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1.
J Biol Chem ; 298(9): 102349, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35934050

RESUMO

Many transcription factors contain intrinsically disordered transcription activation domains (TADs), which mediate interactions with coactivators to activate transcription. Historically, DNA-binding domains and TADs have been considered as modular units, but recent studies have shown that TADs can influence DNA binding. Whether these results can be generalized to more TADs is not clear. Here, we biophysically characterized the NFκB p50/RelA heterodimer including the RelA TAD and investigated the TAD's influence on NFκB-DNA interactions. In solution, we show the RelA TAD is disordered but compact, with helical tendency in two regions that interact with coactivators. We determined that the presence of the TAD increased the stoichiometry of NFκB-DNA complexes containing promoter DNA sequences with tandem κB recognition motifs by promoting the binding of NFκB dimers in excess of the number of κB sites. In addition, we measured the binding affinity of p50/RelA for DNA containing tandem κB sites and single κB sites. While the presence of the TAD enhanced the binding affinity of p50/RelA for all κB sequences tested, it also increased the affinity for nonspecific DNA sequences by over 10-fold, leading to an overall decrease in specificity for κB DNA sequences. In contrast, previous studies have generally reported that TADs decrease DNA-binding affinity and increase sequence specificity. Our results reveal a novel function of the RelA TAD in promoting binding to nonconsensus DNA, which sheds light on previous observations of extensive nonconsensus DNA binding by NFκB in vivo in response to strong inflammatory signals.


Assuntos
Subunidade p50 de NF-kappa B , Fator de Transcrição RelA , Ativação Transcricional , Sequência de Bases , DNA/química , Subunidade p50 de NF-kappa B/química , Subunidade p50 de NF-kappa B/genética , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Fator de Transcrição RelA/química , Fator de Transcrição RelA/genética
2.
Arch Microbiol ; 203(2): 861-864, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33040182

RESUMO

Widely distributed among prokaryotes, short chain fatty acid kinases provide a path for fatty acid entry into central metabolic pathways. These enzymes catalyze the reversible, ATP-dependent synthesis of acyl-phosphates, which leads to the production of acyl-CoA derivatives by a coordinate acyltransferase. To date, characterized representatives of short chain fatty acid kinases exhibit relatively narrow substrate specificity. In this work, biochemical characterization of a predicted acetate kinase from Rhodobacter sphaeroides reveals a novel enzyme with broad substrate specificity for primary fatty acids of varying lengths (C2--C8).


Assuntos
Acetato Quinase/metabolismo , Rhodobacter sphaeroides/enzimologia , Acil Coenzima A/metabolismo , Aciltransferases/metabolismo , Ácidos Graxos/metabolismo , Especificidade por Substrato
3.
Commun Biol ; 6(1): 560, 2023 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-37231125

RESUMO

Mutations in ASAH1 have been linked to two allegedly distinct disorders: Farber disease (FD) and spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME). We have previously reported FD-like phenotypes in mice harboring a single amino acid substitution in acid ceramidase (ACDase), P361R, known to be pathogenic in humans (P361R-Farber). Here we describe a mouse model with an SMA-PME-like phenotype (P361R-SMA). P361R-SMA mice live 2-3-times longer than P361R-Farber mice and have different phenotypes including progressive ataxia and bladder dysfunction, which suggests neurological dysfunction. We found profound demyelination, loss of axons, and altered sphingolipid levels in P361R-SMA spinal cords; severe pathology was restricted to the white matter. Our model can serve as a tool to study the pathological effects of ACDase deficiency on the central nervous system and to evaluate potential therapies for SMA-PME.


Assuntos
Lipogranulomatose de Farber , Atrofia Muscular Espinal , Epilepsias Mioclônicas Progressivas , Humanos , Camundongos , Animais , Lipogranulomatose de Farber/genética , Lipogranulomatose de Farber/metabolismo , Lipogranulomatose de Farber/patologia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Esfingolipídeos/metabolismo , Epilepsias Mioclônicas Progressivas/genética , Epilepsias Mioclônicas Progressivas/patologia , Fenótipo
4.
FEMS Microbiol Lett ; 367(6)2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32166312

RESUMO

Short and branched chain fatty acid kinases participate in both bacterial anabolic and catabolic processes, including fermentation, through the reversible, ATP-dependent synthesis of acyl phosphates. This study reports biochemical properties of a predicted butyrate kinase from Desulfovibrio vulgaris str. Hildenborough (DvBuk) expressed heterologously and purified from Escherichia coli. Gel filtration chromatography indicates purified DvBuk is active as a dimer. The optimum temperature and pH for DvBuk activity is 44°C and 7.5, respectively. The enzyme displays enhanced thermal stability in the presence of substrates as observed for similar enzymes. Measurement of kcat and KM for various substrates reveals DvBuk exhibits the highest catalytic efficiencies for butyrate, valerate and isobutyrate. In particular, these measurements reveal this enzyme's apparent high affinity for C4 fatty acids relative to other butyrate kinases. These results have implications on structure and function relationships within the ASKHA superfamily of phosphotransferases, particularly regarding the acyl binding pocket, as well as potential physiological roles for this enzyme in Desulfovibrio vulgaris str. Hildenborough.


Assuntos
Desulfovibrio vulgaris/enzimologia , Fosfotransferases (Aceptor do Grupo Carboxila)/metabolismo , Proteínas Recombinantes/metabolismo , Cromatografia em Gel , Desulfovibrio vulgaris/genética , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Fosfotransferases (Aceptor do Grupo Carboxila)/genética , Fosfotransferases (Aceptor do Grupo Carboxila)/isolamento & purificação , Proteínas Recombinantes/genética , Relação Estrutura-Atividade , Temperatura
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