Identification of Microprotein‐Protein Interactions via APEX Tagging

Abstract

Microproteins are peptides and small proteins encoded by small open reading frames (smORFs). Genomics and proteomics technologies have led to the recent discovery of hundreds to thousands of new microproteins. Emerging evidence demonstrates that these microproteins have fundamental roles in various biological processes including metabolism, apoptosis, and development, highlighting the value of characterizing these molecules. The identification of microprotein‐protein interactions (MPIs) has proven to be a successful approach to the functional characterization of these genes; however, traditional immunoprecipitation methods result in the enrichment of non‐specific interactions for microproteins. To address this issue, we tested and applied an in situ proximity tagging method that relies on an engineered ascorbate peroxidase (APEX) to elucidate MPIs. The results demonstrated that APEX tagging is superior to traditional immunoprecipitation methods for microproteins. Furthermore, the application of APEX‐tagging to two uncharacterized microproteins, C11orf98‐MP and MIEF‐MP revealed that they interact with nucleolar proteins nucleophosmin and nucleolin (C11orf98‐MP) and acyl carrier protein in mitochondria (MIEF‐MP) respectively, demonstrating the ability of this approach to identify novel hypothesis‐generating MPIs.Support or Funding InformationQ.C. is a postdoctoral fellow funded by the George E. Hewitt Foundation for medical research. This study was supported by the NIH with an NCI Cancer Center Support Grant P30 (CA014195 MASS core, A.S.) and R01 (GM102491, A.S.), the Leona M. and Harry B. Helmsley Charitable Trust grant (#2012‐PG‐MED002, A.S.), and Dr. Frederick Paulsen Chair/Ferring Pharmaceuticals (A.S.). Imaging work in this study was supported by the Waitt Advanced Biophotonics Core Facility of the Salk Institute with funding from NIH‐NCI CCSG: P30 014195, NINDS Neuroscience Core Grant: NS072031 and the Waitt Foundation. We also want to thank the Cancer Center Support Grant P30 CA014195 for purchasing and providing access to the MGC collection.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.