RESUMO
The oxidative phosphorylation (OXPHOS) system is a dynamic system in which the respiratory complexes coexist with super-assembled quaternary structures called supercomplexes (SCs). The physiological role of SCs is still disputed. Here, we used zebrafish to study the relevance of respiratory SCs. We combined immunodetection analysis and deep data-independent proteomics to characterize these structures and found similar SCs to those described in mice, as well as novel SCs including III2 + IV2 , I + IV, and I + III2 + IV2 . To study the physiological role of SCs, we generated two null allele zebrafish lines for supercomplex assembly factor 1 (scaf1). scaf1-/- fish displayed altered OXPHOS activity due to the disrupted interaction of complexes III and IV. scaf1-/- fish were smaller in size and showed abnormal fat deposition and decreased female fertility. These physiological phenotypes were rescued by doubling the food supply, which correlated with improved bioenergetics and alterations in the metabolic gene expression program. These results reveal that SC assembly by Scaf1 modulates OXPHOS efficiency and allows the optimization of metabolic resources.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Fatores de Processamento de Serina-Arginina/metabolismo , Peixe-Zebra , Animais , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético/genética , Feminino , Camundongos , Membranas Mitocondriais/metabolismo , Fosforilação Oxidativa , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Chemotherapeutic drugs kill cancer cells, but it is unclear why this happens in responding patients but not in non-responders. Proteomic profiles of patients with oesophageal adenocarcinoma may be helpful in predicting response and selecting more effective treatment strategies. In this study, pretherapeutic oesophageal adenocarcinoma biopsies were analysed for proteomic changes associated with response to chemotherapy by MALDI imaging mass spectrometry. Resulting candidate proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and investigated for functional relevance in vitro. Clinical impact was validated in pretherapeutic biopsies from an independent patient cohort. Studies on the incidence of these defects in other solid tumours were included. We discovered that clinical response to cisplatin correlated with pre-existing defects in the mitochondrial respiratory chain complexes of cancer cells, caused by loss of specific cytochrome c oxidase (COX) subunits. Knockdown of a COX protein altered chemosensitivity in vitro, increasing the propensity of cancer cells to undergo cell death following cisplatin treatment. In an independent validation, patients with reduced COX protein expression prior to treatment exhibited favourable clinical outcomes to chemotherapy, whereas tumours with unchanged COX expression were chemoresistant. In conclusion, previously undiscovered pre-existing defects in mitochondrial respiratory complexes cause cancer cells to become chemosensitive: mitochondrial defects lower the cells' threshold for undergoing cell death in response to cisplatin. By contrast, cancer cells with intact mitochondrial respiratory complexes are chemoresistant and have a high threshold for cisplatin-induced cell death. This connection between mitochondrial respiration and chemosensitivity is relevant to anticancer therapeutics that target the mitochondrial electron transport chain.
Assuntos
Adenocarcinoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Neoplasias Esofágicas/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Idoso , Biomarcadores Tumorais/genética , Biópsia , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Cromatografia Líquida , Cisplatino/administração & dosagem , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Complexo IV da Cadeia de Transporte de Elétrons/genética , Neoplasias Esofágicas/enzimologia , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Fluoruracila/administração & dosagem , Humanos , Pessoa de Meia-Idade , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Terapia Neoadjuvante , Medicina de Precisão , Proteômica/métodos , Interferência de RNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Transfecção , Resultado do TratamentoRESUMO
The oxidative phosphorylation (OXPHOS) system is intricately organized, with respiratory complexes forming super-assembled quaternary structures whose assembly mechanisms and physiological roles remain under investigation. Cox7a2l, also known as Scaf1, facilitates complex III and complex IV (CIII-CIV) super-assembly, enhancing energetic efficiency in various species. We examined the role of Cox7a1, another Cox7a family member, in supercomplex assembly and muscle physiology. Zebrafish lacking Cox7a1 exhibited reduced CIV2 formation, metabolic alterations, and non-pathological muscle performance decline. Additionally, cox7a1-/- hearts displayed a pro-regenerative metabolic profile, impacting cardiac regenerative response. The distinct phenotypic effects of cox7a1-/- and cox7a2l-/- underscore the diverse metabolic and physiological consequences of impaired supercomplex formation, emphasizing the significance of Cox7a1 in muscle maturation within the OXPHOS system.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Coração , Músculo Esquelético , Fosforilação Oxidativa , Regeneração , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Músculo Esquelético/metabolismo , Regeneração/fisiologia , Coração/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Miocárdio/metabolismo , Multimerização ProteicaRESUMO
Rationale: Mitochondria generate ATP via the oxidative phosphorylation system, which mainly comprises five respiratory complexes found in the inner mitochondrial membrane. A high-order assembly of respiratory complexes is called a supercomplex. COX7A2L is a supercomplex assembly factor that has been well-investigated for studying supercomplex function and assembly. To date, the effects of mitochondrial supercomplexes on cell metabolism have not been elucidated. Methods: We depleted COX7A2L or Cox7a2l in human and mouse cells to generate cell models lacking mitochondrial supercomplexes as well as in DBA/2J mice as animal models. We tested the effect of impaired supercomplex assembly on cell proliferation with different nutrient supply. We profiled the metabolic features in COX7A2L-/- cells and Cox7a2l-/- mice via the combined use of targeted and untargeted metabolic profiling and metabolic flux analysis. We further tested the role of mitochondrial supercomplexes in pancreatic ductal adenocarcinoma (PDAC) through PDAC cell lines and a nude mouse model. Results: Impairing mitochondrial supercomplex assembly by depleting COX7A2L in human cells reprogrammed metabolic pathways toward anabolism and increased glutamine metabolism, cell proliferation and antioxidative defense. Similarly, knockout of Cox7a2l in DBA/2J mice promoted the use of proteins/amino acids as oxidative carbon sources. Mechanistically, impaired supercomplex assembly increased electron flux from CII to CIII/CIV and promoted CII-dependent respiration in COX7A2L-/- cells which further upregulated glutaminolysis and glutamine oxidation to accelerate the reactions of the tricarboxylic acid cycle. Moreover, the proliferation of PDAC cells lacking COX7A2L was inhibited by glutamine deprivation. Conclusion: Our results reveal the regulatory role of mitochondrial supercomplexes in glutaminolysis which may fine-tune the fate of cells with different nutrient availability.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Glutamina , Camundongos , Humanos , Animais , Glutamina/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Camundongos Endogâmicos DBA , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Mamíferos/metabolismoRESUMO
Lung cancer is the most common in incidence and mortality worldwide. With the development of next generation sequencing (NGS) detection technology, more and more patients with rare anaplastic lymphoma kinase (ALK) fusion mutations were detected. A case of advanced lung adenocarcinoma with rare COX7A2L-ALK (C2:A20) fusion detected by NGS was reported in Peking Union Medical College Hospital, and all cases with rare ALK fusion mutations were searched from medical datebase from January 1, 2014 to March 31, 2021, to investigate the treatment of rare ALK fusion mutations with ALK inhibitors. The best response of the patient was assessed as partial response (PR) with Ceritinib treatment. By literature review, 22 cases of rare ALK fusion were reported in 19 articles. Combined with this case, 23 cases were analyzed. The objective response rate (ORR) was 82.6% (19/23) and disease control rate (DCR) was 95.7% (22/23) for rare ALK fusions patients treated with ALK inhibitors. Lung adenocarcinoma patients with rare ALK fusion could benefit from ALK inhibitors.â©.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Humanos , Quinase do Linfoma Anaplásico/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/diagnóstico , Crizotinibe , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Fusão Oncogênica/genéticaRESUMO
The structural and functional organization of the mitochondrial respiratory chain (MRC) remains intensely debated. Here, we show the co-existence of two separate MRC organizations in human cells and postmitotic tissues, C-MRC and S-MRC, defined by the preferential expression of three COX7A subunit isoforms, COX7A1/2 and SCAFI (COX7A2L). COX7A isoforms promote the functional reorganization of distinct co-existing MRC structures to prevent metabolic exhaustion and MRC deficiency. Notably, prevalence of each MRC organization is reversibly regulated by the activation state of the pyruvate dehydrogenase complex (PDC). Under oxidative conditions, the C-MRC is bioenergetically more efficient, whereas the S-MRC preferentially maintains oxidative phosphorylation (OXPHOS) upon metabolic rewiring toward glycolysis. We show a link between the metabolic signatures converging at the PDC and the structural and functional organization of the MRC, challenging the widespread notion of the MRC as a single functional unit and concluding that its structural heterogeneity warrants optimal adaptation to metabolic function.
Assuntos
Glicólise , Fosforilação Oxidativa , Humanos , Transporte de Elétrons , Membranas Mitocondriais/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Isoformas de Proteínas/metabolismoRESUMO
The study of the mitochondrial respiratory chain (MRC) function in relation with its structural organization is of great interest due to the central role of this system in eukaryotic cell metabolism. The complexome profiling technique has provided invaluable information for our understanding of the composition and assembly of the individual MRC complexes, and also of their association into larger supercomplexes (SCs) and respirasomes. The formation of the SCs has been highly debated, and their assembly and regulation mechanisms are still unclear. Previous studies demonstrated a prominent role for COX7A2L (SCAFI) as a structural protein bridging the association of individual MRC complexes III and IV in the minor SC III2 + IV, although its relevance for respirasome formation and function remains controversial. In this work, we have used SILAC-based complexome profiling to dissect the structural organization of the human MRC in HEK293T cells depleted of SCAFI (SCAFIKO) by CRISPR-Cas9 genome editing. SCAFI ablation led to a preferential loss of SC III2 + IV and of a minor subset of respirasomes without affecting OXPHOS function. Our data suggest that the loss of SCAFI-dependent respirasomes in SCAFIKO cells is mainly due to alterations on early stages of CI assembly, without impacting the biogenesis of complexes III and IV. Contrary to the idea of SCAFI being the main player in respirasome formation, SILAC-complexome profiling showed that, in wild-type cells, the majority of respirasomes (ca. 70%) contained COX7A2 and that these species were present at roughly the same levels when SCAFI was knocked-out. We thus demonstrate the co-existence of structurally distinct respirasomes defined by the preferential binding of complex IV via COX7A2, rather than SCAFI, in human cultured cells.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Marcação por Isótopo/métodos , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Sistemas CRISPR-Cas , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/genética , Células HEK293 , Humanos , Espectrometria de MassasRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive fibrosis and hypovascularization, resulting in significant intratumoral hypoxia (low oxygen) that contributes to its aggressiveness, therapeutic resistance, and high mortality. Despite oxygen being a fundamental requirement for many cellular and metabolic processes, and the severity of hypoxia in PDAC, the impact of oxygen deprivation on PDAC biology is poorly understood. Investigating how PDAC cells survive in the near absence of oxygen, we find that PDAC cell lines grow robustly in oxygen tensions down to 0.1%, maintaining mitochondrial morphology, membrane potential, and the oxidative metabolic activity required for the synthesis of key metabolites for proliferation. Disrupting electron transfer efficiency by targeting mitochondrial respiratory supercomplex assembly specifically affects hypoxic PDAC proliferation, metabolism, and in vivo tumor growth. Collectively, our results identify a mechanism that enables PDAC cells to thrive in severe, oxygen-limited microenvironments.
Assuntos
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Respiração Celular/fisiologia , Mitocôndrias/metabolismo , Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/patologia , Hipóxia Celular , HumanosRESUMO
Cumulative evidence has indicated that mitochondrial respiration dysfunction plays important roles in tumorigenesis. However, the role of COX7RP, a critical regulator in the formation of mitochondrial respiratory supercomplex that has been suggested to be over-expressed in hepatocellular carcinoma (HCC) by our bioinformatic analysis of TCGA data, in tumor progression remains largely unclear. In this study, we found that COX7RP is frequently over-expressed in HCC mainly due to the down-regulation of miR-130a-3p and predicts poor prognosis of HCC patients. Functional experiments revealed that COX7RP promoted both growth and metastasis of HCC through induction of cell cycle progression and epithelial to mesenchymal transition (EMT), and suppression of cell apoptosis. Mechanistically, increased generation of reactive oxygen species (ROS) and subsequently activated nuclear transcription factor-κB (NF-κB) signaling was found to contribute to the promotion of HCC cell growth and metastasis by COX7RP. Collectively, COX7RP plays a critical oncogenic role in hepatocellular carcinogenesis, supporting COX7RP as a novel prognostic factor and therapeutic target in HCC.
RESUMO
The mitochondrial respiratory chain enzymes are organized as individual complexes and supercomplexes, whose biogenesis remains to be fully understood. To disclose the role of the human Hypoxia Inducible Gene Domain family proteins HIGD1A and HIGD2A in these processes, we generate and characterize HIGD-knockout (KO) cell lines. We show that HIGD2A controls and coordinates the modular assembly of isolated and supercomplexed complex IV (CIV) by acting on the COX3 assembly module. In contrast, HIGD1A regulates CIII and CIII-containing supercomplex biogenesis by supporting the incorporation of UQCRFS1. HIGD1A also clusters with COX4-1 and COX5A CIV subunits and, when overexpressed, suppresses the CIV biogenesis defect of HIGD2A-KO cells. We conclude that HIGD1A and HIGD2A have both independent and overlapping functions in the biogenesis of respiratory complexes and supercomplexes. Our data illuminate the existence of multiple pathways to assemble these structures by dynamic HIGD-mediated CIV biogenesis, potentially to adapt to changing environmental and nutritional conditions.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , HumanosRESUMO
The mitochondrial respiratory chain is organized in a dynamic set of supercomplexes (SCs). The COX7A2L protein is essential for mammalian SC III2+IV assembly. However, its function in respirasome (SCs I+III2+IVn) biogenesis remains controversial. To unambiguously determine the COX7A2L role, we generated COX7A2L-knockout (COX7A2L-KO) HEK293T and U87 cells. COX7A2L-KO cells lack SC III2+IV but have enhanced complex III steady-state levels, activity, and assembly rate, normal de novo complex IV biogenesis, and delayed respirasome formation. Nonetheless, the KOs have normal respirasome steady-state levels, and only larger structures (SCs I1-2+III2+IV2-n or megacomplexes) were undetected. Functional substrate-driven competition assays showed normal mitochondrial respiration in COX7A2L-KO cells in standard and nutritional-, environmental-, and oxidative-stress-challenging conditions. We conclude that COX7A2L establishes a regulatory checkpoint for the biogenesis of CIII2 and specific SCs, but the COX7A2L-dependent MRC remodeling is essential neither to maintain mitochondrial bioenergetics nor to cope with acute cellular stresses.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético , Mitocôndrias/metabolismo , Animais , Carbono/farmacologia , Linhagem Celular Tumoral , Respiração Celular , Células HEK293 , Humanos , Cinética , Camundongos Endogâmicos C57BL , Modelos Biológicos , Mutação/genética , Fosforilação Oxidativa , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/metabolismo , Deleção de Sequência , Estresse Fisiológico , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismoRESUMO
Lung cancer is the most common in incidence and mortality worldwide. With the development of next generation sequencing (NGS) detection technology, more and more patients with rare anaplastic lymphoma kinase (ALK) fusion mutations were detected. A case of advanced lung adenocarcinoma with rare COX7A2L-ALK (C2:A20) fusion detected by NGS was reported in Peking Union Medical College Hospital, and all cases with rare ALK fusion mutations were searched from medical datebase from January 1, 2014 to March 31, 2021, to investigate the treatment of rare ALK fusion mutations with ALK inhibitors. The best response of the patient was assessed as partial response (PR) with Ceritinib treatment. By literature review, 22 cases of rare ALK fusion were reported in 19 articles. Combined with this case, 23 cases were analyzed. The objective response rate (ORR) was 82.6% (19/23) and disease control rate (DCR) was 95.7% (22/23) for rare ALK fusions patients treated with ALK inhibitors. Lung adenocarcinoma patients with rare ALK fusion could benefit from ALK inhibitors. .
Assuntos
Humanos , Quinase do Linfoma Anaplásico/genética , Neoplasias Pulmonares/diagnóstico , Crizotinibe , Adenocarcinoma de Pulmão/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Fusão Oncogênica/genéticaRESUMO
Mitochondria are cellular organelles that harvest energy in the form of ATP through a process termed oxidative phosphorylation (OXPHOS), which occurs via the protein complexes of the electron transport chain (ETC). In recent years it has become unequivocally clear that mitochondrial complexes of the ETC are not static entities in the inner mitochondrial membrane. These complexes are dynamic and in mammals they aggregate in different stoichiometric combinations to form supercomplexes (SCs) or respirasomes. It has been proposed that the net respiration is more efficient via SCs than via isolated complexes. However, it still needs to be determined whether the activity of a particular SC is associated with a disease etiology. Here we describe a simplified method to visualize and assess in-gel activity of SCs and the individual complexes with good resolution using blue native polyacrylamide gel electrophoresis (BN-PAGE).
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Eletroforese em Gel de Poliacrilamida Nativa/métodos , Animais , Western Blotting , Complexo de Proteínas da Cadeia de Transporte de Elétrons/isolamento & purificação , CamundongosRESUMO
Objective To construct Cox7a2 fluorescent vector and study its effect on cytochrome C oxidase (COX) activity in mouse Sertoli cell line TM4. Methods The coding region of Cox7a2 was amplified from mouse Sertoli cell line TM4 by RT-PCR. The PCR product was inserted into pEYFP-C1 vector with BamH I and EcoR I restriction site, and confirmed by DNA sequencing. The recombinant fusion protein vector was amplified by transforming into DH5a and transfected into TM4 cells. The protein expression was identified by Western blot. COX activity was measured by spectrophotometer 6, 12, 24 and 48 h after the transfection of recombinant vector into the TM4 cell line. Results The entire coding sequence of Cox7a2 was cloned with 252 bp length. Plasmid pEYFP-C1-Cox7a2 vector was constructed and the positive clones were verified by restriction enzymes digestion and DNA sequencing. The transfection efficiency of the TM4 cell line was about 70% and 37000 D fusion protein was obtained. The COX activities were (0.642±0.051), (0.542±0.049), (0.311±0.021) and (0.216±0.010) U/mg 6, 12, 24 and 48 h after the transfection of recombinant vector in the TM4 cell line. Meanwhile, the COX activities were (0.714±0.064) and (0.653±0.031) U/mg in non-tranfected and naked vector group respectively. Compared with the non-tranfected group, COX activity decreased significantly 12, 24 and 48 h after the transfection. Conclusions The recombint plasmid vector was successfully constructed. Cox7a2 gene has an inhibiting effect on COX activity and may play an important role in the regulation of COX activity in mouse Sertoli cell line TM4.