RESUMO
Fumarate hydratase (FH) is an enzyme of the tricarboxylic acid (TCA) cycle mutated in hereditary and sporadic cancers. Despite recent advances in understanding its role in tumorigenesis, the effects of FH loss on mitochondrial metabolism are still unclear. Here, we used mouse and human cell lines to assess mitochondrial function of FH-deficient cells. We found that human and mouse FH-deficient cells exhibit decreased respiration, accompanied by a varying degree of dysfunction of respiratory chain (RC) complex I and II. Moreover, we show that fumarate induces succination of key components of the iron-sulfur cluster biogenesis family of proteins, leading to defects in the biogenesis of iron-sulfur clusters that affect complex I function. We also demonstrate that suppression of complex II activity is caused by product inhibition due to fumarate accumulation. Overall, our work provides evidence that the loss of a single TCA cycle enzyme is sufficient to cause combined RC activity dysfunction.
Assuntos
Complexo II de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Fumarato Hidratase/metabolismo , Animais , Linhagem Celular Tumoral , Respiração Celular , Fumarato Hidratase/deficiência , Fumarato Hidratase/genética , Fumaratos/metabolismo , Humanos , Proteínas Ferro-Enxofre/metabolismo , CamundongosRESUMO
Following the in vivo investigation of thiotetronate assembly in Lentzea sp. and in S. thiolactonus NRRL 15439 (Havemann et al., Chem. Commun., 2017, DOI: 10.1039/c6cc09933e), the minimal set of genes required for thiolactomycin production was determined through heterologous expression and the mechanism for polyketide assembly was established in vitro through incubation of recombinant TlmB with its substrates in the presence of either nonhydrolysable or hydrolysable chemical probes. The results presented here constitute unequivocal evidence of enzymatic processing by an unusual iterative polyketide synthase.
Assuntos
Actinomycetales/enzimologia , Antibacterianos/metabolismo , Policetídeo Sintases/metabolismo , Actinomycetales/genética , Actinomycetales/metabolismo , Antibacterianos/análise , Vias Biossintéticas , Família Multigênica , Policetídeo Sintases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiofenos/análise , Tiofenos/metabolismoRESUMO
Chemical 'chain termination' probes were utilised for the investigation of thiotetronate antibiotic biosynthesis in the filamentous bacteria Lentzea sp. and Streptomyces thiolactonus NRRL 15439. The use of these tools led to the capture of biosynthetic intermediates involved in the thiotetronate polyketide backbone assembly, providing first insights into substrate specificity and in vivo intermediate processing by unusual iterative synthases.