RESUMEN
Telomeres are nucleoprotein complexes that play essential roles in protecting chromosome ends. Mammalian telomeres consist of repetitive DNA sequences bound by the shelterin complex. In this complex, the POT1-TPP1 heterodimer binds to single-stranded telomeric DNAs, while TRF1 and TRF2-RAP1 interact with double-stranded telomeric DNAs. TIN2, the linchpin of this complex, simultaneously interacts with TRF1, TRF2, and TPP1 to mediate the stable assembly of the shelterin complex. However, the molecular mechanism by which TIN2 interacts with these proteins to orchestrate telomere protection remains poorly understood. Here, we report the crystal structure of the N-terminal domain of TIN2 in complex with TIN2-binding motifs from TPP1 and TRF2, revealing how TIN2 interacts cooperatively with TPP1 and TRF2. Unexpectedly, TIN2 contains a telomeric repeat factor homology (TRFH)-like domain that functions as a protein-protein interaction platform. Structure-based mutagenesis analyses suggest that TIN2 plays an important role in maintaining the stable shelterin complex required for proper telomere end protection.
Asunto(s)
Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Serina Proteasas/metabolismo , Complejo Shelterina/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/química , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Animales , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/química , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/aislamiento & purificación , Humanos , Ratones , Conformación Proteica , Serina Proteasas/química , Serina Proteasas/aislamiento & purificación , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/aislamiento & purificación , Proteína 2 de Unión a Repeticiones Teloméricas/química , Proteína 2 de Unión a Repeticiones Teloméricas/aislamiento & purificaciónRESUMEN
Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter, we describe a comprehensive methodology that uses our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we demonstrate the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.