Identification of Novel SCIRR69-Interacting Proteins During ER Stress Using SILAC-Immunoprecipitation Quantitative Proteomics Approach

Abstract

Spinal cord injury and regeneration-related protein #69 (SCIRR69),also known as cAMP-responsive element-binding protein 3-like 2, belongs to the CREB/ATF family, some members of which play significant roles in ER stress. However, it is still not fully elucidated whether SCIRR69 involves in ER stress and its biochemical and functional roles during ER stress. In this study, we firstly treated fetal rat spinal cord neuron cells (SCN) and PC12 cells with ER stress activator thapsigargin (TG) or tunicamycin (TM) and then detected the expression pattern of SCIRR69 in response to ER stress at mRNA and protein levels using real-time PCR assay and immunoblotting. Results showed that the expression pattern of SCIRR69 was largely consistent with those of ER stress marker (ATF6, BIP and CHOP) at either mRNA level or protein level, implying that SCIRR69 may play important roles in ER stress. Subsequently, we used stable isotope labeling by amino acids in cell culture (SILAC)-immunoprecipitation quantitative proteomics to identify interaction partners of SCIRR69 during TG-induced ER stress in PC12 cells and found that transitional endoplasmic reticulum ATPase (TERA) and sideroflexin-1 (SFXN1) were potential SCIRR69-interacting proteins. The interaction between SCIRR69 and TERA or SFXN1 was validated using co-immunoprecipitation. Those results provide some clues for novel signaling nexuses that made by interactions between SCIRR69 and TERA or SFXN1. Our findings may facilitate a better understanding of the fundamental functions of SCIRR69 during ER stress.

Keywords: ER stress; Protein interaction; Quantitative proteomics; SCIRR69; SILAC.