RESUMO
Adenosine nucleotide translocases (ANTs) are a family of proteins abundant in the inner mitochondrial membrane, primarily responsible for shuttling ADP and ATP across the mitochondrial membrane. Additionally, ANTs are key players in balancing mitochondrial energy metabolism and regulating cell death. ANT2 isoform, highly expressed in undifferentiated and proliferating cells, is implicated in the development and drug resistance of various tumors. We conduct a detailed analysis of the potential mechanisms by which ANT2 may influence tumorigenesis and drug resistance. Notably, the significance of ANT2 extends beyond oncology, with roles in non-tumor cell processes including blood cell development, gastrointestinal motility, airway hydration, nonalcoholic fatty liver disease, obesity, chronic kidney disease, and myocardial development, making it a promising therapeutic target for multiple pathologies. To better understand the molecular mechanisms of ANT2, this review summarizes the structural properties, expression patterns, and basic functions of the ANT2 protein. In particular, we review and analyze the controversy surrounding ANT2, focusing on its role in transporting ADP/ATP across the inner mitochondrial membrane, its involvement in the composition of the mitochondrial permeability transition pore, and its participation in apoptosis.
Assuntos
Translocador 2 do Nucleotídeo Adenina , Humanos , Animais , Translocador 2 do Nucleotídeo Adenina/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Apoptose , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
Bidirectional transcription of mammalian mitochondrial DNA generates overlapping transcripts that are capable of forming double-stranded RNA (dsRNA) structures. Release of mitochondrial dsRNA into the cytosol activates the dsRNA-sensing immune signaling, which is a defense mechanism against microbial and viral attack and possibly cancer, but could cause autoimmune diseases when unchecked. A better understanding of the process is vital in therapeutic application of this defense mechanism and treatment of cognate human diseases. In addition to exporting dsRNAs, mitochondria also export and import a variety of non-coding RNAs. However, little is known about how these RNAs are transported across mitochondrial membranes. Here we provide direct evidence showing that adenine nucleotide translocase-2 (ANT2) functions as a mammalian RNA translocon in the mitochondrial inner membrane, independent of its ADP/ATP translocase activity. We also show that mitochondrial dsRNA efflux through ANT2 triggers innate immunity. Inhibiting this process alleviates inflammation in vivo, providing a potential therapeutic approach for treating autoimmune diseases.
Assuntos
Translocador 2 do Nucleotídeo Adenina , Mitocôndrias , Membranas Mitocondriais , RNA de Cadeia Dupla , Animais , Translocador 2 do Nucleotídeo Adenina/metabolismo , Translocador 2 do Nucleotídeo Adenina/genética , Humanos , RNA de Cadeia Dupla/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Camundongos , Imunidade Inata , Transporte de RNA , Células HEK293 , Camundongos Endogâmicos C57BLRESUMO
Successful completion of spermatogenesis is crucial for the perpetuation of the species. In Drosophila, spermatid individualization, a process involving changes in mitochondrial structure and function is critical to produce functional mature sperm. Ant2, encoding a mitochondrial adenine nucleotide translocase, is highly expressed in male testes and plays a role in energy metabolism in the mitochondria. However, its molecular function remains unclear. Here, we identified an important role of Ant2 in spermatid individualization. In Ant2 knockdown testes, spermatid individualization complexes composed of F-actin cones exhibited a diffuse distribution, and mature sperms were absent in the seminal vesicle, thus leading to male sterility. The most striking effects in Ant2-knockdown spermatids were decrease in tubulin polyglycylation and disruption of proper mitochondria derivatives function. Excessive apoptotic cells were also observed in Ant2-knockdown testes. To further investigate the phenotype of Ant2 knockdown in testes at the molecular level, complementary transcriptome and proteome analyses were performed. At the mRNA level, 868 differentially expressed genes were identified, of which 229 genes were upregulated and 639 were downregulated induced via Ant2 knockdown. iTRAQ-labeling proteome analysis revealed 350 differentially expressed proteins, of which 117 proteins were upregulated and 233 were downregulated. The expression of glutathione transferase (GstD5, GstE5, GstE8, and GstD3), proteins involved in reproduction were significantly regulated at both the mRNA and protein levels. These results indicate that Ant2 is crucial for spermatid maturation by affecting mitochondrial morphogenesis.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Espermatogênese , Animais , Masculino , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Testículo/metabolismo , Translocador 2 do Nucleotídeo Adenina/metabolismo , Translocador 2 do Nucleotídeo Adenina/genética , Espermátides/metabolismoRESUMO
Adenine Nucleotide Translocator isoforms (ANTs) exchange ADP/ATP across the inner mitochondrial membrane, are also voltage-activated proton channels and regulate mitophagy and apoptosis. The ANT1 isoform predominates in heart and muscle while ANT2 is systemic. Here, we report the creation of Ant mutant mouse myoblast cell lines with normal Ant1 and Ant2 genes, deficient in either Ant1 or Ant2, and deficient in both the Ant1 and Ant2 genes. These cell lines are immortal under permissive conditions (IFN-γ + serum at 32 °C) permitting expansion but return to normal myoblasts that can be differentiated into myotubes at 37 °C. With this system we were able to complement our Ant1 mutant studies by demonstrating that ANT2 is important for myoblast to myotube differentiation and myotube mitochondrial respiration. ANT2 is also important in the regulation of mitochondrial biogenesis and antioxidant defenses. ANT2 is also associated with increased oxidative stress response and modulation for Ca++ sequestration and activation of the mitochondrial permeability transition (mtPTP) pore during cell differentiation.
Assuntos
Translocador 2 do Nucleotídeo Adenina , Nucleotídeos de Adenina , Translocador 2 do Nucleotídeo Adenina/genética , Translocador 2 do Nucleotídeo Adenina/metabolismo , Nucleotídeos de Adenina/metabolismo , Animais , Camundongos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Desenvolvimento Muscular/genéticaRESUMO
Marine organisms are a rich source of bioactive secondary metabolites. Although many marine natural products with bioactivities have been isolated, successful elucidation of their mechanisms of action remains limited. In this study, we prepared a probe molecule based on the marine cyclic peptide kapakahine A (1) by introducing a linker with an azide terminal group, which enables the introduction of fluorescent groups for the effective monitoring of subcellular localization, or coupling to affinity beads for the pull-down of target proteins. The results of LC/MS/MS measurements, ProteinPilot analysis, and Western blotting suggest that kapakahine A interacts with the mitochondrial inner membrane proteins PHB1, PHB2, and ANT2, which is consistent with the results of the subcellular localization analysis using a fluorescent probe.
Assuntos
Translocador 2 do Nucleotídeo Adenina/metabolismo , Organismos Aquáticos/química , Corantes Fluorescentes/química , Peptídeos Cíclicos/farmacologia , Proibitinas/metabolismo , Animais , Linhagem Celular , Cromatografia Líquida , Camundongos , Estrutura Molecular , Peptídeos Cíclicos/química , Metabolismo Secundário , Espectrometria de Massas em TandemRESUMO
Macrophage proinflammatory activation is an important etiologic component of the development of insulin resistance and metabolic dysfunction in obesity. However, the underlying mechanisms are not clearly understood. Here, we demonstrate that a mitochondrial inner membrane protein, adenine nucleotide translocase 2 (ANT2), mediates proinflammatory activation of adipose tissue macrophages (ATMs) in obesity. Ant2 expression was increased in ATMs of obese mice compared with lean mice. Myeloid-specific ANT2-knockout (ANT2-MKO) mice showed decreased adipose tissue inflammation and improved insulin sensitivity and glucose tolerance in HFD/obesity. At the molecular level, we found that ANT2 mediates free fatty acid-induced mitochondrial permeability transition, leading to increased mitochondrial reactive oxygen species production and damage. In turn, this increased HIF-1α expression and NF-κB activation, leading to proinflammatory macrophage activation. Our results provide a previously unknown mechanism for how obesity induces proinflammatory activation of macrophages with propagation of low-grade chronic inflammation (metaflammation).
Assuntos
Translocador 2 do Nucleotídeo Adenina/metabolismo , Inflamação/genética , Ativação de Macrófagos/genética , Obesidade/genética , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , CamundongosRESUMO
Long-term studies have shown that virus infection affects the energy metabolism of host cells, which mainly affects the function of mitochondria and leads to the hydrolysis of ATP in host cells, but it is not clear how virus infection participates in mitochondrial energy metabolism in host cells. In our study, HUVEC cells were infected with HSV-1, and the differentially expressed genes were obtained by microarray analysis and data analysis. The viral gene encoding protein UL16 was identified to interact with host protein ANT2 by immunoprecipitation and mass spectrometry. We also reported that UL16 transfection promoted oxidative phosphorylation of glucose and significantly increased intracellular ATP content. Furthermore, UL16 was transfected into the HUVEC cell model with mitochondrial dysfunction induced by D-Gal, and it was found that UL16 could restore the mitochondrial function of cells. It was first discovered that viral protein UL16 could enhance mitochondrial function in mammalian cells by promoting mitochondrial metabolism. This study provides a theoretical basis for the prevention and treatment of mitochondrial dysfunction or the pathological process related to mitochondrial dysfunction.
Assuntos
Translocador 2 do Nucleotídeo Adenina/metabolismo , Metabolismo Energético , Herpes Simples/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Mitocôndrias/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Biomarcadores , Linhagem Celular , Perfilação da Expressão Gênica , Herpes Simples/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Potencial da Membrana Mitocondrial , Oxirredução , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismoRESUMO
Following a prolonged exposure to hypoxia-reoxygenation, a partial disruption of the ER-mitochondria tethering by mitofusin 2 (MFN2) knock-down decreases the Ca2+ transfer between the two organelles limits mitochondrial Ca2+ overload and prevents the Ca2+-dependent opening of the mitochondrial permeability transition pore, i.e., limits cardiomyocyte cell death. The impact of the metabolic changes resulting from the alteration of this Ca2+crosstalk on the tolerance to hypoxia-reoxygenation injury remains partial and fragmented between different field of expertise. >In this study, we report that MFN2 loss of function results in a metabolic switch driven by major modifications in energy production by mitochondria. During hypoxia, mitochondria maintain their ATP concentration and, concomitantly, the inner membrane potential by importing cytosolic ATP into mitochondria through an overexpressed ANT2 protein and by decreasing the expression and activity of the ATP hydrolase via IF1. This adaptation further blunts the detrimental hyperpolarisation of the inner mitochondrial membrane (IMM) upon re-oxygenation. These metabolic changes play an important role to attenuate cell death during a prolonged hypoxia-reoxygenation challenge.
Assuntos
Translocador 2 do Nucleotídeo Adenina/metabolismo , Trifosfato de Adenosina/metabolismo , Hipóxia/metabolismo , Mitocôndrias/metabolismo , Animais , Cálcio/metabolismo , Morte Celular/fisiologia , Linhagem Celular , Potencial da Membrana Mitocondrial/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , RatosRESUMO
Duchenne muscular dystrophy (DMD) is a severe hereditary disease caused by a lack of dystrophin, a protein essential for myocyte integrity. Mitochondrial dysfunction is reportedly responsible for DMD. This study examines the effect of glucocorticoid deflazacort on the functioning of the skeletal-muscle mitochondria of dystrophin-deficient mdx mice and WT animals. Deflazacort administration was found to improve mitochondrial respiration of mdx mice due to an increase in the level of ETC complexes (complexes III and IV and ATP synthase), which may contribute to the normalization of ATP levels in the skeletal muscle of mdx animals. Deflazacort treatment improved the rate of Ca2+ uniport in the skeletal muscle mitochondria of mdx mice, presumably by affecting the subunit composition of the calcium uniporter of organelles. At the same time, deflazacort was found to reduce the resistance of skeletal mitochondria to MPT pore opening, which may be associated with a change in the level of ANT2 and CypD. In this case, deflazacort also affected the mitochondria of WT mice. The paper discusses the mechanisms underlying the effect of deflazacort on the functioning of mitochondria and contributing to the improvement of the muscular function of mdx mice.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Mitocôndrias Musculares/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Distrofia Muscular de Duchenne/tratamento farmacológico , Pregnenodionas/farmacologia , Translocador 2 do Nucleotídeo Adenina/genética , Translocador 2 do Nucleotídeo Adenina/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Peptidil-Prolil Isomerase F/genética , Peptidil-Prolil Isomerase F/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologiaRESUMO
Tilapia piscidin (TP) 4 is an antimicrobial peptide derived from Nile tilapia (Oreochromis niloticus), which shows broad-spectrum antibacterial activity and excellent cancer-killing ability in vitro and in vivo. Like many other antimicrobial peptides, TP4 treatment causes mitochondrial toxicity in cancer cells. However, the molecular mechanisms underlying TP4 targeting of mitochondria remain unclear. In this study, we used a pull-down assay on A549 cell lysates combined with LC-MS/MS to discover that TP4 targets adenine nucleotide translocator (ANT) 2, a protein essential for adenine nucleotide exchange across the inner membrane. We further showed that TP4 accumulates in mitochondria and colocalizes with ANT2. Moreover, molecular docking studies showed that the interaction requires Phe1, Ile2, His3, His4, Ser11, Lys14, His17, Arg21, Arg24 and Arg25 residues in TP4 and key residues within the cavity of ANT2. These findings suggest a mechanism by which TP4 may induce mitochondrial dysfunction to disrupt cellular energy metabolism.
Assuntos
Translocador 2 do Nucleotídeo Adenina/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , Ciclídeos/metabolismo , Proteínas de Peixes/farmacologia , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Células A549 , Translocador 2 do Nucleotídeo Adenina/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/metabolismo , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Metabolismo Energético/efeitos dos fármacos , Proteínas de Peixes/isolamento & purificação , Proteínas de Peixes/metabolismo , Humanos , Células MCF-7 , Microscopia Confocal , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Simulação de Acoplamento Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Ligação ProteicaRESUMO
BACKGROUND: The aberrant expression of genes involved in androgen metabolism and genetic contribution are unclear in hypospadias. METHODS: We compared gene expression profiles by RNA sequencing from five non-hypospadiac foreskins, five mild hypospadiac foreskins, and five severe hypospadiac foreskins. In addition, to identify rare coding variants with large effects on hypospadias risk, we carried out whole exome sequencing in three patients in a hypospadias family. RESULTS: The average expression of androgen receptor (AR) and CYP19A1 were significantly decreased in severe hypospadias (p < .01) and mild hypospadias (p < .05), whereas expression of several other androgen metabolism enzymes, including CYP3A4, HSD17B14, HSD3B7, HSD17B7, CYP11A1 were exclusively significantly expressed in severe hypospadias (p < .05). Compound rare damaging mutants of AR gene with HSD3B1 and SLC25A5 genes were identified in the different severe hypospadias. CONCLUSIONS: In conclusion, our findings demonstrated that dysregulation of AR and CYP19A1 could play a crucial role in the development of hypospadias. Inconsistent AR expression may be caused by the feedback loop of ESR1 signaling or combined genetic effects with other risk genes. This findings complement the possible role of AR triggered mechanism in the development of hypospadias.
Assuntos
Androgênios/genética , Hipospadia/genética , Mutação , Transcriptoma , 17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/metabolismo , Translocador 2 do Nucleotídeo Adenina/genética , Translocador 2 do Nucleotídeo Adenina/metabolismo , Androgênios/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Criança , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Exoma , Humanos , Hipospadia/patologia , Masculino , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Progesterona Redutase/genética , Progesterona Redutase/metabolismo , Proteína A6 Ligante de Cálcio S100/genética , Proteína A6 Ligante de Cálcio S100/metabolismo , Esteroide Isomerases/genética , Esteroide Isomerases/metabolismoRESUMO
Rationale: P21-activated kinase 6 (PAK6) is a member of the class II PAKs family, which is a conserved family of serine/threonine kinases. Although the effects of PAK6 on many malignancies, especially in prostate cancer, have been studied for a long time, the role of PAK6 in mitochondria remains unknown. Methods: The expression of PAK6, SIRT4 and ANT2 in prostate cancer and adjacent non-tumor tissues was detected by immunohistochemistry. Immunofuorescence and immunoelectron microscopy were used to determine the subcellular localization of PAK6. Immunoprecipitation, immunofuorescence and ubiquitination assays were performed to determine how PAK6 regulates SIRT4, how SIRT4 regulates ANT2, and how PAK6 regulates ANT2. Flow cytometry detection and xenograft models were used to evaluate the impact of ANT2 mutant expression on the prostate cancer cell cycle and apoptosis regulation. Results: The present study revealed that the PAK6-SIRT4-ANT2 complex is involved in mitochondrial apoptosis in prostate cancer cells. It was found that PAK6 is mainly located in the mitochondrial inner membrane, in which PAK6 promotes SIRT4 ubiquitin-mediated proteolysis. Furthermore, SIRT4 deprives the ANT2 acetylation at K105 to promote its ubiquitination degradation. Hence, PAK6 adjusts the acetylation level of ANT2 through the PAK6-SIRT4-ANT2 pathway, in order to regulate the stability of ANT2. Meanwhile, PAK6 directly phosphorylates ANT2 atT107 to inhibit the apoptosis of prostate cancer cells. Therefore, the phosphorylation and deacetylation modifications of ANT2 are mutually regulated, leading to tumor growth in vivo. Consistently, these clinical prostate cancer tissue evaluations reveal that PAK6 is positively correlated with ANT2 expression, but negatively correlated with SIRT4. Conclusion: These present findings suggest the pivotal role of the PAK6-SIRT4-ANT2 complex in the apoptosis of prostate cancer. This complex could be a potential biomarker for the treatment and prognosis of prostate cancer.
Assuntos
Translocador 2 do Nucleotídeo Adenina/metabolismo , Adenocarcinoma/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neoplasias da Próstata/metabolismo , Sirtuínas/metabolismo , Quinases Ativadas por p21/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Células PC-3RESUMO
Duchenne muscular dystrophy (DMD) is characterized by a pronounced and progressive degradation of the structure of skeletal muscles, which decreases their strength and lowers endurance of the organism. At muscular dystrophy, mitochondria are known to undergo significant functional changes, which is manifested in a decreased efficiency of oxidative phosphorylation and impaired energy metabolism of the cell. It is believed that the DMD-induced functional changes of mitochondria are mainly associated with the dysregulation of Ca2+ homeostasis. This work examines the kinetic parameters of Ca2+ transport and the opening of the Ca2+-dependent MPT pore in the skeletal-muscle mitochondria of the dystrophin-deficient C57BL/10ScSn-mdx mice. As compared to the organelles of wild-type animals, skeletal-muscle mitochondria of mdx mice have been found to be much less efficient in respect to Ca2+ uniport, with the kinetics of Na+-dependent Ca2+ efflux not changing. The data obtained indicate that the decreased rate of Ca2+ uniport in the mitochondria of mdx mice may be associated with the increased level of the dominant negative subunit of Ca2+ uniporter (MCUb). The experiments have also shown that in mdx mice, skeletal-muscle mitochondria have low resistance to the induction of MPT, which may be related to a significantly increased expression of adenylate translocator (ANT2), a possible structural element of the MPT pore. The paper discusses how changes in the expression of calcium uniporter and putative components of the MPT pore caused by the development of DMD can affect Ca2+ homeostasis of skeletal-muscle mitochondria.
Assuntos
Cálcio/metabolismo , Mitocôndrias Musculares/patologia , Necrose Dirigida por Permeabilidade Transmembrânica da Mitocôndria/genética , Distrofia Muscular de Duchenne/patologia , Translocador 2 do Nucleotídeo Adenina/genética , Translocador 2 do Nucleotídeo Adenina/metabolismo , Animais , Cátions Bivalentes/metabolismo , Modelos Animais de Doenças , Distrofina/genética , Distrofina/metabolismo , Humanos , Transporte de Íons/genética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos mdx , Microscopia Eletrônica , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Distrofia Muscular de Duchenne/genética , Fosforilação OxidativaRESUMO
In addition to skeletal muscle dysfunction, cancer cachexia is a systemic disease involving remodeling of nonmuscle organs such as adipose and liver. Impairment of mitochondrial function is associated with multiple chronic diseases. The tissue-specific control of mitochondrial function in cancer cachexia is not well defined. This study determined mitochondrial respiratory capacity and coupling control of skeletal muscle, white adipose tissue (WAT), and liver in colon-26 (C26) tumor-induced cachexia. Tissues were collected from PBS-injected weight-stable mice, C26 weight-stable mice and C26 mice with moderate (10% weight loss) and severe cachexia (20% weight loss). The respiratory control ratio [(RCR) an index of oxidative phosphorylation (OXPHOS) coupling efficiency] was low in WAT during the induction of cachexia because of high nonphosphorylating LEAK respiration. Liver RCR was low in C26 weight-stable and moderately cachexic mice because of reduced OXPHOS. Liver RCR was further reduced with severe cachexia, where Ant2 but not Ucp2 expression was increased. Ant2 was inversely correlated with RCR in the liver (r = -0.547, P < 0.01). Liver cardiolipin increased in moderate and severe cachexia, suggesting this early event may also contribute to mitochondrial uncoupling. Impaired skeletal muscle mitochondrial respiration occurred predominantly in severe cachexia, at complex I. These findings suggest that mitochondrial function is subject to tissue-specific control during cancer cachexia, whereby remodeling in WAT and liver arise early and may contribute to altered energy balance, followed by impaired skeletal muscle respiration. We highlight an under-recognized role of liver and WAT mitochondrial function in cancer cachexia and suggest mitochondrial function of multiple tissues to be therapeutic targets.
Assuntos
Caquexia/metabolismo , Mitocôndrias Musculares/metabolismo , Neoplasias Experimentais/metabolismo , Consumo de Oxigênio/fisiologia , Translocador 2 do Nucleotídeo Adenina/genética , Translocador 2 do Nucleotídeo Adenina/metabolismo , Animais , Cardiolipinas/metabolismo , Neoplasias do Colo , Fígado/metabolismo , Masculino , Camundongos , Músculo Esquelético/metabolismo , Acoplamento Oxidativo , Distribuição Aleatória , Espécies Reativas de Oxigênio , Redução de PesoRESUMO
The nucleosome remodelling and deacetylase complex (NuRD) is a widely conserved regulator of gene expression. The determination of the subunit composition of the complex and identification of its binding partners are important steps towards understanding its architecture and function. The question of how these properties of the complex vary across different cell types has not been addressed in detail to date. Here, we set up a two-step purification protocol coupled to liquid chromatography-tandem mass spectrometry to assess NuRD composition and interaction partners in three different cancer cell lines, using label-free intensity-based absolute quantification (iBAQ). Our data indicate that the stoichiometry of the NuRD complex is preserved across our three different cancer cell lines. In addition, our interactome data suggest ZNF219 and SLC25A5 as possible interaction partners of the complex. To corroborate this latter finding, in vitro and cell-based pull-down experiments were carried out. These experiments indicated that ZNF219 can interact with RBBP4, GATAD2A/B and chromodomain helicase DNA binding 4, whereas SLC25A5 might interact with MTA2 and GATAD2A.
Assuntos
Linhagem Celular Tumoral/química , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Proteínas de Neoplasias/metabolismo , Mapas de Interação de Proteínas , Translocador 2 do Nucleotídeo Adenina/metabolismo , Centrifugação com Gradiente de Concentração , Cromatografia de Afinidade , Cromatografia Líquida , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/química , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Mapeamento de Interação de Proteínas/métodos , Subunidades Proteicas , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em Tandem , Dedos de ZincoAssuntos
Translocador 2 do Nucleotídeo Adenina/metabolismo , Tecido Adiposo/metabolismo , Hipóxia/metabolismo , Inflamação/metabolismo , Obesidade/metabolismo , Adipócitos/citologia , Adipócitos/fisiologia , Tecido Adiposo/patologia , Aminoácidos/fisiologia , Animais , Biomarcadores/metabolismo , Cromo/fisiologia , Modelos Animais de Doenças , Humanos , Hipóxia/prevenção & controle , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/prevenção & controle , Resistência à Insulina/fisiologia , Camundongos , Camundongos Knockout , Ácidos Nicotínicos/fisiologia , Obesidade/fisiopatologia , Consumo de Oxigênio/fisiologia , Sensibilidade e EspecificidadeRESUMO
Cellular senescence is a form of cell cycle arrest that limits the proliferative potential of cells, including tumour cells. However, inability of immune cells to subsequently eliminate senescent cells from the organism may lead to tissue damage, inflammation, enhanced carcinogenesis and development of age-related diseases. We found that the anticancer agent mitochondria-targeted tamoxifen (MitoTam), unlike conventional anticancer agents, kills cancer cells without inducing senescence in vitro and in vivo. Surprisingly, it also selectively eliminates both malignant and non-cancerous senescent cells. In naturally aged mice treated with MitoTam for 4 weeks, we observed a significant decrease of senescence markers in all tested organs compared to non-treated animals. Mechanistically, we found that the susceptibility of senescent cells to MitoTam is linked to a very low expression level of adenine nucleotide translocase-2 (ANT2), inherent to the senescent phenotype. Restoration of ANT2 in senescent cells resulted in resistance to MitoTam, while its downregulation in non-senescent cells promoted their MitoTam-triggered elimination. Our study documents a novel, translationally intriguing role for an anticancer agent targeting mitochondria, that may result in a new strategy for the treatment of age-related diseases and senescence-associated pathologies.
Assuntos
Translocador 2 do Nucleotídeo Adenina/metabolismo , Antineoplásicos Hormonais/farmacologia , Senescência Celular/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Tamoxifeno/farmacologia , Translocador 2 do Nucleotídeo Adenina/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Técnicas de Silenciamento de Genes , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Mitocôndrias/metabolismo , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Death associated protein kinase (DAPK)-related apoptosis-inducing protein kinase (DRAK)-1 is a positive apoptosis regulator. However, the molecular mechanisms underlying the DRAK1-mediated apoptotic pathway remain unclear. In this study, we demonstrated the intracellular localization and binding partners of DRAK1. In human osteosarcoma cell line U2OS cells, DRAK1 was mainly localized in the nucleus and translocated outside the nucleus through Ser395 phosphorylation by protein kinase C. In the nucleus, DRAK1 associated with tumor suppressor p53 and positively regulated p53 transcriptional activity in response to DNA-damaging agent cisplatin. On the other hand, DRAK1 interacted with the mitochondrial inner-membrane protein, adenine nucleotide translocase (ANT)-2, an anti-apoptotic oncoprotein, outside the nucleus. These findings suggest that DRAK1 translocates in response to stimuli and induces apoptosis through its interaction with specific binding partners, p53 and/or ANT2.
Assuntos
Translocador 2 do Nucleotídeo Adenina/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Frações Subcelulares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Humanos , Ligação Proteica , Proteínas Serina-Treonina Quinases , Distribuição TecidualRESUMO
Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disorder in obese individuals. Adenine nucleotide translocase (ANT) exchanges ADP/ATP through the mitochondrial inner membrane, and Ant2 is the predominant isoform expressed in the liver. Here we demonstrate that targeted disruption of Ant2 in mouse liver enhances uncoupled respiration without damaging mitochondrial integrity and liver functions. Interestingly, liver specific Ant2 knockout mice are leaner and resistant to hepatic steatosis, obesity and insulin resistance under a lipogenic diet. Protection against fatty liver is partially recapitulated by the systemic administration of low-dose carboxyatractyloside, a specific inhibitor of ANT. Targeted manipulation of hepatic mitochondrial metabolism, particularly through inhibition of ANT, may represent an alternative approach in NAFLD and obesity treatment.