"A biochemical approach to define the interactome for calpain2 in endot . . ." by Dorothy E. Croall PhD, Jordan R. Hoffman MS et al.

Molecular & Biomedical Sciences Faculty Scholarship

Document Type

Poster

Title

A biochemical approach to define the interactome for calpain2 in endothelial cells

Authors

Dorothy E. Croall PhD, University of MaineFollow
Jordan R. Hoffman MS, University of Maine
Calvin PH Vary PhD, Maine Medical Center Research Institute
Sharon L. Ashworth PhD, University of Maine
Billy R. Chase, University of Maine
Jay Beaudoin II, University of Maine

Publication Date

7-2016

Place of conference

Big Sky Montana

Conference Sponsor

FASEB

Abstract/ Summary

Current repositories for protein-protein interactions and high throughput screening methods focus on individual gene products and do not consider the significance of calcium induced conformational changes. These limitations suggest the need for alternative strategies to better define the calpain2 interactome. Affinity capture coupled with LC-MS/MS and proteomic analysis of the recovered proteins provides a powerful approach to identify protein-protein interactions for the heterodimeric calpain2. CAPN2 (rat) was modified to be catalytically incompetent (C105A) and fused with a C-terminal 15 residue peptide optimized for biotinylation by the biotin protein ligase, BirA. The resulting CAPN2*, heterodimerized with truncated CAPNS1, was purified from E. coli, and biotinylated in vitro. Biotinylated calpain2* served as ‘bait’ for streptavidin affinity capture of calpain2 and its interacting proteins from lysates of bovine aortic (BAEC) and human umbilical vein (HUVEC) endothelial cells (ECs). Protein-calpain2 complexes were formed in the presence of calcium to allow EGTA elution of interacting proteins and LC-MS/MS analysis in the absence of an abundance of bait peptides. Capture of the well characterized calpain inhibitor protein calpastatin (CAST), and a known substrate, vimentin provide proof of concept and validates the conformational integrity of the bait calpain2*. Significant overlap between datasets (two from BAEC and one HUVEC) is also encouraging. Of numerous other proteins including several annexins, ANXA1 was confirmed as a substrate for calpain2. Findings are expected to contribute to continuing efforts in the field to better characterize calpain2’s selection of substrates and may reveal other important clues to calpain’s localization and regulation.

Repository Citation

Croall, Dorothy E. PhD; Hoffman, Jordan R. MS; Vary, Calvin PH PhD; Ashworth, Sharon L. PhD; Chase, Billy R.; and Beaudoin, Jay II, "A biochemical approach to define the interactome for calpain2 in endothelial cells" (2016). Molecular & Biomedical Sciences Faculty Scholarship. 2.
https://digitalcommons.library.umaine.edu/mbms_facpub/2