RESUMEN
Mitochondria are cellular powerhouses that generate energy through the electron transport chain (ETC). The mitochondrial genome (mtDNA) encodes essential ETC proteins in a compartmentalized manner, however, the mechanism underlying metabolic regulation of mtDNA function remains unknown. Here, we report that expression of tricarboxylic acid cycle enzyme succinate-CoA ligase SUCLG1 strongly correlates with ETC genes across various TCGA cancer transcriptomes. Mechanistically, SUCLG1 restricts succinyl-CoA levels to suppress the succinylation of mitochondrial RNA polymerase (POLRMT). Lysine 622 succinylation disrupts the interaction of POLRMT with mtDNA and mitochondrial transcription factors. SUCLG1-mediated POLRMT hyposuccinylation maintains mtDNA transcription, mitochondrial biogenesis, and leukemia cell proliferation. Specifically, leukemia-promoting FMS-like tyrosine kinase 3 (FLT3) mutations modulate nuclear transcription and upregulate SUCLG1 expression to reduce succinyl-CoA and POLRMT succinylation, resulting in enhanced mitobiogenesis. In line, genetic depletion of POLRMT or SUCLG1 significantly delays disease progression in mouse and humanized leukemia models. Importantly, succinyl-CoA level and POLRMT succinylation are downregulated in FLT3-mutated clinical leukemia samples, linking enhanced mitobiogenesis to cancer progression. Together, SUCLG1 connects succinyl-CoA with POLRMT succinylation to modulate mitochondrial function and cancer development.
Asunto(s)
Biogénesis de Organelos , Succinato-CoA Ligasas , Animales , Humanos , Ratones , Acilcoenzima A/metabolismo , Acilcoenzima A/genética , Línea Celular Tumoral , Proliferación Celular , Progresión de la Enfermedad , ADN Mitocondrial/metabolismo , ADN Mitocondrial/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Leucemia/metabolismo , Leucemia/genética , Leucemia/patología , Mitocondrias/metabolismo , Mitocondrias/genética , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Succinato-CoA Ligasas/metabolismo , Succinato-CoA Ligasas/genéticaRESUMEN
The Succinate-CoA ligase (SUCL1) gene family is involved in energy metabolism, phytohormone signaling, and plant growth, development, and tolerance to stress. This is the first study to analyze the SUCL1 gene family in wheat (Triticum aestivum). 17 TaSUCL1 genes were identified in the complete genome sequence and classified into five subfamilies based on related genes found in three other species. The 17 TaSUCL1 genes were unevenly distributed across 11 chromosomes, and the collinearity of these genes was further investigated. Through using real-time qPCR (RT-qPCR) analysis, we identified the expression patterns of the TaSUCL1 genes under various tissues and different heavy metal stress conditions. The functions of selected TaSUCL1-1 gene were investigated by RNA interference (RNAi). This study provided a comprehensive analysis of the TaSUCL1 gene family. Within the TaSUCL1 genes, the exon-intron structure and motif composition exhibited significant similarity among members of the same evolutionary branch. Homology analysis and phylogenetic comparison of the SUCL1 genes in different plants offered valuable insights for studying the evolutionary characteristics of the SUCL1 genes. The expression levels of the TaSUCL1 genes in different tissues and under various metal stress conditions reveal its important role in plant growth and development. Gene function analysis demonstrated that TaSUCL1-1 silenced wheat plants exhibited a decrease in the total cadmium (Cd) concentrations and gene expression levels compared to the wild type (WT). Additionally, TaSUCL1-1 belonging to class c physically interacts with the ß-amylase protein TaBMY1 as verified by yeast two-hybridization. This research provides a useful resource for further study of the function and molecular genetic mechanism of the SUCL1 gene family members.
Asunto(s)
Cadmio , Regulación de la Expresión Génica de las Plantas , Familia de Multigenes , Filogenia , Proteínas de Plantas , Succinato-CoA Ligasas , Triticum , Triticum/genética , Triticum/metabolismo , Cadmio/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Succinato-CoA Ligasas/genética , Succinato-CoA Ligasas/metabolismo , Estrés Fisiológico/genética , Cromosomas de las Plantas/genéticaRESUMEN
Lysine succinylation is a subtype of protein acylation associated with metabolic regulation of succinyl-CoA in the tricarboxylic acid cycle. Deficiency of succinyl-CoA synthetase (SCS), the tricarboxylic acid cycle enzyme catalyzing the interconversion of succinyl-CoA to succinate, results in mitochondrial encephalomyopathy in humans. This report presents a conditional forebrain-specific knockout (KO) mouse model of Sucla2, the gene encoding the ATP-specific beta isoform of SCS, resulting in postnatal deficiency of the entire SCS complex. Results demonstrate that accumulation of succinyl-CoA in the absence of SCS leads to hypersuccinylation within the murine cerebral cortex. Specifically, increased succinylation is associated with functionally significant reduced activity of respiratory chain complex I and widescale alterations in chromatin landscape and gene expression. Integrative analysis of the transcriptomic data also reveals perturbations in regulatory networks of neuronal transcription in the KO forebrain. Together, these findings provide evidence that protein succinylation plays a significant role in the pathogenesis of SCS deficiency.
Asunto(s)
Mitocondrias , Succinato-CoA Ligasas , Humanos , Animales , Ratones , Mitocondrias/metabolismo , Acilcoenzima A/metabolismo , Succinato-CoA Ligasas/genética , Succinato-CoA Ligasas/metabolismo , Ratones NoqueadosRESUMEN
Biallelic pathogenic variants in subunits of succinyl-CoA synthetase (SCS), a tricarboxylic acid (TCA) cycle enzyme, are associated with mitochondrial encephalomyopathy in humans. SCS catalyzes the interconversion of succinyl-CoA to succinate, coupled to substrate-level phosphorylation of either ADP or GDP, within the TCA cycle. SCS-deficient encephalomyopathy typically presents in infancy and early childhood, with many patients succumbing to the disease during childhood. Common symptoms include abnormal brain MRI, basal ganglia lesions and cerebral atrophy, severe hypotonia, dystonia, progressive psychomotor regression, and growth deficits. Although subunits of SCS were first identified as causal genes for progressive metabolic encephalomyopathy in the early 2000s, recent investigations are now beginning to unravel the pathomechanisms underlying this metabolic disorder. This article reviews the current understanding of SCS function within and outside the TCA cycle as it relates to the complex and multifactorial mechanisms underlying SCS-related mitochondrial encephalomyopathy.
Asunto(s)
Encefalomiopatías Mitocondriales , Succinato-CoA Ligasas , Preescolar , Humanos , Encefalomiopatías Mitocondriales/genética , Encefalomiopatías Mitocondriales/metabolismo , Mitocondrias/metabolismo , Succinato-CoA Ligasas/genética , Succinato-CoA Ligasas/metabolismo , Estrés OxidativoRESUMEN
BACKGROUND: Succinate-CoA ligase/synthetase (SCS) deficiency is responsible for encephalomyopathy with mitochondrial DNA depletion and mild methylmalonic aciduria. Variants in SUCLG1, the nuclear gene encoding the alpha subunit of the SCS enzyme playing a pivotal role in maintaining mtDNA integrity and stability, are associated with mitochondrial DNA depletion syndrome 9 (MTDPS9). METHODS: In this study, we reported an infant with clinical features of MTDPS9 from China. Whole exome sequencing (WES) was used to identify the genetic cause. Bioinformatic analysis and mtDNA level detection were performed to assess pathogenicity. RESULTS: The proband manifested with hypotonia, lactic acidosis, mild methylmalonic aciduria, hearing loss and psychomotor retardation. WES identified new compound heterozygous SUCLG1 variants of c.601A>G (p.R201G) in exon 6 and c.871G>C (p.A291P) in exon 8. Computational analysis predicted that these missense variants might alter structure stability and mitochondrial translocation of SUCLG1. qRT-PCR showed 68% depletion of mtDNA content in proband as compared to controls. CONCLUSION: Novel compound heterozygous variants c.601A>G (p.R201G) and c.871G>C (p.A291P) in SUCLG1 may cause MTDPS9 in this family. Our finding should be helpful for molecular diagnosis, genetic counseling and clinical management of SCS deficiency disorders.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos , Succinato-CoA Ligasas , Errores Innatos del Metabolismo de los Aminoácidos/genética , ADN Mitocondrial/genética , Humanos , Lactante , Mitocondrias/genética , Succinato-CoA Ligasas/química , Succinato-CoA Ligasas/genéticaRESUMEN
Glutaminase regulates glutaminolysis to promote cancer cell proliferation. However, the mechanism underlying glutaminase activity regulation is largely unknown. Here, we demonstrate that kidney-type glutaminase (GLS) is highly expressed in human pancreatic ductal adenocarcinoma (PDAC) specimens with correspondingly upregulated glutamine dependence for PDAC cell proliferation. Upon oxidative stress, the succinyl-coenzyme A (CoA) synthetase ADP-forming subunit ß (SUCLA2) phosphorylated by p38 mitogen-activated protein kinase (MAPK) at S79 dissociates from GLS, resulting in enhanced GLS K311 succinylation, oligomerization, and activity. Activated GLS increases glutaminolysis and the production of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione, thereby counteracting oxidative stress and promoting tumor cell survival and tumor growth in mice. In addition, the levels of SUCLA2 pS79 and GLS K311 succinylation, which were mutually correlated, were positively associated with advanced stages of PDAC and poor prognosis for patients. Our findings reveal critical regulation of GLS by SUCLA2-coupled GLS succinylation regulation and underscore the regulatory role of metabolites in glutaminolysis and PDAC development.
Asunto(s)
Carcinoma Ductal Pancreático/genética , Glutaminasa/genética , Neoplasias Pancreáticas/genética , Succinato-CoA Ligasas/genética , Animales , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/enzimología , Carcinoma Ductal Pancreático/mortalidad , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Glutaminasa/metabolismo , Glutamina/metabolismo , Glutatión/metabolismo , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Desnudos , NADP/metabolismo , Estrés Oxidativo , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/mortalidad , Fosforilación , Pronóstico , Procesamiento Proteico-Postraduccional , Transducción de Señal , Succinato-CoA Ligasas/metabolismo , Ácido Succínico/metabolismo , Análisis de Supervivencia , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Many bacteria and other organisms carry out fermentations forming acetate. These fermentations have broad importance for foods, agriculture, and industry. They also are important for bacteria themselves because they often generate ATP. Here, we found a biochemical pathway for forming acetate and synthesizing ATP that was unknown in fermentative bacteria. We found that the bacterium Cutibacterium granulosum formed acetate during fermentation of glucose. It did not use phosphotransacetylase or acetate kinase, enzymes found in nearly all acetate-forming bacteria. Instead, it used a pathway involving two different enzymes. The first enzyme, succinyl coenzyme A (succinyl-CoA):acetate CoA-transferase (SCACT), forms acetate from acetyl-CoA. The second enzyme, succinyl-CoA synthetase (SCS), synthesizes ATP. We identified the genes encoding these enzymes, and they were homologs of SCACT and SCS genes found in other bacteria. The pathway resembles one described in eukaryotes, but it uses bacterial, not eukaryotic, gene homologs. To find other instances of the pathway, we analyzed sequences of all biochemically characterized homologs of SCACT and SCS (103 enzymes from 64 publications). Homologs with similar enzymatic activity had similar sequences, enabling a large-scale search for them in genomes. We searched nearly 600 genomes of bacteria known to form acetate, and we found that 6% encoded homologs with SCACT and SCS activity. This included >30 species belonging to 5 different phyla, showing that a diverse range of bacteria encode the SCACT/SCS pathway. This work suggests the SCACT/SCS pathway is important for acetate formation in many branches of the tree of life. IMPORTANCE Pathways for forming acetate during fermentation have been studied for over 80 years. In that time, several pathways in a range of organisms, from bacteria to animals, have been described. However, one pathway (involving succinyl-CoA:acetate CoA-transferase and succinyl-CoA synthetase) has not been reported in prokaryotes. Here, we discovered enzymes for this pathway in the fermentative bacterium Cutibacterium granulosum. We also found >30 other fermentative bacteria that encode this pathway, demonstrating that it could be common. This pathway represents a new way for bacteria to form acetate from acetyl-CoA and synthesize ATP via substrate-level phosphorylation. It could be a target for controlling yield of acetate during fermentation, with relevance for foods, agriculture, and industry.
Asunto(s)
Acetatos/metabolismo , Adenosina Trifosfato/metabolismo , Propionibacteriaceae/metabolismo , Succinato-CoA Ligasas/metabolismo , Acetilcoenzima A/metabolismo , Coenzima A Transferasas/genética , Coenzima A Transferasas/metabolismo , Fermentación , Genoma Bacteriano , Propionibacteriaceae/genética , Succinato-CoA Ligasas/genéticaRESUMEN
Substrate specificity of an enzyme is an important characteristic of its mechanism of action. Investigation of the nucleotide specificity of Plasmodium falciparum succinyl-CoA synthetase (SCS; PfSCS) would provide crucial insights of its substrate recognition. Charged gatekeeper residues have been shown to alter the substrate specificity via electrostatic interactions with approaching substrates. The enzyme kinetics of recombinant PfSCS (wild-type), generated by refolding of the individual P. falciparum SCSß and Blastocystis SCSα subunits, demonstrated ADP-forming activity (KmATP = 48 µm). Further, the introduction of charged gatekeeper residues, either positive (Lys and Lys) or negative (Glu and Asp), resulted in significant reductions in the ATP affinity of PfSCS. It is interesting to note that the recombinant PfSCSß subunit can be refolded to a functional enzyme conformation using Blastocystis SCSα, indicating the possibility of subunits swapping among different organisms. These results concluded that electrostatic interactions at the gatekeeper region alone are insufficient to alter the substrate specificity of PfSCS, and further structural analysis with a particular focus on binding site architecture is required.
Asunto(s)
Mutación , Plasmodium falciparum/enzimología , Succinato-CoA Ligasas/química , Succinato-CoA Ligasas/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Blastocystis/enzimología , Nucleótidos/metabolismo , Plasmodium falciparum/química , Unión Proteica , Dominios Proteicos , Pliegue de Proteína , Electricidad Estática , Especificidad por Sustrato , Succinato-CoA Ligasas/genéticaRESUMEN
Growth of Geobacter sulfurreducens PCA on lactate was enhanced by laboratory adaptive evolution. The enhanced growth was considered to be attributed to increased expression of the sucCD genes, encoding a succinyl-coenzyme A (CoA) synthetase. To further investigate the function of the succinyl-CoA synthetase, the sucCD genes were deleted from G. sulfurreducens The mutant showed defective growth on lactate but not on acetate. Introduction of the sucCD genes into the mutant restored the full potential to grow on lactate. These results verify the importance of the succinyl-CoA synthetase in growth on lactate. Genome analysis of Geobacter species identified candidate genes, GSU1623, GSU1624, and GSU1620, for lactate dehydrogenase. Deletion mutants of the identified genes for d-lactate dehydrogenase (ΔGSU1623 ΔGSU1624 mutant) or l-lactate dehydrogenase (ΔGSU1620 mutant) could not grow on d-lactate or l-lactate but could grow on acetate and l- or d-lactate, respectively. Introduction of the respective genes into the mutants allowed growth on the corresponding lactate stereoisomer. These results suggest that the identified genes were essential for d- or l-lactate utilization. The lacZ reporter assay demonstrated that the putative promoter regions were more active during growth on lactate than during growth on acetate, indicating that the genes for the lactate dehydrogenases were expressed more during growth on lactate than during growth on acetate. The gene deletion phenotypes and the expression profiles indicate that there are metabolic switches between lactate and acetate. This study advances the understanding of anaerobic lactate utilization in G. sulfurreducensIMPORTANCE Lactate is a microbial fermentation product as well as a source of carbon and electrons for microorganisms in the environment. Furthermore, lactate is a common amendment for stimulation of microbial growth in environmental biotechnology applications. However, anaerobic metabolism of lactate has been poorly studied for environmentally relevant microorganisms. Geobacter species are found in various environments and environmental biotechnology applications. By employing genomic and genetic approaches, succinyl-CoA synthetase and lactate dehydrogenase were identified as key enzymes in anaerobic metabolism of lactate in Geobacter sulfurreducens, a representative Geobacter species. Differential gene expression during growth on lactate and acetate was observed, demonstrating that G. sulfurreducens could metabolically switch to adapt to available substrates in the environment. The findings provide new insights into basic physiology in lactate metabolism as well as cellular responses to growth conditions in the environment and can be informative for the application of lactate in environmental biotechnology.
Asunto(s)
Proteínas Bacterianas/metabolismo , Geobacter/enzimología , L-Lactato Deshidrogenasa/metabolismo , Ácido Láctico/metabolismo , Succinato-CoA Ligasas/metabolismo , Anaerobiosis , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Geobacter/genética , Geobacter/metabolismo , L-Lactato Deshidrogenasa/genética , Succinato-CoA Ligasas/genéticaRESUMEN
SUCLA2 is a component of mitochondrial succinate-CoA ligase and nucleotide diphosphokinase activities. Its absence results in Krebs cycle failure, mitochondrial DNA depletion, and a childhood-fatal encephalomyopathy. We describe a purely neurologic allelic form of the disease consisting of deafness, putamenal hyperintensity on MRI and a myoclonic-dystonic movement disorder unchanging from childhood into, so far, the late fourth decade. We show that succinate supplementation circumvents the Krebs cycle block, but does not correct the neurologic disease. Our patients' Arg407Trp mutation has been reported in children with (yet) no MRI abnormalities. It remains possible that early succinate supplementation could impact the disease.
Asunto(s)
Sordera/genética , Trastornos del Movimiento/genética , Succinato-CoA Ligasas/genética , Sordera/tratamiento farmacológico , Femenino , Humanos , Masculino , Trastornos del Movimiento/tratamiento farmacológico , Mutación Missense , Linaje , Ácido Succínico/uso terapéuticoRESUMEN
The mitochondrial encephalomyopathies represent a clinically heterogeneous group of neurodegenerative disorders. The clinical phenotype of patients could be explained by mutations of mitochondria-related genes, notably SUCLG1 and SUCLA2. Here, we presented a 5-year-old boy with clinical features of mitochondrial encephalomyopathy from Iran. Also, a systematic review was performed to explore the involvement of SUCLG1 mutations in published mitochondrial encephalomyopathies cases. Genotyping was performed by implementing whole-exome sequencing. Moreover, quantification of the mtDNA content was performed by real-time qPCR. We identified a novel, homozygote missense variant chr2: 84676796 A > T (hg19) in the SUCLG1 gene. This mutation substitutes Cys with Ser at the 60-position of the SUCLG1 protein. Furthermore, the in-silico analysis revealed that the mutated position in the genome is well conserved in mammalians, that implies mutation in this residue would possibly result in phenotypic consequences. Here, we identified a novel, homozygote missense variant chr2: 84676796 A > T in the SUCLG1 gene. Using a range of experimental and in silico analysis, we found that the mutation might explain the observed phenotype in the family.
Asunto(s)
ADN Mitocondrial/genética , Mitocondrias/genética , Encefalomiopatías Mitocondriales/genética , Succinato-CoA Ligasas/genética , Preescolar , Homocigoto , Humanos , Irán , Masculino , Mutación MissenseRESUMEN
Mitochondrial acyl-coenzyme A species are emerging as important sources of protein modification and damage. Succinyl-CoA ligase (SCL) deficiency causes a mitochondrial encephalomyopathy of unknown pathomechanism. Here, we show that succinyl-CoA accumulates in cells derived from patients with recessive mutations in the tricarboxylic acid cycle (TCA) gene succinyl-CoA ligase subunit-ß (SUCLA2), causing global protein hyper-succinylation. Using mass spectrometry, we quantify nearly 1,000 protein succinylation sites on 366 proteins from patient-derived fibroblasts and myotubes. Interestingly, hyper-succinylated proteins are distributed across cellular compartments, and many are known targets of the (NAD+)-dependent desuccinylase SIRT5. To test the contribution of hyper-succinylation to disease progression, we develop a zebrafish model of the SCL deficiency and find that SIRT5 gain-of-function reduces global protein succinylation and improves survival. Thus, increased succinyl-CoA levels contribute to the pathology of SCL deficiency through post-translational modifications.
Asunto(s)
Acilcoenzima A/metabolismo , Enfermedades Mitocondriales/patología , Succinato-CoA Ligasas/genética , Animales , Células Cultivadas , Femenino , Humanos , Lactante , Lisina/metabolismo , Masculino , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Mutación , Proteómica , Sirtuinas/deficiencia , Sirtuinas/genética , Sirtuinas/metabolismo , Succinato-CoA Ligasas/deficiencia , Succinato-CoA Ligasas/metabolismo , Análisis de Supervivencia , Pez CebraRESUMEN
Neuroendocrine (NE) differentiation is a well-recognized phenotypic change of prostate cancer after androgen deprivation therapy (ADT), and it ultimately develops into an aggressive subset of this disease. However, the contribution of signaling pathways that lead to metabolic disorders and NE differentiation of prostate cancer remains unclear. In this study, we identified that ADT induced upregulation of the succinate-CoA ligase GDP-forming beta subunit (SUCLG2), which regulates succinate metabolism and NE differentiation of prostate cancer. We demonstrated a connection that upregulation of epidermal growth factor receptor (EGFR)-leukemia inhibitory factor receptor (LIFR) signaling induced SUCLG2 expression in prostate cancer cells. The LIFR is upregulated by nuclear EGFR, which acts as a transcriptional regulator, directly binds to the LIFR promoter, and drives NE differentiation and glycolysis of prostate cancer. LIFR upregulation is associated with SUCLG2, which increased succinate synthesis and enzymatic activities of mitochondrial nucleoside diphosphate kinase (NDPK) in prostate cancer cells. Knockdown of SUCLG2 suppressed NE differentiation in cultured cells and reduced prostate tumor growth in a xenograft model. Analysis of prostate tissue samples showed increased intensity of nuclear EGFR associated with the LIFR and SUCLG2 in castration-resistant prostate cancer tumors. Our study provides a mechanism whereby ADT upregulates EGFR-LIFR signaling that activates SUCLG2, which subsequently stimulates the metabolic changes associated with NE differentiation and aggressive prostate cancer phenotype.
Asunto(s)
Antagonistas de Andrógenos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia/genética , Tumores Neuroendocrinos/genética , Neoplasias de la Próstata Resistentes a la Castración/genética , Succinato-CoA Ligasas/metabolismo , Antagonistas de Andrógenos/uso terapéutico , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular Tumoral , Núcleo Celular/patología , Transdiferenciación Celular/efectos de los fármacos , Transdiferenciación Celular/genética , Receptores ErbB/metabolismo , Técnicas de Silenciamiento del Gen , Glucólisis/efectos de los fármacos , Glucólisis/genética , Humanos , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia/metabolismo , Masculino , Ratones , Tumores Neuroendocrinos/tratamiento farmacológico , Tumores Neuroendocrinos/patología , Regiones Promotoras Genéticas , Próstata/efectos de los fármacos , Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Androgénicos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Succinato-CoA Ligasas/genética , Regulación hacia Arriba/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Acetate:succinate CoA transferase (ASCT) is a mitochondrial enzyme that catalyzes the production of acetate and succinyl-CoA, which is coupled to ATP production with succinyl-CoA synthetase (SCS) in a process called the ASCT/SCS cycle. This cycle has been studied in Trypanosoma brucei (T. brucei), a pathogen of African sleeping sickness, and is involved in (i) ATP and (ii) acetate production and proceeds independent of oxygen and an electrochemical gradient. Interestingly, knockout of ASCT in procyclic form (PCF) of T. brucei cause oligomycin A-hypersensitivity phenotype indicating that ASCT/SCS cycle complements the deficiency of ATP synthase activity. In bloodstream form (BSF) of T. brucei, ATP synthase works in reverse to maintain the electrochemical gradient by hydrolyzing ATP. However, no information has been available on the source of ATP, although ASCT/SCS cycle could be a potential candidate. Regarding mitochondrial acetate production, which is essential for fatty acid biosynthesis and growth of T. brucei, ASCT or acetyl-CoA hydrolase (ACH) are known to be its source. Despite the importance of this cycle, direct evidence of its function is lacking, and there are no comprehensive biochemical or structural biology studies reported so far. Here, we show that in vitro-reconstituted ASCT/SCS cycle is highly specific towards acetyl-CoA and has a higher kcat than that of yeast and bacterial ATP synthases. Our results provide the first biochemical basis for (i) rescue of ATP synthase-deficient phenotype by ASCT/SCS cycle in PCF and (ii) a potential source of ATP for the reverse reaction of ATP synthase in BSF.
Asunto(s)
Acetatos/metabolismo , Adenosina Trifosfato/metabolismo , Coenzima A Transferasas/metabolismo , Mitocondrias/metabolismo , Succinato-CoA Ligasas/metabolismo , Trypanosoma brucei brucei/metabolismo , Acilcoenzima A/metabolismo , Coenzima A Transferasas/química , Coenzima A Transferasas/genética , Mutación , Fosforilación Oxidativa , Succinato-CoA Ligasas/química , Succinato-CoA Ligasas/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
RB1 gene is often homozygously deleted or mutated in prostate adenocarcinomas following acquirement of castration resistance and/or metastatic ability. We found that SUCLA2 gene is frequently involved in the deletion of the RB1 gene region in advanced prostate cancer. SUCLA2 constitutes the ß-subunit of succinate CoA ligase heterodimer that reversibly converts succinyl CoA into succinate. We sought the possibility that deletion of SUCLA2 gives rise to a metabolic vulnerability that could be targeted therapeutically. We found a significant metabolic shift in SUCLA2-deleted prostate cancer cells, including lower mitochondrial respiratory activity. By screening a number of libraries for compounds that induce cell death selectively in SUCLA2-deficient prostate cancer cells, we identified thymoquinone (2-isopropyl-5-methylbenzo-1,4-quinone) and PMA (phorbol-12-myristate-13-acetate) from a natural compound library. These findings indicate that the metabolic vulnerability in SUCLA2-deficient prostate cancer cells is pharmacologically targetable.
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Eliminación de Gen , Neoplasias de la Próstata/genética , Proteína de Retinoblastoma/genética , Succinato-CoA Ligasas/genética , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Benzoquinonas/farmacología , Línea Celular Tumoral , Células HEK293 , Humanos , Masculino , Ratones Noqueados , Ratones Desnudos , Ratones SCID , Células PC-3 , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteína de Retinoblastoma/deficiencia , Succinato-CoA Ligasas/deficiencia , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Nucleoside diphosphate kinase (NDK), a ubiquitous enzyme, catalyses reversible transfer of the γ phosphate from nucleoside triphosphates to nucleoside diphosphates and functions to maintain the pools of ribonucleotides and deoxyribonucleotides in the cell. As even a minor imbalance in the nucleotide pools can be mutagenic, NDK plays an antimutator role in maintaining genome integrity. However, the mechanism of the antimutator roles of NDK is not completely understood. In addition, NDKs play important roles in the host-pathogen interactions, metastasis, gene regulation, and various cellular metabolic processes. To add to these diverse roles of NDK in cells, a recent study now reveals that NDK may even confer mutator phenotypes to the cell by acting on the damaged deoxyribonucleoside diphosphates that may be formed during the oxidative stress. In this review, we discuss the roles of NDK in homeostasis of the nucleotide pools and genome integrity, and its possible implications in conferring growth/survival fitness to the organisms in the changing environmental niches.
Asunto(s)
Inestabilidad Genómica , Nucleósido-Difosfato Quinasa/genética , Nucleósido-Difosfato Quinasa/metabolismo , Animales , Escherichia coli/genética , Humanos , Mutación , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo , Succinato-CoA Ligasas/genética , Succinato-CoA Ligasas/metabolismo , Uracilo/metabolismoRESUMEN
The tumor microenvironment (TME) plays a pivotal role in cancer progression, and, in ovarian cancer (OvCa), the primary TME is the omentum. Here, we show that the diabetes drug metformin alters mesothelial cells in the omental microenvironment. Metformin interrupts bidirectional signaling between tumor and mesothelial cells by blocking OvCa cell TGF-ß signaling and mesothelial cell production of CCL2 and IL-8. Inhibition of tumor-stromal crosstalk by metformin is caused by the reduced expression of the tricarboxylic acid (TCA) enzyme succinyl CoA ligase (SUCLG2). Through repressing this TCA enzyme and its metabolite, succinate, metformin activated prolyl hydroxylases (PHDs), resulting in the degradation of hypoxia-inducible factor 1α (HIF1α) in mesothelial cells. Disruption of HIF1α-driven IL-8 signaling in mesothelial cells by metformin results in reduced OvCa invasion in an organotypic 3D model. These findings indicate that tumor-promoting signaling between mesothelial and OvCa cells in the TME can be targeted using metformin.
Asunto(s)
Carcinogénesis/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Metformina/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Células del Estroma/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Femenino , Humanos , Hipoglucemiantes/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones Endogámicos C57BL , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Prolil Hidroxilasas/genética , Prolil Hidroxilasas/metabolismo , Células del Estroma/patología , Succinato-CoA Ligasas/genética , Succinato-CoA Ligasas/metabolismo , Células Tumorales CultivadasRESUMEN
To date, tripartite tricarboxylate transport (TTT) systems are not well characterized in most organisms. To investigate which carbon sources are transported by the TTT system of A. mimigardefordensis DPN7T, single deletion mutants were generated lacking either completely both sets of genes encoding for these transport systems tctABCDE1 and tctABDE2 in the organism or the two genes encoding for the regulatory components of the third chosen TTT system, tctDE3. Deletion of tctABCDE1 (MIM_c39170-MIM_c39210) in Advenella mimigardefordensis strain DPN7T led to inhibition of growth of the cells with citrate indicating that TctABCDE1 is the transport system for the uptake of citrate. Because of the negative phenotype, it was concluded that this deletion cannot be substituted by other transporters encoded in the genome of strain DPN7T. A triple deletion mutant of A. mimigardefordensis lacking both complete TTT transport systems and the regulatory components of the third chosen system (ΔTctABCDE1 ΔTctABDE2 ΔTctDE3) showed a leaky growth with alpha-ketoglutarate in comparison with the wild type. The other investigated TTT (TctABDE3, MIM_c17190-MIM_c17220) is most probably involved in the transport of alpha-ketoglutarate. Additionally, thermoshift assays with TctC1 (MIM_c39190) showed a significant shift in the melting temperature of the protein in the presence of citrate whereas no shift occurred with alpha-ketoglutarate. A dissociation constant Kd for citrate of 41.7 μM was determined. Furthermore, alternative alpha-ketoglutarate transport was investigated via in silico analysis
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Asunto(s)
Ácidos Tricarboxílicos/metabolismo , Bordetella/genética , Betaproteobacteria/enzimología , Propionatos/metabolismo , Succinato-CoA Ligasas/metabolismo , Transportadores de Ácidos Dicarboxílicos/genética , Ácidos Dicarboxílicos/metabolismo , Espectrometría de Masas/métodos , Ciclo del Ácido Cítrico , Betaproteobacteria/clasificación , Propionatos/química , Succinato-CoA Ligasas/genéticaRESUMEN
Succinyl-CoA synthetase (SCS) catalyzes the only step of the tricarboxylic acid cycle that leads to substrate-level phosphorylation. Some forms of SCS are specific for ADP/ATP or for GDP/GTP, while others can bind all of these nucleotides, generally with different affinities. The theory of `gatekeeper' residues has been proposed to explain the nucleotide-specificity. Gatekeeper residues lie outside the binding site and create specific electrostatic interactions with incoming nucleotides to determine whether the nucleotides can enter the binding site. To test this theory, the crystal structure of the nucleotide-binding domain in complex with Mg2+-ADP was determined, as well as the structures of four proteins with single mutations, K46ßE, K114ßD, V113ßL and L227ßF, and one with two mutations, K46ßE/K114ßD. The crystal structures show that the enzyme is specific for ADP/ATP because of interactions between the nucleotide and the binding site. Nucleotide-specificity is provided by hydrogen-bonding interactions between the adenine base and Gln20ß, Gly111ß and Val113ß. The O atom of the side chain of Gln20ß interacts with N6 of ADP, while the side-chain N atom interacts with the carbonyl O atom of Gly111ß. It is the different conformations of the backbone at Gln20ß, of the side chain of Gln20ß and of the linker that make the enzyme ATP-specific. This linker connects the two subdomains of the ATP-grasp fold and interacts differently with adenine and guanine bases. The mutant proteins have similar conformations, although the L227ßF mutant shows structural changes that disrupt the binding site for the magnesium ion. Although the K46ßE/K114ßD double mutant of Blastocystis hominis SCS binds GTP better than ATP according to kinetic assays, only the complex with Mg2+-ADP was obtained.
Asunto(s)
Adenosina Trifosfato/metabolismo , Blastocystis hominis/enzimología , Modelos Moleculares , Succinato-CoA Ligasas/química , Succinato-CoA Ligasas/genética , Succinato-CoA Ligasas/metabolismo , Sitios de Unión , Cristalografía por Rayos X/métodos , Escherichia coli/genética , Fluorometría/métodos , Enlace de Hidrógeno , Cinética , Mutación , Unión Proteica , Dominios ProteicosRESUMEN
The enzymes pyruvate ferredoxin oxidoreductase (PFO), malic enzyme (ME), and the α- and ß-subunits of succinyl-CoA synthetase (SCS) catalyze key steps of energy metabolism in Trichomonas vaginalis hydrogenosomes. These proteins have also been characterized as the adhesins AP120 (PFO), AP65 (ME), AP33, and AP51 (α- and ß-SCS), which are localized on the cell surface and mediate the T. vaginalis cytoadherence. However, the mechanisms that facilitate the targeting of these proteins to the cell surface via the secretory pathway and/or to hydrogenosomes are not known. Here we adapted an in vivo biotinylation system to perform highly sensitive tracing of protein trafficking in T. vaginalis. We showed that α- and ß-SCS are biotinylated in the cytosol and imported exclusively into the hydrogenosomes. Neither α- nor ß-SCS is biotinylated in the endoplasmic reticulum and delivered to the cell surface via the secretory pathway. In contrast, two surface proteins, tetratricopeptide domain-containing membrane-associated protein and tetraspanin family surface protein, as well as soluble-secreted ß-amylase-1 are biotinylated in the endoplasmic reticulum and delivered through the secretory pathway to their final destinations. Taken together, these results demonstrate that the α- and ß-SCS subunits are targeted only to the hydrogenosomes, which argues against their putative moonlighting function.