Functional Study Of Two Novel Human Cell-growth Associated Genes: HOLFML1 And C10ORF58

Secreted proteins play important roles in many fundamental cellular and organism functions,such as signaling pathway,morphogenesis,apoptosis,cell differentiation and immune defense.Moreover,secreted proteins have properties that let themselves to be particularly utilized as therapeutic agents or targets.It is interesting and important to search for novel secreted proteins and study their biological functions.The strategy of bioinformatic prediction combined with experimental confirmation was used to isolate and identify novel potential human secreted protein genes which are associated with cell growth from the public protein database in this work.In the first part,we researched a novel member of human olfactomedin family, called hOLFML1(human Olfactomedin-like protein 1),which encodes 402 aa with an OLF domain in its C-terminus.hOLFML1 protein could be secreted into culture medium and was subjected to the N-linked glycosylational modification.Northern blot analysis indicated that hOLFML1 was mainly expressed in small intestine,liver, lung,and heart.Furthermore,immunohistochemical study on human small intestine demonstrated that it localized preferentially surrounding the intestinal villi. Interestingly,when overexpressed in HeLa cell lines.The recombinant hOLFML1 protein can promote the proliferation of cells and increase the percentage of S phase. Moreover,the rabbit anti-hOLFML1 antibody could block growth and cell cycle progression.Surprisingly,in human hepatocarcinoma Huh7 cells,knockdown of hOLFML1 negatively altered the effect of hOLFML1 on the cell growth and cell cycle profile.Together,our findings suggest that hOLFML1 is primarily expressed as an extracellular glycoprotein which could enhance the growth of cells and facilitate cell cycle progression in vitro.In the second part,we report a novel human gene C100RF58(chromosome10 open-reading framework 58,Q9BRX8,short for RX8),with a signal peptide but cannot secreted into serum.C10ORF58 is a very conserved gene in evolution,and there is a orthologuous gene existing in C.elegans.Realtime PCR assay indicated that RX8 ubiquitously expressed in adult human tissues,especially in brain,heart,kidney, pancreas and testis.In HepG2-Teton cell lines,the induced expression of RX8 inhibited cell growth and the formation of cell colones,and deduced S phase cell number and the de novo protein synthesis.Moreover,RX8 can specifically enhance the apoptosis-stimulating activity of BFA(Brefeldin A).Furthermore,subcellular fractionation assay showed that RX8 is an integrated membrane protein in endoplasmic reticulum.We found the interacting protein of RX8:CAML (calcium-modulating cyclophilin ligand) via yeast two-hybrid experiment.Then,we confirmed the interaction of RX8 and CAML thought co-immunoprecipitation, GST-pulldown and immunofluorescence assay.Importantly,we found that RXS, CAML and EGFR(EGF Receptor) co-existed in a complex.Interestingly,the Overexpression of RX8 significantly reduced the phosphorylation level and inhibited the activation of EGF signaling pathway downstream molecules:ERK1/2.On the other hand,the knockdown of RX8 by RNAi promoted the phosphorylation of ERK1/2,and the promotion could be block by the specific inhibitor of MEK1/2: PD98059.Finally,We provide a hypothetic molecular model.The normal recycling of EGFR depended on CAML which integrated in the endoplasmic reticulum.When the RX8 is overexpressed,RX8 could interfere or competitively inhibit CAML and EGFR interaction,therefore,the activation of ERK1/2 and cell growth were been inhibited.In the third part,we describe a novel secreted protein,named C12ORF39 (chromosome12 open-reading framework 39),in the middle of which contains a typical amidation-proteolytic processing signal(Gly-Arg-Arg motif).Western blotting assay indicated that the c-myc tagged C12ORF39 could be secreted into culture medium in transfected HeLa cells.Interestingly,C12ORF39 protein is not been hydrolysated,but is a full-length sequence protein without signal peptides.Our current research results about the three novel human genes will help to understand more deeply their biological functions and discovery their applied Prospects.