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Enlace disulfuro intramolecular entre cisteínas catalíticas en un precursor de inteína

Wen Chen¹, Lingyun Li, Zhenming Du

¹Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.

Journal of the American Chemical Society

|January 28, 2012

Resumen

Los investigadores descubrieron un enlace disulfuro en la inteína Pyrococcus abyssi PolII que inhibe el empalme de proteínas. Los agentes reductores rompen este enlace, permitiendo el empalme y sugiriendo el control redox de la función intein.

Área de la Ciencia:

La bioquímica es la bioquímica.
Biología Molecular Biología Molecular
Biología Estructural Biología estructural.

Sus antecedentes:

El empalme de proteínas es una modificación crucial después de la traducción mediada por inteínas.
Las enteínas catalizan su propia escisión y ligadura de las enteínas flanqueantes.
Comprender la regulación de la inteína es clave para controlar la función de las proteínas.

Objetivo del estudio:

Investigar las implicaciones estructurales y funcionales de un nuevo enlace disulfuro intramolecular en un intein hipertermófilo.
Para dilucidar el mecanismo por el cual este enlace disulfuro afecta la actividad de empalme de proteínas.
Para explorar el papel potencial de este enlace disulfuro en la regulación mediada por redox de la función inteína.

Principales métodos:

Mutagénesis dirigida al sitio para investigar las funciones de la cisteína.
Tratamiento con agentes reductores como la tris(2-carboxieetil) fosfina (TCEP).
Cromatografía líquida-espectrometría de masas (LC-MS/MS) para la identificación de enlaces disulfuro.

Principales resultados:

Primer descubrimiento de un enlace disulfuro intramolecular entre las cisteínas del sitio activo (Cys1 y Cys+1) en el precursor de la inteína Pyrococcus abyssi PolII.
El enlace disulfuro fue confirmado utilizando estudios de LC-MS/MS y mutagénesis.
La formación de enlaces disulfuro inhibe significativamente el empalme de proteínas, que puede ser restaurado por reducción.
La estabilidad del enlace disulfuro está influenciada por las interacciones de la extein y la temperatura.

Conclusiones:

Un enlace disulfuro intramolecular actúa como un potente inhibidor del empalme de proteínas en la proteína estudiada.
Las condiciones de reducción pueden superar esta inhibición, lo que sugiere un mecanismo de interruptor redox.
Las inteínas pueden poseer elementos reguladores sensibles al redox que controlan su actividad catalítica y la función de las exteinas.

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