Systematic Proteogenomic Approach To Exploring a Novel Function for NHERF1 in Human Reproductive Disorder: Lessons for Exploring Missing Proteins

One
of the major goals of the Chromosome-Centric Human Proteome
Project (C-HPP) is to fill the knowledge gaps between human genomic
information and the corresponding proteomic information. These gaps
are due to “missing” proteins (MPs)predicted
proteins with insufficient evidence from mass spectrometry (MS), biochemical,
structural, or antibody analysesthat currently account for
2579 of the 19587 predicted human proteins (neXtProt, 2017-01).
We address some of the lessons learned from the inconsistent annotations
of missing proteins in databases (DB) and demonstrate a systematic
proteogenomic approach designed to explore a potential new function
of a known protein. To illustrate a cautious and strategic approach
for characterization of novel function in vitro and in vivo, we present
the case of Na­(+)/H­(+) exchange regulatory cofactor 1 (NHERF1/SLC9A3R1, located at chromosome 17q25.1;
hereafter NHERF1), which was mistakenly labeled as an MP in one DB
(Global Proteome Machine Database; GPMDB, 2011-09 release) but was
well known in another public DB and in the literature. As a first
step, NHERF1 was determined by MS and immunoblotting for its molecular
identity. We next investigated the potential new function of NHERF1
by carrying out the quantitative MS profiling of placental trophoblasts
(PXD004723) and functional study of cytotrophoblast JEG-3 cells. We
found that NHERF1 was associated with trophoblast differentiation
and motility. To validate this newly found cellular function of NHERF1,
we used the Caenorhabditis elegans mutant of nrfl-1 (a nematode ortholog of NHERF1),
which exhibits a protruding vulva (Pvl) and egg-laying-defective phenotype,
and performed genetic complementation work. The nrfl-1 mutant was almost fully rescued by the transfection of the recombinant transgenic construct that contained human NHERF1. These results suggest that NHERF1 could have a previously unknown function in pregnancy and in the development of human embryos. Our study outlines a stepwise experimental platform to explore new functions of ambiguously denoted candidate proteins and scrutinizes the mandated DB search for the selection of MPs to study in the future.