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Biochemistry. 2017 Jul 5;56(26):3299-3306. doi: 10.1021/acs.biochem.7b00265. Epub 2017 Jun 7.

Identification of Microprotein-Protein Interactions via APEX Tagging.

Author information

1
Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies , 10010 North Torrey Pines Road, La Jolla, California 92037, United States.
2
Division of Biological Sciences, University of California, San Diego , 9500 Gilman Drive, La Jolla, California 92093, United States.
3
Department of Chemical Physiology, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

Abstract

Microproteins are peptides and small proteins encoded by small open reading frames (smORFs). Newer technologies have led to the recent discovery of hundreds to thousands of new microproteins. The biological functions of a few microproteins have been elucidated, and these microproteins have fundamental roles in biology ranging from limb development to muscle function, 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 nonspecific interactions for microproteins. Here, we test and apply an in situ proximity tagging method that relies on an engineered ascorbate peroxidase 2 (APEX) to elucidate MPIs. The results demonstrate that APEX tagging is superior to traditional immunoprecipitation methods for microproteins. Furthermore, the application of APEX tagging to an uncharacterized microprotein called C11orf98 revealed that this microprotein interacts with nucleolar proteins nucleophosmin and nucleolin, demonstrating the ability of this approach to identify novel hypothesis-generating MPIs.

PMID:
28589727
PMCID:
PMC5499098
DOI:
10.1021/acs.biochem.7b00265
[Indexed for MEDLINE]
Free PMC Article

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