| Apoptosis is a highly conserved process in multicellular organisms that plays an important role in development and survival. Recent studies have shown that lysosomes participate in apoptosis under certain pathologic condition as well as physiologic situation such as during normal aging. Lysosomal membrane permeabilization (LMP) is an early event in apoptosis, which precedes changes of mitochondria, and is responsible for activation of downstream signal pathway, indicating a dominant role of lysosomes in apoptosis. In the current study, we isolated a novel molecule from human bone marrow stromal cells (BMSC), which could induce apoptosis of L929 cells concomitant with its translocation to lysosomes and thus permeabilization of lysosomal membrane. Given the structural characteristic of containing a PH domain and a FYVE domain, it was designated as LAPF (lysosome-associated and apoptosis-inducing protein containing PH and FYVE domains).Part I Identification of a novel family Phafins and characterization of LAPFA novel full-length gene hLAPF, isolated from a human BMSC cDNA library by large-scale random sequencing, potentially encodes a 279-amino-acid protein. The gene has been deposited to GenBank (GenBank accession Number AY037145). A mouse 279-amino-acid protein (GenBank accession Number NP077724), sharing 90.3 % overallidentity to hLAPF, was also cloned and designated as mouse LAPF (mLAPF).Homology analysis revealed that LAPF shares close similarities (about 34-91 % similarity) to 12 unknown proteins originating from various species. Structural analysis based on multiple alignment showed that all of these proteins were predicted to confain two common structural characteristics; an N-terminal PH domain and a C-terminal FYVE domain. We thus classified these proteins as a novel family, designated as the Phafins (protein containing both PH and FYVE domains). Members are 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+XCG. Based on homology comparison and extent of evolutionary conservation, members of the Phafin family can be grouped into two subfamilies: Phafin-1, Phafin-2, and an unclassified group.The mRNA expression of hLAPF in human tissues was examined by Northern blot analysis. An approximately 1.8 kb hLAPF transcript was abundant in heart and skeletal muscle, whereas expression in brain, thymus, spleen, kidney, liver, small intestine, placenta, lung and PBL was weak. mLAPF was also widely expressed in adult mouse tissues, and its expression in heart and brain was strong. hLAPF expression was observed in freshly isolated human BMSC, human dendritic cells (DC) and a variety of leukemia cell lines, but not in solid tumor cell lines. In contrast, mLAPF mRNA could also be detected in some solid tumor cell lines, in addition to leukemia cell lines, mDC and mBMSC.The effects of hTNFa, LPS and PMA treatments on LAPF expression in U937 and L929 cells, which express low levels of LAPF, were examined. Expression of hLAPF mRNA in U937 cells increased 12 h after an apoptosis-triggering dose (50 ng/ml) of hTNFa, and was maintained at high levels until 24 h, whereas mLAPF mRNA levels increased within 8 h in hTNFa-treated L929 cells. In parallel with mRNA expression profiles, protein expression of hLAPF and mLAPF in U937 and L929 cells was induced byhTNFa at 36 h and 24 h respectively, and maintained until at least 48 h. However, neither LPS nor PMA up-regulated LAPF gene expression. The time-dependent increase in LAPF expression in response to hTNFa indicates that LAPF may participate in TNFa-induced apoptosis.PartII LAPF induces apoptosis via a lysosomal-mitochondrial pathwayTo determine whether LAPF is involved in apoptosis, TNFa-sensitive L929 cells were transiently transfected with hLAPF or mLAPF expression vectors. Transient overexpression of hLAPF or mLAPF induced apoptosis of L929 cells, in t...
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