Cloning And Functional Identification Of A Novel Protein EAPF Involved In Apoptosis

Apoptosis is a regulated biological program that selectively eliminates unwanted cells to keep the delicate balance between cell proliferation and death. Apoptosis plays an important role in development and survival. Recently it has been established that endoplasmic reticulum (ER) is another important organelle involved in apoptosis besides mitochondria and lysosomes. ER initiates apoptosis by at least two different mechanisms, namely the unfolded protein response (UPR) and Ca²⁺ signaling. In the current study, we isolated a novel molecule from a human bone marrow stromal cells (BMSC) cDNA library. Stable overexpression of this novel molecule could increase sensitivity of L929 cells to hTNF-a-induced apoptosis. We speculate that this novel molecule may participate in apoptosis via following steps: it partially translocates to ER and then might cause apoptotic changes of ER, which might directly or indirectly trigger the changes of mitochondria including collapse of mitochondrial membrane potential (ΔΨm) and the release of apoptosis inducing factor (AIF), resulting in caspase-independent apoptosis. Based on the structural characteristics of containing a PH domain and a FYVE domain, this novel molecule was designated as EAPF (endoplasmic reticulum-associated apoptosis-involved protein containing PH and FYVE domains).Part I Characterization and expression pattern of EAPFA novel full-length gene hLAPF (GenBank accession Number AY037145) had been isolated from a human BMSC cDNA library by large-scale random sequencing and a novel family Phafins (protein containing both PH and FYVE domains) had been identified by our laboratory. According to homology analysis of 14 members in the Phafins protein family, we found that an unknown protein (GenBank accession Number AY037145) from homosapiens shared 56% identity with hLAPF. We cloned the full-length cDNA of this protein from the human bone marrow stromal cells (BMSC) by RT-PCR using primers corresponding to the sequence NP₀78889. In view of the structural characteristics of containing a PH domain and a FYVE domain and its translocalization to ER and pro-apoptotic activity, it was designated as hEAPF (Human endoplasraic reticulum-associated apoptosis involved protein containing PH and FYVE domains). The mouse homologue (GenBank accession Number XP₁31308) of hEAPF was derived from the mouse BMSC and was named as mEAPF accordingly. hEAPF and mEAPF potentially encode a 249-amino-acid protein. Based on the primary amino acid sequence, mEAPF protein shares 97.6% overall identity with hEAPF protein. hEAPF is located on human chromosome 8q22.1 and mEAPF is located on mouse chromosome 4A1.The Phafin-2 subfamily of Phafins contains the following four members: hEAPF (Homo sapiens, NP₀78889), mEAPF (Mus Musculus, XP₁31308), dEAPF (Danio rerio, AAH47820), fEAPF (Fugu rubripes, SINFRUP00000161271). Based on overall protein sequence comparison, hEAPF shared 97.6% identity with mEAPF, 83.2% identity with dEAPF, and 83.9% identity with fEAPF. All these proteins were predicted to have common structural characteristics, an N-terminal PH domain and a C-terminal FYVE domain. Members were predicted to contain six conserved blocks within the PH domain, which comprise a highly conserved three-dimensional organization, and eight conserved cysteine residues within FYVE domain, two of these residues are part of the core motif R+HHC+XCGThe mRNA expression of hEAPF in human tissues was examined by RT-PCR analysis. We found that the expression of hEAPF was high in placenta, ovary and small intestine, whereas its expression in heart and pancreas was weak. mEAPF was expressed abundantly in brain, stomach and thymus and weakly in spleen, kidney and skeletal muscle. However, the expression of mEAPF was not detected in some tissues including liver, lung, small intestine, colon and heart.By RT-PCR analysis, hEAPF expression was observed in freshly isolated human BMSC, human dendritic cells (DC) and a variety of leukemia cell lines, including K562, TF-1, KG-1, HL-60, NaMAL, Molt-4, TOPI and NB4 cell lines...