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

Abstract

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.

Reflections on the HUPO Human Proteome Project, the Flagship Project of the Human Proteome Organization, at 10 Years

“…In 2017, the C-HPP launched the CP50 Initiative to stimulate experimental studies in support of functional annotation and characterization of these uPE1 proteins ( 50 ). Elaborate studies have been conducted to characterize individual proteins ( 51 ). In parallel, a computational approach utilizing I-TASSER/COFACTOR algorithms for protein folding and protein function prediction is now available upon request via the neXtProt community button on protein-specific pages ( 52 ).…”

ERM-1 phosphorylation and NRFL-1 redundantly control lumen formation in the C. elegans intestine

“…Previous studies using partial deletion alleles of nrfl-1 indicated that loss of NRFL-1 alone does not cause defects in the formation of the intestine (Hagiwara et al, 2012;Na et al, 2017). To rule out the possibility that the lack of severe defects is due to the production of truncated NRFL-1 proteins, we used CRISPR/Cas9 genome engineering to generate the nrfl-1(mib59) deletion allele.…”

Launching the C-HPP neXt-CP50 Pilot Project for Functional Characterization of Identified Proteins with No Known Function

“…For genetics-based functional complementation assays, Lane’s group in the Ch 2 team performed cross-species functional prediction analysis ( e.g ., zebrafish, human), gene knockdown (or overexpression) and gene complementation assays to assign C2orf62 and its interacting partner as key proteins involved in primary ciliogenesis in human cells and modulation of actin polymerization 53 . Paik’s group also employed genetic complementation assay ( e.g ., mutant of C. elegans nrf-2 −/− ), manipulation of cellular expression ( e.g ., RNAi knock-down) and biochemical verification in vitro and in vivo to characterize a new function of NHERF1, which is involved in human reproduction 34 . Thus, a combination of two or three screens is promising start points to characterize the function of the dark uPE1 proteins.…”