MASS SPECTROMETRY ANALYSIS OF CALCINEURIN-PERK INTERFACE

VILCHEZ ARUANI, JUAN; ROSANO, GERMÁN L.; GOMEZ, GABRIELA; BOLLO, MARIANA

Mendoza

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2022

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

A common event in many neurodegenerative diseases and brain injuries, such as ischemic stroke, is the accumulation of misfoldedproteins in the lumen of the ER. This causes ER stress, and activates the Unfolded Protein Response (UPR), a complex signalingpathway that can lead either to cell recovery or to programmed cell death if the damage cannot be resolved. A key ER stress sensingprotein and activator of the UPR is PERK, an ER transmembrane kinase. It´s activation leads to a global reduction in proteinsynthesis. PERK is inactivated if the ER stress is resolved in the acute phase. However, in chronic ER stress PERK remains active,and contributes to the transition to apoptosis.Calcineurin (CN) is a heterodimeric calcium/calmodulin-dependent phosphatase. Previous results from our lab showed a noncanonical cytoprotective function of CNAβ/B in astrocytes, by direct interaction with PERK. We have observed that thecytoprotective effect of CNAβ/B takes place during the acute phase of ER stress, and it is independent of CN phosphatase activity.This non-canonical function is related to PERK activity regulation. The aim of this work is to identify the interface between CNand PERK binding, using crosslinked recombinant proteins (6xHis-CNAβ/B and GST-cPERK) and mass spectrometry (MS)analysis. Our long-term goal is to design peptides derived from the contact surface of CNAβ/B that can modulate PERK signaling.MS spectra of tryptic fragments after reaction with crosslinker disuccinimidyl suberate (DSS) shows 3 main products suggestinginter-molecular links between lysine pair of CN-Aβ/B and cPERK fragments. The analysis suggests that disordered regions of bothproteins form part of the interface and that the calmodulin-binding domain of CN may interact with the juxtamembrane domain ofPERK. These findings give insights into the structural features of the CN/PERK complex, and help characterize the non-canonicalfunction of CN.