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1.
J Biol Chem ; 298(9): 102349, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-35934050

RESUMEN

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.


Asunto(s)
Subunidad p50 de NF-kappa B , Factor de Transcripción ReIA , Activación Transcripcional , Secuencia de Bases , ADN/química , Subunidad p50 de NF-kappa B/química , Subunidad p50 de NF-kappa B/genética , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Factor de Transcripción ReIA/química , Factor de Transcripción ReIA/genética
2.
Sci Adv ; 9(48): eadk6191, 2023 12.
Artículo en Inglés | MEDLINE | ID: mdl-38039358

RESUMEN

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial Parkinson's disease (PD) and a risk factor for the sporadic form. Increased kinase activity was shown in patients with both familial and sporadic PD, making LRRK2 kinase inhibitors a major focus of drug development efforts. Although much progress has been made in understanding the structural biology of LRRK2, there are no available structures of LRRK2 inhibitor complexes. To this end, we solved cryo-electron microscopy structures of LRRK2, wild-type and PD-linked mutants, bound to the LRRK2-specific type I inhibitor MLi-2 and the broad-spectrum type II inhibitor GZD-824. Our structures revealed an active-like LRRK2 kinase in the type I inhibitor complex, and an inactive DYG-out in the type II inhibitor complex. Our structural analysis also showed how inhibitor-induced conformational changes in LRRK2 are affected by its autoinhibitory N-terminal repeats. The structures provide a template for the rational development of LRRK2 kinase inhibitors covering both canonical inhibitor binding modes.


Asunto(s)
Enfermedad de Parkinson , Humanos , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Microscopía por Crioelectrón , Fosforilación , Mutación
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