NADPH Oxidase 2 , Oxirredução , Espécies Reativas de Oxigênio , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP , Humanos , NADPH Oxidase 2/metabolismo , NADPH Oxidase 2/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Espécies Reativas de Oxigênio/metabolismo , Peróxido de Hidrogênio/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico/metabolismo , Movimento Celular , Fosforilação , Senescência Celular , Técnicas de Silenciamento de Genes
17.
J Biol Chem ; 287(41): 34445-54, 2012 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-22904319

RESUMO

In pancreatic ß-cells, uptake of Ca(2+) into mitochondria facilitates metabolism-secretion coupling by activation of various matrix enzymes, thus facilitating ATP generation by oxidative phosphorylation and, in turn, augmenting insulin release. We employed an siRNA-based approach to evaluate the individual contribution of four proteins that were recently described to be engaged in mitochondrial Ca(2+) sequestration in clonal INS-1 832/13 pancreatic ß-cells: the mitochondrial Ca(2+) uptake 1 (MICU1), mitochondrial Ca(2+) uniporter (MCU), uncoupling protein 2 (UCP2), and leucine zipper EF-hand-containing transmembrane protein 1 (LETM1). Using a FRET-based genetically encoded Ca(2+) sensor targeted to mitochondria, we show that a transient knockdown of MICU1 or MCU diminished mitochondrial Ca(2+) uptake upon both intracellular Ca(2+) release and Ca(2+) entry via L-type channels. In contrast, knockdown of UCP2 and LETM1 exclusively reduced mitochondrial Ca(2+) uptake in response to either intracellular Ca(2+) release or Ca(2+) entry, respectively. Therefore, we further investigated the role of MICU1 and MCU in metabolism-secretion coupling. Diminution of MICU1 or MCU reduced mitochondrial Ca(2+) uptake in response to d-glucose, whereas d-glucose-triggered cytosolic Ca(2+) oscillations remained unaffected. Moreover, d-glucose-evoked increases in cytosolic ATP and d-glucose-stimulated insulin secretion were diminished in MICU1- or MCU-silenced cells. Our data highlight the crucial role of MICU1 and MCU in mitochondrial Ca(2+) uptake in pancreatic ß-cells and their involvement in the positive feedback required for sustained insulin secretion.


Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Canais de Cálcio/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Endopeptidases/genética , Endopeptidases/metabolismo , Técnicas de Silenciamento de Genes , Glucose/genética , Glucose/metabolismo , Humanos , Insulina/genética , Secreção de Insulina , Células Secretoras de Insulina/citologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Ubiquitina Tiolesterase
18.
J Biol Chem ; 287(25): 21110-20, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22556413

RESUMO

Accumulation of palmitic acid (PA) in cells from nonadipose tissues is known to induce lipotoxicity resulting in cellular dysfunction and death. The exact molecular pathways of PA-induced cell death are still mysterious. Here, we show that PA triggers autophagy, which did not counteract but in contrast promoted endothelial cell death. The PA-induced cell death was predominantly necrotic as indicated by annexin V and propidium iodide (PI) staining, absence of caspase activity, low levels of DNA hypoploidy, and an early ATP depletion. In addition PA induced a strong elevation of mRNA levels of ubiquitin carboxyl-terminal hydrolase (CYLD), a known mediator of necroptosis. Moreover, siRNA-mediated knockdown of CYLD significantly antagonized PA-induced necrosis of endothelial cells. In contrast, inhibition and knockdown of receptor interacting protein kinase 1 (RIPK1) had no effect on PA-induced necrosis, indicating the induction of a CYLD-dependent but RIPK1-independent cell death pathway. PA was recognized as a strong and early inducer of autophagy. The inhibition of autophagy by both pharmacological inhibitors and genetic knockdown of the autophagy-specific genes, vacuolar protein sorting 34 (VPS34), and autophagy-related protein 7 (ATG7), could rescue the PA-induced death of endothelial cells. Moreover, the initiation of autophagy and cell death by PA was reduced in endothelial cells loaded with the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-(acetoxymethyl) ester (BAPTA-AM), indicating that Ca(2+) triggers the fatal signaling of PA. In summary, we introduce an unexpected mechanism of lipotoxicity in endothelial cells and provide several novel strategies to counteract the lipotoxic signaling of PA.


Assuntos
Autofagia/efeitos dos fármacos , Células Endoteliais/metabolismo , Inibidores Enzimáticos/farmacocinética , Ácido Palmítico/farmacologia , Proteína 7 Relacionada à Autofagia , Sinalização do Cálcio , Células Cultivadas , Quelantes/farmacologia , Classe III de Fosfatidilinositol 3-Quinases/genética , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Enzima Desubiquitinante CYLD , Ácido Egtázico/análogos & derivados , Células Endoteliais/patologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Necrose , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas Supressoras de Tumor , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo
19.
Nat Commun ; 14(1): 3094, 2023 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-37248315

RESUMO

Oxidative stress is associated with cardiovascular and neurodegenerative diseases. Here we report studies of neurovascular oxidative stress in chemogenetic transgenic mouse lines expressing yeast D-amino acid oxidase (DAAO) in neurons and vascular endothelium. When these transgenic mice are fed D-amino acids, DAAO generates hydrogen peroxide in target tissues. DAAO-TGCdh5 transgenic mice express DAAO under control of the putatively endothelial-specific Cdh5 promoter. When we provide these mice with D-alanine, they rapidly develop sensory ataxia caused by oxidative stress and mitochondrial dysfunction in neurons within dorsal root ganglia and nodose ganglia innervating the heart. DAAO-TGCdh5 mice also develop cardiac hypertrophy after chronic chemogenetic oxidative stress. This combination of ataxia, mitochondrial dysfunction, and cardiac hypertrophy is similar to findings in patients with Friedreich's ataxia. Our observations indicate that neurovascular oxidative stress is sufficient to cause sensory ataxia and cardiac hypertrophy. Studies of DAAO-TGCdh5 mice could provide mechanistic insights into Friedreich's ataxia.


Assuntos
Ataxia de Friedreich , Camundongos , Animais , Camundongos Transgênicos , Cardiomegalia , Estresse Oxidativo , Ataxia/complicações
20.
J Biol Chem ; 286(32): 28444-55, 2011 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-21613221

RESUMO

Cytosolic Ca(2+) signals are transferred into mitochondria over a huge concentration range. In our recent work we described uncoupling proteins 2 and 3 (UCP2/3) to be fundamental for mitochondrial uptake of high Ca(2+) domains in mitochondria-ER junctions. On the other hand, the leucine zipper EF hand-containing transmembrane protein 1 (Letm1) was identified as a mitochondrial Ca(2+)/H(+) antiporter that achieved mitochondrial Ca(2+) sequestration at small Ca(2+) increases. Thus, the contributions of Letm1 and UCP2/3 to mitochondrial Ca(2+) uptake were compared in endothelial cells. Knock-down of Letm1 did not affect the UCP2/3-dependent mitochondrial uptake of intracellularly released Ca(2+) but strongly diminished the transfer of entering Ca(2+) into mitochondria, subsequently, resulting in a reduction of store-operated Ca(2+) entry (SOCE). Knock-down of Letm1 and UCP2/3 did neither impact on cellular ATP levels nor the membrane potential. The enhanced mitochondrial Ca(2+) signals in cells overexpressing UCP2/3 rescued SOCE upon Letm1 knock-down. In digitonin-permeabilized cells, Letm1 exclusively contributed to mitochondrial Ca(2+) uptake at low Ca(2+) conditions. Neither the Letm1- nor the UCP2/3-dependent mitochondrial Ca(2+) uptake was affected by a knock-down of mRNA levels of mitochondrial calcium uptake 1 (MICU1), a protein that triggers mitochondrial Ca(2+) uptake in HeLa cells. Our data indicate that Letm1 and UCP2/3 independently contribute to two distinct, mitochondrial Ca(2+) uptake pathways in intact endothelial cells.


Assuntos
Sinalização do Cálcio/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Células Endoteliais/metabolismo , Canais Iônicos/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Células Endoteliais/citologia , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Canais Iônicos/genética , Proteínas de Membrana/genética , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Desacopladora 2 , Proteína Desacopladora 3
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