RESUMEN
An important property of NEMO, the core element of the IKK complex involved in NF-kappaB activation, resides in its ability to specifically recognize poly-ubiquitin chains. A small domain called NOA/UBAN has been suggested to be responsible for this property. We recently demonstrated that the C-terminal Zinc Finger (ZF) of NEMO is also able to bind ubiquitin. We show here by ZF swapping and mutagenesis that this represents its only function. While neither NOA nor ZF shows any preference for K63-linked chains, we demonstrate that together they form a bipartite high-affinity K63-specific ubiquitin-binding domain. A similar domain can be found in two other proteins, Optineurin and ABIN2, and can be freely exchanged with that of NEMO without interfering with its activity. This suggests that the main function of the C-terminal half of NEMO is to specifically bind K63-linked poly-ubiquitin chains. We also demonstrate that the recently described binding of NEMO to linear poly-ubiquitin chains is dependent on the NOA alone and does not require the presence of the ZF.
Asunto(s)
Quinasa I-kappa B/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Poliubiquitina/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Regulación hacia Abajo , Humanos , Quinasa I-kappa B/química , Quinasa I-kappa B/genética , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Células Jurkat , Ratones , Datos de Secuencia Molecular , Poliubiquitina/química , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Dedos de ZincRESUMEN
Tissue engineering requires the response of the cells to different stimuli inducing the synthesis of the extracellular matrix (ECM). It was been shown that mechanical and biochemical stimuli acted on the synthesis of ECM, particularly type I and III collagens. Growth factors implied in transduction pathways are multiple, but the main is TGF-beta. Member of the transforming growth factor-beta (TGF-beta) family bind to type II and type I serine/threonine kinase receptors, which initiate intracellular signals through activation of SMADs proteins. Nevertheless, the effects of mechanical stress of this pathway remain unknown. The aim of this work was to study the pathway of TGF-beta via the SMADs proteins under mechanical (stretching) and biochemical (TGF-beta) stimulations. Endogenous SMADs expression and its modulation by biochemical and mechanical stimulations were evaluated by both flow cytometry and confocal microscopy. Our results demonstrate that 10 ng of TGF-beta and stretching (5%, 1 Hz) applied during 15 min induced a negative feed back loop which blocks the signalling pathway to control TGF-beta activity. This inhibition effect was raised after 1 h of stimulation. Nevertheless, these preliminary studies should be continued by study of expression and localization of inhibitory SMADs (SMAD7).