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1.
Adv Exp Med Biol ; 1232: 183-190, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31893409

RESUMO

Oxygen delivery to tissue mitochondria relies on simple diffusion in the target cells and tissues. As such, intracellular availability of O2 in tissue depends on its solubility and diffusivity in complex and heterogeneous macromolecular environments. The path of oxygen diffusion is key to its rate of transfer, especially where pathways of differing favorability are present. Most commonly, aqueous media, such as interstitial fluid and cytoplasm, are assumed to provide the dominant diffusion path. Here, the 'hydrophobic channeling' hypothesis is revisited, and several lines of evidence pointing toward lipid-accelerated oxygen diffusion pathways are discussed. The implications of hydrophobic channeling are considered in light of extended membrane networks in cells and tissues.


Assuntos
Células , Difusão , Metabolismo dos Lipídeos , Oxigênio , Células/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Lipídeos/química , Mitocôndrias/metabolismo , Oxigênio/metabolismo
2.
Cell Physiol Biochem ; 53(S1): 52-62, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31854954

RESUMO

Kv1.3 is a voltage gated potassium channel located in the plasma membrane, as well as at intracellular levels, such as mitochondria (mitoKv1.3), nucleus and Golgi apparatus. The plasma membrane channel has been shown to be important for cell proliferation, while the mitochondrial counterpart has been related to modulation of cell death. Moreover, altered expression of Kv1.3 was observed in various tumors and Kv1.3 seems to be involved in development and progression of various cancerous forms. Recent experimental evidences have proved that pharmacological inhibition of the mitoKv1.3 succeeded in reducing up to 90% of tumor volume in vivo in orthotopic mouse model. Furthermore, mitoKv1.3 modulation could impact on cell proliferation as well as on regulation of intracellular signaling pathways. Indeed, the treatment with sub-lethal doses of mitoKv1.3 inhibitors can downregulate Wnt-ß catenin signaling by reducing mitochondrial ATP production and triggering ER-stress. In this review, we describe the role of the mitoKv1.3 in cell death, cancer and intracellular signaling. We will discuss how pharmacological modulation of mitochondrial potassium fluxes impact on mitochondrial membrane potential, reactive oxygen species production and ATP synthesis. All these changes in mitochondrial fitness are related to cell proliferation as well as to cell death and finally on cancer development and progression, so Kv1.3 (and mitoKv1.3) could be now considered a new oncological target.


Assuntos
Canal de Potássio Kv1.3/metabolismo , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Animais , Apoptose , Proliferação de Células , Estresse do Retículo Endoplasmático , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/genética , Neoplasias/patologia , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
3.
Cell Physiol Biochem ; 53(S1): 63-78, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31860207

RESUMO

Mitochondria play a central role in cancer development, by contributing to most of the classical hallmarks of cancer, including sustained proliferation, metabolic re-programming, apoptosis resistance, invasion and induction of angiogenesis [1]. In addition, mitochondria affect also the function of anti- and pro-tumoral immune cells in the tumor microenvironment. Mitochondria harbor a plethora of regulated ion channels whose function is related to ion/ metabolite transport and to fine-tuning of mitochondrial membrane potential as well as of reactive oxygen species release. As a consequence, growing evidence link ion channels located both in the outer and inner mitochondrial membranes to several cancer hallmarks. The present review summarizes our recent knowledge about the participation and role of mitochondrial channels leading to acquisition of cancer hallmarks and thus to cancer progression.


Assuntos
Canais Iônicos/metabolismo , Mitocôndrias/patologia , Neoplasias/patologia , Animais , Apoptose , Proliferação de Células , Progressão da Doença , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/patologia , Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia
4.
Biochemistry (Mosc) ; 84(10): 1143-1150, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31694510

RESUMO

Mitochondria are essential organelles of eukaryotic cell that provide its respiratory function by means of the electron transfer chain. Expression of mitochondrial genes is organized in a bacterial-like manner; however multiple evolutionary differences are observed between the two systems, including translation initiation machinery. This review is dedicated to the mitochondrial translation initiation factor 3 (IF3mt), which plays a key role in the protein synthesis in mitochondria. Involvement of IF3mt in human health and disease is discussed.


Assuntos
Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Doença de Parkinson/metabolismo , Humanos , Mitocôndrias/metabolismo
5.
Biochemistry (Mosc) ; 84(11): 1359-1374, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31760923

RESUMO

The review summarizes the data of our research and published studies on the ubiquitination of brain mitochondrial proteins and its changes during the development of experimental parkinsonism and administration of the neuroprotector isatin (indole-2,3-dione) with special attention to the mitochondrial ubiquitin-conjugating system and location of ubiquitinated proteins in these organelles. Incubation of brain mitochondrial fraction with biotinylated ubiquitin in vitro resulted in the incorporation of biotinylated ubiquitin in both mitochondrial and mitochondria-associated proteins. According to the interactome analysis, the identified non-ubiquitinated proteins are able to form tight complexes with ubiquitinated proteins or their partners and components of mitochondrial membranes, in which interactions of ubiquitin chains with the ubiquitin-binding protein domains play an important role. The studies of endogenous ubiquitination in the total brain mitochondrial fraction of C57Bl mice performed in different laboratories have shown that mitochondrial proteins represent about 30% of all ubiquitinated proteins. However, comparison of brain subproteomes of mitochondrial ubiquitinated proteins reported in the literature revealed significant differences both in their composition and involvement of identified ubiquitinated proteins in biological processes listed in the Gene Ontology database. The development of experimental parkinsonism in C57Bl mice induced by a single-dose administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) resulted in a decrease in the total number of mitochondrial ubiquitinated proteins and increase in the number of oxidized mitochondrial proteins containing the ubiquitin signature (K-ε-GG). Comparison of ubiquitinated proteins associated with the mouse brain mitochondrial fraction and mouse brain mitochondrial proteins bound to the proteasome ubiquitin receptor (Rpn10 subunit) did not reveal any common proteins. This suggests that ubiquitination of brain mitochondrial proteins is not directly related to their degradation in the proteasomes. Proteomic profiling of brain isatin-binding proteins identified enzymes involved in the ubiquitin-conjugating system functioning. Mapping of the identified isatin-binding proteins to known metabolic pathways indicates their participation in the parkin (E3 ubiquitin ligase)-associated pathway (CH000000947). The functional links involving brain mitochondrial ubiquitinated proteins were found only in the group of animals with the MPTP-induced parkinsonism, but not in animals treated with MPTP/isatin or isatin only. This suggests that the neuroprotective effect of isatin may be associated with the impaired functional relationships of proteins targeted to subsequent degradation.


Assuntos
Encéfalo/metabolismo , Transtornos Parkinsonianos/patologia , Ubiquitina/metabolismo , Animais , Autofagia , Redes e Vias Metabólicas , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/veterinária , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação
6.
Biochemistry (Mosc) ; 84(11): 1411-1423, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31760927

RESUMO

Ischemic stroke and neonatal hypoxic-ischemic encephalopathy are two of the leading causes of disability in adults and infants. The energy demands of the brain are provided by mitochondrial oxidative phosphorylation. Ischemia/reperfusion (I/R) affects the production of ATP in brain mitochondria, leading to energy failure and death of the affected tissue. Among the enzymes of the mitochondrial respiratory chain, mitochondrial complex I is the most sensitive to I/R; however, the mechanisms of its inhibition are poorly understood. This article reviews some of the existing data on the mitochondria impairment during I/R and proposes two distinct mechanisms of complex I damage emerging from recent studies. One mechanism is a reversible dissociation of natural flavin mononucleotide cofactor from the enzyme I after ischemia. Another mechanism is a modification of critical cysteine residue of complex I involved into the active/deactive conformational transition of the enzyme. I describe potential effects of these two processes in the development of mitochondrial I/R injury and briefly discuss possible neuroprotective strategies to ameliorate I/R brain injury.


Assuntos
Encéfalo/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Traumatismo por Reperfusão/patologia , Animais , Flavinas/química , Flavinas/metabolismo , Humanos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Compostos de Sulfidrila/química
7.
J Agric Food Chem ; 67(41): 11428-11435, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31589037

RESUMO

Diosgenin and diosgenyl saponins as the major bioactive compounds isolated from dietary fenugreek seeds, yam roots, etc. possessed strong antitumor effects. To understand their detailed antitumor mechanisms, a fluorophore-appended derivative of diosgenin [Glc/CNHphth-diosgenin (GND)] was synthesized, starting from diosgenin and glucosamine hydrochloride in overall yields of 7-12% over 7-10 steps. Co-localization of GND with organelle-specific stains, transmission electron microscopy, and relative protein analyses demonstrated that GND crossed the plasma membrane through organic anion-transporting polypeptide 1B1 and distributed in the endoplasmic reticulum (ER), lysosome, and mitochondria. In this process, GND induced ER swelling, mitochondrial damage, and autophagosome and upregulating IRE-1α to induce autophagy and apoptosis. Furthermore, autophagy inhibitor chloroquine delayed the appearance of cleaved poly(ADP-ribose) polymerase and inhibited cleaved caspase 8, which indicated that GND induced autophagy to activate caspase-8-dependent apoptosis. These observations suggested that diosgenyl saponin was a potent anticancer agent that elicited ER stress and mitochondria-mediated apoptotic pathways in liver cancer.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Autofagia/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Neoplasias Hepáticas/fisiopatologia , Extratos Vegetais/farmacologia , Saponinas/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/genética , Lisossomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo
8.
Life Sci ; 237: 116941, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31606382

RESUMO

AIMS: Podocytes play an important role in the development of diabetic kidney disease (DKD). Mitochondria are the source of energy for cell survival, and mitochondrial abnormalities have been shown to contribute to podocyte injury in DKD. In high glucose (HG)-treated podocytes, mitochondrial function and dynamics are abnormal, and intracellular metabolism is often disrupted. However, the molecular mechanism is still unclear. Mitochondrial pyruvate carrier 2 (MPC2) mediates pyruvate transport from the cytoplasm to the mitochondrial matrix, which determines the cellular energy supply and cell survival. Here, we hypothesize that MPC2 damages mitochondria and induces apoptosis in HG-treated podocytes. MAIN METHODS: We used Western blotting, immunofluorescence and immunoprecipitation to detect the expression of MPC2 in HG-treated podocytes. Pyruvate levels were measured to evaluate metabolic station. Mitochondrial membrane potential (MMP) was measured by inverted fluorescence microscopy and flow cytometry. Mitochondrial morphology was assayed by MitoTracker Red staining, and cellular apoptosis was examined by flow cytometry. Furthermore, we treated podocytes with UK5099 and MPC2 siRNA to assess the outcomes of UK5099 treatment and MPC2 knockdown. KEY FINDINGS: Intracellular pyruvate accumulated, the mitochondria were damaged and cellular apoptosis increased in podocytes cultured with HG compared to that in control podocytes. MPC2 acetylation was significantly increased in HG-treated podocytes. Furthermore, the mitochondrial morphology changed, the MMP decreased, and cellular apoptosis increased. Inhibition of MPC2 function by UK5099 or MPC2 knockdown by siRNA produced the same abnormal effects observed following treatment with HG. SIGNIFICANCE: MPC2 may mediate mitochondrial dysfunction in HG-treated podocytes, ultimately leading to cell apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Glucose/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Podócitos/patologia , Ácido Pirúvico/metabolismo , Células Cultivadas , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Podócitos/efeitos dos fármacos , Podócitos/metabolismo
9.
Yi Chuan ; 41(10): 893-904, 2019 Oct 20.
Artigo em Chinês | MEDLINE | ID: mdl-31624052

RESUMO

Mitochondrion is the metabolic center and powerhouse of cells producing cellular energy which plays an important role in various physiological and pathophysiological processes. Recent research demonstrates that mitochondrial energy metabolism mediates the transmission of mitochondrial-nuclear signals through intermediate products which regulates epigenetic presentation of the chromatin and thereby affects gene expression. Epigenetic modification, a genetic regulatory model, is independent of DNA sequence and plays a major role in establishing and maintaining a specific gene's expression profile. Disorders of mitochondrial metabolism can induce epigenetic reprogramming which in turn initiates aging phenotypes and degenerative diseases. This review introduces recent research progress on the relationship between mitochondrial metabolism and chromatin-related epigenetic modification, discusses the role of mitochondrial stress in chromatin recombination, and suggests future research directions and their application in the study of age-related diseases such as cognitive dysfunction.


Assuntos
Envelhecimento , Cromatina , Epigênese Genética , Mitocôndrias/metabolismo , Humanos
10.
Int J Nanomedicine ; 14: 7309-7322, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31571855

RESUMO

Introduction: The only treatment for aseptic loosening is the replacement of the prosthesis through revision surgery. A preventive approach, achieved through anti-inflammatory drugs released from the device, has shown to be a viable strategy; however, the performance of these devices is not yet satisfactory thus further improvements are necessary. Methods: We used titanium nanoparticles as a model for implant surfaces and developed a coating containing dexamethasone (DEX) using layer-by-layer deposition. Results: The amount of deposited drug depended on the number of layers and the release was sustained for months. The efficiency of the released DEX in reducing inflammation markers (tumor necrosis factor alpha and IL-6) produced by human monocytes and macrophages was similar to the pure drug at the same concentration without negative impacts on the viability and morphology of these cells. Conclusion: These coatings were not inferior to medical grade titanium (the standard material used in uncemented devices) regarding their ability to sustain osteoblasts and fibroblasts growth.


Assuntos
Anti-Inflamatórios/farmacologia , Cimentos para Ossos/farmacologia , Materiais Revestidos Biocompatíveis/farmacologia , Liberação Controlada de Fármacos , Nanopartículas/química , Falha de Prótese , Linhagem Celular , Forma Celular/efeitos dos fármacos , Dexametasona/farmacologia , Fibroblastos/efeitos dos fármacos , Humanos , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Monócitos/efeitos dos fármacos , Nanopartículas/ultraestrutura , Osteoblastos/efeitos dos fármacos , Osteoblastos/patologia , Tamanho da Partícula , Termogravimetria
11.
Int J Nanomedicine ; 14: 7173-7190, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31564872

RESUMO

Background: Nanotechnology proposes the use of gold nanoparticles (AuNPs) for drug delivery, diagnosis, and treatment of cancer. Leukemia is a type of hematopoietic cancer that results from the malignant transformation of white blood cells. Chitosan-coated AuNPs (CH-AuNPs) are cell death inductors in HeLa and MCF-7 cancer cells without affecting peripheral blood mononuclear cells (PBMC). Considering the selectivity and versatile cytotoxicity of CH-AuNPs, we evaluated whether their selectivity is due to the cell lineage or the characteristics of the cancer cells, by assessing its cytotoxicity in leukemic cells. Moreover, we further examined the cell death mechanism and assessed the implication of nuclear damage, autophagosome formation, and the cell death mechanism induced in leukemic cells. Materials and methods: We synthesized CH-AuNPs by chemical methods and analyzed their cell death capacity in a T-acute lymphocytic leukemia cell line (CEM), in a chronic myeloid leukemia cell line (K562), and in healthy cells from the same lineage (PBMC and bone marrow, BM, cells). Then, we assessed ROS generation and mitochondrial and nuclear damage. Finally, we evaluated whether cell death occurred by autophagy, apoptosis, or necroptosis, and the role of ROS in this mechanism. Results: We found that CH-AuNPs did not affect PBMC and BM cells, whereas they are cytotoxic in a dose-dependent manner in leukemic cells. ROS production leads to mitochondrial and nuclear damage, and cell death. We found that CH-AuNPs induce apoptosis in CEM and necroptosis in K562, both undergoing autophagy as a pro-survival mechanism. Conclusion: CH-AuNPs are selective cell death inductors in hematologic cancer cells, without affecting their healthy counterparts. Cell death induced by CH-AuNPs is independent of the cancer cell type; however, its mechanism is different depending on the type of leukemic cells.


Assuntos
Apoptose , Quitosana/química , Ouro/química , Leucemia/patologia , Nanopartículas Metálicas/química , Espécies Reativas de Oxigênio/metabolismo , Animais , Autofagossomos/metabolismo , Autofagia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Núcleo Celular/metabolismo , Sobrevivência Celular , Ativação Enzimática , Humanos , Leucemia/enzimologia , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Mitocôndrias/metabolismo , Necrose , Fosfatidilserinas/metabolismo
12.
Toxicol Lett ; 317: 92-101, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31593750

RESUMO

Cigarette smoke (CS) is known to cause mitochondrial dysfunction leading to cellular senescence in lung cells. We determined the mechanism of mitochondrial dysfunction by CS in lung epithelial cells. CS extract (CSE) treatment differentially affected mitochondrial function, such as membrane potential, mitochondrial reactive oxygen species (mtROS) and mitochrondrial mass as analyzed by FACS, and were associated with altered oxidative phosphorylation (OXPHOS) protein levels (Complexes I-IV) in primary lung epithelial cells (SAEC and NHBE), and (complexes I and II) in BEAS2B cells. There were dose- and time-dependent changes in mitochondrial respiration (oxygen consumption rate parameters i.e. maximal respiration, ATP production and spare capacity, measured by the Seahorse analyzer) in control vs. CSE treated BEAS2B and NHBE/DHBE cells. Electron microscopy (EM) analysis revealed perinuclear clustering by localization and increased mitochondrial fragmentation by fragement length analysis. Immunoblot analysis revealed CS-mediated increase in Drp1 and decrease in Mfn2 levels that are involved in mitochondrial fission/fusion process. CSE treatment reduced Miro1 and Pink1 abundance that play a crucial role in the intercellular transfer mechanism and mitophagy process. Overall, these findings highlight the role of Miro1 in context of CS-induced mitochondrial dysfunction in lung epithelial cells that may contribute to the pathogenesis of chronic inflammatory lung diseases.


Assuntos
Fumar Cigarros/efeitos adversos , Células Epiteliais/metabolismo , Pulmão/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Doença Pulmonar Obstrutiva Crônica/etiologia , Fumaça/efeitos adversos , Proteínas rho de Ligação ao GTP/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Regulação para Baixo , Metabolismo Energético , Células Epiteliais/ultraestrutura , Humanos , Pulmão/ultraestrutura , Mitocôndrias/ultraestrutura , Estresse Oxidativo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Transdução de Sinais
13.
Nat Neurosci ; 22(10): 1731-1742, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31501572

RESUMO

Mitochondria vary in morphology and function in different tissues; however, little is known about their molecular diversity among cell types. Here we engineered MitoTag mice, which express a Cre recombinase-dependent green fluorescent protein targeted to the outer mitochondrial membrane, and developed an isolation approach to profile tagged mitochondria from defined cell types. We determined the mitochondrial proteome of the three major cerebellar cell types (Purkinje cells, granule cells and astrocytes) and identified hundreds of mitochondrial proteins that are differentially regulated. Thus, we provide markers of cell-type-specific mitochondria for the healthy and diseased mouse and human central nervous systems, including in amyotrophic lateral sclerosis and Alzheimer's disease. Based on proteomic predictions, we demonstrate that astrocytic mitochondria metabolize long-chain fatty acids more efficiently than neuronal mitochondria. We also characterize cell-type differences in mitochondrial calcium buffering via the mitochondrial calcium uniporter (Mcu) and identify regulator of microtubule dynamics protein 3 (Rmdn3) as a determinant of endoplasmic reticulum-mitochondria proximity in Purkinje cells. Our approach enables exploring mitochondrial diversity in many in vivo contexts.


Assuntos
Encéfalo/citologia , Mitocôndrias/metabolismo , Neurônios/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Astrócitos/metabolismo , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Células Cultivadas , Cerebelo/citologia , Ácidos Graxos/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Membranas Mitocondriais/metabolismo , Proteômica , Células de Purkinje/metabolismo
15.
Cell Physiol Biochem ; 53(3): 550-572, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31529928

RESUMO

BACKGROUND/AIMS: Atherosclerosis underlies the majority of cardiovascular events, consequent to non-resolving inflammation. Considerable evidence implicates autophagy dysfunction at the core of this inflammatory condition, but the basis of this dysfunction is not fully understood. METHODS: Using an in vitro model of lipid-laden macrophages, activity-based probes and high-throughput techniques, we studied the role of the cysteine proteases cathepsins in autophagy. RESULTS: We showed that cathepsin activity is suppressed by oxidized lipids and that cathepsin has an indispensable role in the autophagy-lysosomal degradation pathway. Accordingly, loss of cathepsin function resulted in autophagy derangement. Shotgun proteomics confirmed autophagy dysfunction and unveiled a pivotal role of cathepsin L in a putative cathepsin degradation network. At the physiological level, cathepsin inhibition resulted in mitochondrial stress, which translated into impaired oxidative metabolism, excessive production of reactive oxygen species and activation of the cellular stress response, driven by ATF4-CHOP transcription factors. In addition, transcriptomic analysis of these cells uncovered some genetic similarities with the inflammatory macrophage phenotype (a.k.a M1 macrophages) and increased expression of inflammatory cytokines. CONCLUSION: Our data highlight the importance of cathepsins for mitochondrial quality control mechanisms and amelioration of vascular inflammation.


Assuntos
Anti-Inflamatórios/farmacologia , Catepsina B/metabolismo , Catepsina L/metabolismo , Catepsinas/metabolismo , Macrófagos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Células da Medula Óssea/citologia , Catepsina B/antagonistas & inibidores , Catepsina L/antagonistas & inibidores , Células Cultivadas , Colesterol/metabolismo , Humanos , Macrófagos/efeitos dos fármacos , Masculino , Espectrometria de Massas , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Estresse Oxidativo/efeitos dos fármacos , Proteômica/métodos , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo
16.
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
17.
Bratisl Lek Listy ; 120(9): 630-635, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31475544

RESUMO

OBJECTIVES: To test the hypothesis if mitochondrial bioenergetic function analyzed in circulating platelets may represent peripheral signature of mitochondrial dysfunction in nephropathy associated to non-communicable human diseases such as cardiovascular diseases, diabetes and with statins treatment. METHODS: High-resolution respirometry was used for analysis of mitochondrial bioenergetics in human platelets isolated from peripheral blood. This method is less-invasively compared to skeletal muscle biopsy. Patients with nephropathies and in combination with non-communicable diseases were included in the study. RESULTS: This pilot study showed platelet mitochondrial bioenergy dysfunction in patients with nephropathies and non-communicable diseases. Positive effect of treatment with 10 mg atorvastatin on platelet mitochondrial respiratory chain Complex I-linked respiration and ATP production in patients with nephropathies, diabetes and 80 mg atorvastatin in patient with nephropathy and dialysis was found. Positive effect of 80 mg fluvastatin treatment, and negative effect of thrombocytopenia and renal transplantation on platelet mitochondrial bioenergy was determined. CONCLUSION: High-resolution respirometry allowed detection of small changes in platelet mitochondrial function. This method could be used as a sensitive bioenergetic test of mitochondrial function for diagnosis and monitoring the therapy in patients with nephropathy (Tab. 1, Fig. 3, Ref. 39).


Assuntos
Plaquetas/metabolismo , Metabolismo Energético , Nefropatias/metabolismo , Mitocôndrias/metabolismo , Doenças não Transmissíveis , Respiração Celular , Humanos , Projetos Piloto
18.
Hypertension ; 74(4): 957-966, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31476900

RESUMO

Persistent pulmonary hypertension of the newborn (PPHN) is a failure of pulmonary vascular resistance to decline at birth rapidly. One principal mechanism implicated in PPHN development is mitochondrial oxidative stress. Expression and activity of mitochondrial SOD2 (superoxide dismutase) are decreased in PPHN; however, the mechanism remains unknown. Recently, OLA1 (Obg-like ATPase-1) was shown to act as a critical regulator of proteins controlling cell response to stress including Hsp70, an obligate chaperone for SOD2. Here, we investigated whether OLA1 is causally linked to PPHN. Compared with controls, SOD2 expression is reduced in distal-pulmonary arteries (PAs) from patients with PPHN and fetal-lamb models. Disruptions of the SOD2 gene reproduced PPHN phenotypes, manifested by elevated right ventricular systolic pressure, PA-endothelial cells apoptosis, and PA-smooth muscle cells proliferation. Analyses of SOD2 protein dynamics revealed higher ubiquitinated-SOD2 protein levels in PPHN-lambs, suggesting dysregulated protein ubiquitination. OLA1 controls multiple proteostatic mechanisms and is overexpressed in response to stress. We demonstrated that OLA1 acts as a molecular chaperone, and its activity is induced by stress. Strikingly, OLA1 expression is decreased in distal-PAs from PPHN-patients and fetal-lambs. OLA1 deficiency enhanced CHIP affinity for Hsp70-SOD2 complexes, facilitating SOD2 degradation. Consequently, mitochondrial H2O2 formation is impaired, leading to XIAP (X-linked inhibitor of apoptosis) overexpression that suppresses caspase activity in PA-smooth muscle cells, allowing them to survive and proliferate, contributing to PA remodeling. In-vivo, ola1-/- downregulated SOD2 expression, induced distal-PA remodeling, and right ventricular hypertrophy. We conclude that decreased OLA1 expression accounts for SOD2 downregulation and, therefore, a therapeutic target in PPHN treatments.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Pulmão/metabolismo , Síndrome da Persistência do Padrão de Circulação Fetal/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Superóxido Dismutase/metabolismo , Ubiquitina/metabolismo , Animais , Apoptose , Regulação para Baixo , Feminino , Hemodinâmica/fisiologia , Humanos , Peróxido de Hidrogênio/metabolismo , Recém-Nascido , Masculino , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Ovinos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
19.
Hum Genet ; 138(10): 1183-1200, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31471722

RESUMO

The glutamate pyruvate transaminase 2 (GPT2) gene produces a nuclear-encoded mitochondrial enzyme that catalyzes the reversible transfer of an amino group from glutamate to pyruvate, generating alanine and alpha-ketoglutarate. Recessive mutations in GPT2 have been recently identified in a new syndrome involving intellectual and developmental disability (IDD), postnatal microcephaly, and spastic paraplegia. We have identified additional families with recessive GPT2 mutations and expanded the phenotype to include small stature. GPT2 loss-of-function mutations were identified in four families, nine patients total, including: a homozygous mutation in one child [c.775T>C (p.C259R)]; compound heterozygous mutations in two siblings [c.812A>C (p.N271T)/c.1432_1433delGT (p.V478Rfs*73)]; a novel homozygous, putative splicing mutation [c.1035C>T (p.G345=)]; and finally, a recurrent mutation, previously identified in a distinct family [c.1210C>T (p.R404*)]. All patients were diagnosed with IDD. A majority of patients had remarkably small stature throughout development, many < 1st percentile for height and weight. Given the potential biological function of GPT2 in cellular growth, this phenotype is strongly suggestive of a newly identified clinical susceptibility. Further, homozygous GPT2 mutations manifested in at least 2 of 176 families with IDD (approximately 1.1%) in a Pakistani cohort, thereby representing a relatively common cause of recessive IDD in this population, with recurrence of the p.R404* mutation in this population. Based on variants in the ExAC database, we estimated that approximately 1 in 248 individuals are carriers of moderately or severely deleterious variants in GPT2.


Assuntos
Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/genética , Genes Recessivos , Predisposição Genética para Doença , Mutação , Fenótipo , Transaminases/genética , Adolescente , Alelos , Substituição de Aminoácidos , Deficiências do Desenvolvimento/metabolismo , Ativação Enzimática , Éxons , Feminino , Frequência do Gene , Estudos de Associação Genética , Genética Populacional , Genótipo , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Imagem por Ressonância Magnética , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Moleculares , Linhagem , Conformação Proteica , Sítios de Splice de RNA , Análise de Sequência de DNA , Relação Estrutura-Atividade , Transaminases/química , Transaminases/metabolismo
20.
Life Sci ; 235: 116810, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31472147

RESUMO

AIMS: Previous reports have demonstrated that melatonin exists in multiple extrapineal sites, and higher amounts of melatonin are present in human follicular fluid than in serum, which indicates that it might play key roles in human oocyte maturation and subsequent embryonic development. Melatonin has been shown to be a potent antioxidant and might be beneficial to human oocytes during in vitro maturation (IVM). However, the underlying mechanisms of melatonin action during IVM have not been thoroughly investigated. MAIN METHODS: Immunofluorescence staining, western blotting, and ELISA were applied to investigate whether melatoninergic components are expressed in the cultured human ovarian cumulus cells. TMRE staining and Fluo-4 AM staining were performed to detect the mitochondrial membrane potential and intracellular Ca2+ levels of immature human oocytes respectively. KEY FINDINGS: First, cultured human ovary cumulus cells synthesized melatonin in vitro, and it expressed serotonin (the precursor of melatonin) and the two key enzymes, i.e. N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). Additionally, the results suggest that melatonin maintains the mitochondrial membrane potential and decrease excessive Ca2+ levels in immature human oocytes during IVM. SIGNIFICANCE: In conclusion, we provide evidence that the melatoninergic components were expressed in cultured human ovarian cumulus cells, and melatonin might reduce oxidative stress of human oocytes by ameliorating mitochondrial function. In view of the significant clinical value that immature human oocytes have in assisted reproductive technology (ART), our findings highlight a potential treatment strategy of using melatonin to improve mitochondrial function and to enhance the quality of human oocytes during IVM.


Assuntos
Antioxidantes/farmacologia , Cálcio/metabolismo , Melatonina/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Antioxidantes/análise , Feminino , Humanos , Técnicas de Maturação in Vitro de Oócitos , Melatonina/análise , Mitocôndrias/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Estresse Oxidativo
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