Livin, a novel inhibitor of apoptosis protein family member

A novel human inhibitor of apoptosis protein (IAP) family member termed Livin was identified, containing a single baculoviral IAP repeat (BIR) domain and a COOH-terminal RING finger domain. The mRNA for livin was not detectable by Northern blot in most normal adult tissues with the exception of the placenta, but was present in developmental tissues and in several cancer cell lines. Highest levels were observed in two melanoma-derived cell lines, G361 and SK-Mel29. Transfection of livin in HeLa cells resulted in protection from apoptosis induced by expression of FADD, Bax, RIP, RIP3, and DR6. Similar to other IAP family members, the anti-apoptotic activity of Livin was dependent on the BIR domain. Livin was also capable of inhibiting DEVD-like caspase activity triggered by tumor necrosis factor-alpha. In vitro binding studies demonstrated a direct interaction between Livin and the active form of the downstream caspases, caspase-3 and -7, that was dependent on the BIR domain of Livin. In addition, the unprocessed and cleaved forms of caspase-9 co-immunoprecipitated with Livin in vivo, and recombinant Livin could inhibit the activation of caspase-9 induced by Apaf-1, cytochrome c, and dATP. The subcellular distribution of the transfected Livin was analyzed by immunofluorescence. Both Livin and Survivin were expressed in the nucleus and in a filamentous pattern throughout the cytoplasm. In contrast to the apoptotic activity, the COOH-terminal RING domain mediated its subcellular localization patterning. Further studies found that transfection of an antisense construct against livin could trigger apoptosis specifically in cell lines expressing livin mRNA. This was associated with an increase in DNA fragmentation and in DEVD-like caspase activity. Thus, disruption of Livin may provide a strategy to induce apoptosis in certain cancer cells.     

Molecular cloning of ILP-2, a novel member of the inhibitor of apoptosis protein family

Inhibitor of apoptosis protein (IAP)-like protein-1 (ILP-1) (also known as X-linked IAP [XIAP] and mammalian IAP homolog A [MIHA]) is a potent inhibitor of apoptosis and exerts its effects, at least in part, by the direct association with and inhibition of specific caspases. Here, we describe the molecular cloning and characterization of a human gene related to ILP-1, termed ILP-2. Despite high homology to ILP-1, ILP-2 is encoded by a distinct gene, which in normal tissues is expressed solely in testis. In contrast to ILP-1, overexpression of ILP-2 had no protective effect on apoptosis mediated by Fas (also known as CD95) or tumor necrosis factor. However, ILP-2 potently inhibited apoptosis induced by overexpression of Bax or by coexpression of caspase 9 with Apaf-1, and preincubation of cytosolic extracts with ILP-2 abrogated caspase activation in vitro. A processed form of caspase 9 could be coprecipitated with ILP-2 from cells, suggesting a physical interaction between ILP-2 and caspase 9. Thus, ILP-2 is a novel IAP family member with restricted specificity for caspase 9.     

Role of XIAP protein, a human member of the inhibitor of apoptosis (IAP) protein family, in phytohemagglutinin-induced apoptosis of human T cell lines

A new family of human genes xiap, hiap-1 and hiap-2, which are homologous to the baculovirus iap (inhibitor of apoptosis) genes cp-iap and op-iap, has been recently cloned and shown to suppress apoptosis after serum withdrawal or exposure to a free radical inducer. In order to examine the role of one of these human genes, namely xiap, in lymphoid cells, we studied XIAP expression, after PHA stimulation in three different human T cell lines. We report here that stimulation with PHA resulted in the human T cell lines undergoing apoptosis, as assessed by DNA fragmentation and by propidiumiodide (PI) staining and flow cytometry. When XIAP protein expression was evaluated by Western blot, we observed that the induction of apoptosis by PHA was associated with a parallel decrease of XIAP expression. We also provide evidence that stably transfected Jurkat cells containing the xiap open reading frame became resistant to PHA-induced apoptosis. These data suggest a role for XIAP protein in the regulation of apoptosis in lymphoid cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nephrocan, a novel member of the small leucine-rich repeat protein family, is an inhibitor of transforming growth factor-beta signaling

In a search of new, small leucine-rich repeat proteoglycan/protein (SLRP) family members, a novel gene, nephrocan (NPN), has been identified. The gene consists of three exons, and based on the deduced amino acid sequence, NPN has 17 leucine-rich repeat motifs and unique cysteine-rich clusters both in the N and C termini, indicating that this gene belongs to a new class of SLRP family. NPN mRNA was predominantly expressed in kidney in adult mice, and during mouse embryogenesis, the expression was markedly increased in 11-day-old embryos at a time when early kidney development takes place. In the adult mouse kidney, NPN protein was located in distal tubules and collecting ducts. When NPN was overexpressed in cell culture, the protein was detected in the cultured medium, and upon treatment with N-glycosidase F, the molecular mass was lowered by approximately 14 kDa, indicating that NPN is a secreted N-glycosylated protein. Furthermore, transforming growth factor-beta (TGF-beta)-responsive 3TP promoter luciferase activity was down-regulated, and TGF-beta-induced Smad3 phosphorylation was also inhibited by NPN, suggesting that NPN suppresses TGF-beta/Smad signaling. Taken together, NPN is a novel member of the SLRP family that may play important roles in kidney development and pathophysiology by functioning as an endogenous inhibitor of TGF-beta signaling.     

Cell division regulation by BIR1, a member of the inhibitor of apoptosis family in yeast

The inhibitor of apoptosis (IAP) gene family comprises molecules that block the activity of pro-apoptotic caspase proteases. Paradoxically, yeasts contain IAP proteins but no caspases and no apoptotic program. To determine the function of these proteins in vivo, we disrupted the BIR1 gene, encoding the only known IAP in yeast Saccharomyces cerevisiae. Sporulation of heterozygous diploids yielded no viable mutant haploids, indicating that BIR1 is an essential gene. By flow cytometry, some heterozygous mutants were polyploid accumulating >4 N DNA content. These cells exhibited a 20-40% reduction in growth rate, which was rescued by plasmid-borne over-expression of BIR1 but not by its human counterpart, survivin. Deletion analysis revealed that the N-terminal domain of Bir1, containing the conserved baculovirus IAP repeat, was able to partially complement the cell growth defect caused by BIR1 deletion. Moreover, the full-length and truncated forms of Bir1 accelerated cell division in wild-type cells. Finally, BIR1 heterozygous mutants exhibited grossly altered cell morphology with misshapen or abnormally long buds connected to an unusually large mother cell. These findings identify a novel function of IAP proteins in the pleiotropic control of cell division, in addition to their role in the suppression of apoptosis.     

hDLG/SAP97, a member of the MAGUK protein family, is a novel caspase target during cell-cell detachment in apoptosis

Cell-cell detachment is one of the hallmarks of apoptosis. To date, several transmembrane and plaque proteins from tight and adherent junctions have been characterised as caspase targets during apoptosis. Human discs large protein (hDLG)/SAP97 is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins, localised at the adherent junctions of epithelial and endothelial cells, that is required for adherens junction assembly and differentiation. Here, hDLG is shown to be a caspase target during UV irradiation and staurosporine (STS)-induced apoptosis in HaCaT and CaCo-2 cells. Immunohistological data show a rapid loss of hDLG localisation at the sites of cell-cell contacts, preceding actual cell-cell detachment. In vitro experiments revealed cleavages at multiple sites located in the N-terminal half of the protein by caspase-3 only. Using Ala scanning mutagenesis, one cleavage site with an unusual recognition sequence for the executioner caspases (QSVD427/N) was identified. These data suggest that caspase-mediated cleavage of hDLG, and other MAGUKs, and their removal from sites of cell-cell contacts is an early step in the disruption of adherens junctions and dismantling of cell-cell contacts during apoptosis.     

Identification of a novel human member of the DEAD box protein family

The cDNA library of human pancreatic islets was screened with sera from patients with insulin-dependent diabetes mellitus (IDDM). From the library screening, we isolated a novel cDNA, RNA helicase-like protein (RHELP), which exhibited strong sequence homology to p68 RNA helicase, a prototypic member of the DEAD (Asp-Glu-Ala-Asp) box protein family. Sequence analysis of the cDNA revealed that RHELP contained DEAD sequence motif and other conserved motifs of the DEAD box protein family, indicating that RHELP is a new member of this family. DEAD box-containing proteins are involved in the RNA processing, ribosome assembly, spermatogenesis, embryogenesis, and cell growth and division. RHELP showed 42% and 44% amino acid sequence identity to human p68 RNA helicase and yeast DBP2 RNA helicase, respectively, among the DEAD box protein family. Northern blot analysis revealed that RHELP is expressed in most tissues including the liver, lung, tonsil, thymus, and muscle in addition to the pancreatic islets. In vivo or in vitro functions of RHELP as a putative RNA helicase and its potential role as a diabetic autoantigen need to be further investigated.     

Identification of a novel testis-specific member of the phosphatidylethanolamine binding protein family,pebp-2

The phosphatidylethanolamine binding proteins (pebps) are an evolutionarily conserved family of proteins recently implicated in mitogen-activated protein (MAP) kinase pathway regulation, where they are called raf kinase inhibitory proteins. Here, we describe the cloning, cellular localization, and partial characterization of a new member, pebp-2, with potential roles in male fertility. Expression data show that pebp-2 is a testis-specific 21-kDa protein found within late meiotic and haploid germ cells in a stage-specific pattern that is temporally distinct from that of pebp-1. Sequence analyses suggest that pebp-2 forms a distinct subset of the pebp family within mammals. Database analyses revealed the existence of a third subset. Analysis suggests that the specificity/regulation of the distinct pebps subsets is likely to be determined by the amino terminal 40 amino acids or the 3' untranslated region, where the majority of sequence differences occur. Protein homology modeling suggests that pebp-2 protein is, however, topologically similar to other pebps and composed of Greek key fold motifs, a dominant beta-sheet formed from five anti-parallel beta strands forming a shallow groove associated with a putative phosphatidylethanolamine binding site. The pebp-2 gene is intronless and data suggest that it is a retrogene derived from pebp-1. Further, pebp-2 colocalizes with members of the MAP kinase pathway in late spermatocytes and spermatids and on the midpiece of epididymal sperm. These data raise the possibility that pebp-2 is a novel participant in the MAP kinase signaling pathway, with a role in spermatogenesis or posttesticular sperm maturation.     

Neuroglycan C, a novel member of the neuregulin family

Neuroglycan C (NGC) is a transmembrane chondroitin sulfate proteoglycan expressed predominantly in the brain that possesses an EGF-like extracellular domain. The goal of the present study was to determine whether NGC may activate ErbB tyrosine kinases. A recombinant human NGC extracellular domain induced tyrosine phosphorylation of ErbB2 and ErbB3 as well as cell growth of the human breast tumor cell lines, T47D and MDA-MB-453. In vitro pull-down assay revealed that NGC could directly bind to a recombinant ErbB3-immunoglobulin Fc fusion protein (ErbB3-Fc) but not to ErbB1-Fc, ErbB2-Fc or ErbB4-Fc. A newly established anti-ErbB3 neutralizing monoclonal antibody (#5C3) almost completely blocked NGC-induced ErbB activation in MDA-MB-453 cells. Taken together, these data indicate that NGC is an active growth factor and a direct ligand for ErbB3 and that NGC transactivates ErbB2. Thus, NGC should be classified as the sixth member (neuregulin-6) of the neuregulin family.     

Angiopoietin-3, a novel member of the angiopoietin family

A cDNA clone encoding angiopoietin-3 protein (Ang3), a novel member of the angiopoietin family, was identified. Ang3 cDNA was cloned from a human aorta cDNA library. Ang3 is a 503 amino acid protein having 45.1% and 44.7% identity with human angiopoietin-1 and human angiopoietin-2, respectively. Ang3 mRNA is expressed in lung and cultured human umbilical vein endothelial cells (HUVECs). Ang3 mRNA expression in HUVECs was slightly decreased by vascular endothelial cell growth factor treatment, suggesting that the regulation of Ang3 mRNA expression is different from that of Ang2.     

Bim: a novel member of the Bcl-2 family that promotes apoptosis.

Certain members of the Bcl-2 family inhibit apoptosis while others facilitate this physiological process of cell death. An expression screen for proteins that bind to Bcl-2 yielded a small novel protein, denoted Bim, whose only similarity to any known protein is the short (nine amino acid) BH3 motif shared by most Bcl-2 homologues. Bim provokes apoptosis, and the BH3 region is required for Bcl-2 binding and for most of its cytotoxicity. Like Bcl-2, Bim possesses a hydrophobic C-terminus and localizes to intracytoplasmic membranes. Three Bim isoforms, probably generated by alternative splicing, all induce apoptosis, the shortest being the most potent. Wild-type Bcl-2 associates with Bim in vivo and modulates its death function, whereas Bcl-2 mutants that lack survival function do neither. Significantly, Bcl-xL and Bcl-w, the two closest homologues of Bcl-2, also bind to Bim and inhibit its activity, but more distant viral homologues, adenovirus E1B19K and Epstein-Barr virus BHRF-1, can do neither. Hence, Bim appears to act as a 'death ligand' which can only neutralize certain members of the pro-survival Bcl-2 sub-family.     

Molecular characterization of CRMP5, a novel member of the collapsin response mediator protein family

The CRMP (collapsin response mediator protein) family is thought to play key roles in growth cone guidance during neural development. The four members (CRMP1-4) identified to date have been demonstrated to form hetero-multimeric structures through mutual associations. In this study, we cloned a novel member of this family, which we call CRMP5, by the yeast two-hybrid method. This protein shares relatively low amino acid identity with the other CRMP members (49-50%) and also with dihydropyrimidinase (51%), whereas CRMP1-4 exhibit higher identity with each other (68-75%), suggesting that CRMP5 might be categorized into a third subfamily. The mouse CRMP5 gene was located at chromosome 5 B1. Northern blot and in situ hybridization analyses indicated that CRMP5 is expressed throughout the nervous system similarly to the other members (especially CRMP1 and CRMP4) with the expression peak in the first postnatal week. Association experiments using the yeast two-hybrid method and co-immunoprecipitation showed that CRMP5 interacts with dihydropyrimidinase and all the CRMPs including itself, except for CRMP1, although the expression profile almost overlaps with that of CRMP1 during development. These results suggest that CRMP complexes in the developing nervous system are classifiable into two populations that contain either CRMP1 or CRMP5. This indicates that different complexes may have distinct functions in shaping the neural networks.     

Bcl-G, a novel pro-apoptotic member of the Bcl-2 family

A new member of the Bcl-2 family was identified, Bcl-G. The human BCL-G gene consists of 6 exons, resides on chromosome 12p12, and encodes two proteins through alternative mRNA splicing, Bcl-G(L) (long) and Bcl-G(S) (short) consisting of 327 and 252 amino acids in length, respectively. Bcl-G(L) and Bcl-G(S) have identical sequences for the first 226 amino acids but diverge thereafter. Among the Bcl-2 homology (BH) domains previously recognized in Bcl-2 family proteins, the BH3 domain is found in both Bcl-G(L) and Bcl-G(S), but only the longer Bcl-G(L) protein possesses a BH2 domain. Bcl-G(L) mRNA is expressed widely in adult human tissues, whereas Bcl-G(S) mRNA was found only in testis. Overexpression of Bcl-G(L) or Bcl-G(S) in cells induced apoptosis although Bcl-G(S) was far more potent than Bcl-G(L). Apoptosis induction by Bcl-G(S) depended on the BH3 domain and was suppressed by coexpression of anti-apoptotic Bcl-X(L) protein. Bcl-X(L) also coimmunoprecipitated with Bcl-G(S) but not with mutants of Bcl-G(S) in which the BH3 domain was deleted or mutated or with Bcl-G(L). Bcl-G(S) was predominantly localized to cytosolic organelles, whereas Bcl-G(L) was diffusely distributed throughout the cytosol. A mutant of Bcl-G(L) in which the BH2 domain was deleted displayed increased apoptotic activity and coimmunoprecipitated with Bcl-X(L), suggesting that the BH2 domain autorepresses Bcl-G(L).     

XIAP, a cellular member of the inhibitor of apoptosis protein family, links the receptors to TAB1-TAK1 in the BMP signaling pathway.

Signals elicited by transforming growth factor-beta (TGF-beta) superfamily ligands are generated following the formation of heteromeric receptor complexes consisting of type I and type II receptors. TAK1, a member of the MAP kinase kinase kinase family, and its activator, TAB1, participate in the bone morphogenetic protein (BMP) signaling pathway involved in mesoderm induction and patterning in early Xenopus embryos. However, the events leading from receptor activation to TAK1 activation remain to be identified. A yeast interaction screen was used to search for proteins that function in the pathway linking the receptors and TAB1-TAK1. The human X-chromosome-linked inhibitor of apoptosis protein (XIAP) was isolated as a TAB1-binding protein. XIAP associated not only with TAB1 but also with the BMP receptors in mammalian cells. Injection of XIAP mRNA into dorsal blastomeres enhanced the ventralization of Xenopus embryos in a TAB1-TAK1-dependent manner. Furthermore, a truncated form of XIAP lacking the TAB1-binding domain partially blocked the expression of ventral mesodermal marker genes induced by a constitutively active BMP type I receptor. These results suggest that XIAP participates in the BMP signaling pathway as a positive regulator linking the BMP receptors and TAB1-TAK1.     

Identification and characterization of rDJL, a novel member of the DnaJ protein family, in rat testis

Applying the method of segmentation of seminiferous tubules combined with DDRT-PCR and cDNA library screening, a novel DnaJ homologue, rDJL was identified in rat testis. The reading frame encodes a protein of 223 amino acid residues containing J domain in the NH2 terminal region. rDJL gene is expressed mainly in testis and rDJL protein was immunolocalized notably in the acrosome region of spermatozoa. Immunoprecipitation experiments showed that rDJL interacted with Hsc70 and clathrin protein. When CHO cells were treated with EGF, rDJL and clathrin protein were found to be colocalized and be concentrated as endosome vesicles. The present findings suggest that rDJL functions as co-chaperone to Hsc70, participates in vesicular trafficking and may play an important role in acrosomogenesis.     

Molecular cloning of PP2Ceta, a novel member of the protein phosphatase 2C family

We have cloned a novel member of the mouse protein phosphatase 2C family, PP2Ceta. Sequence analysis suggests that PP2Ceta, PP2Czeta and NERPP-2C constitute a unique subgroup of the PP2C family. PP2Ceta had extremely low activity against alpha-casein compared with PP2Calpha and was localized mainly in cell nuclei, suggesting that PP2Ceta dephosphorylates a unique nuclear protein(s) in the cells.     

Characterization of ubiquitin-like polypeptide acceptor protein, a novel pro-apoptotic member of the Bcl2 family.

Monoclonal nonspecific suppressor factor (MNSF) is a cytokine with antigen nonspecific suppressive activity. MNSFbeta (a subunit of MNSF) is a 14.5 kDa fusion protein consisting of a protein with 36% identity with ubiquitin and ribosomal protein S30. The ubiquitin-like segment (Ubi-L) may be cleaved from MNSFbeta in the cytosol. Recently, we have observed that Ubi-L covalently binds to intracellular proteins in mitogen-activated murine T-helper type 2 clone, D.10 cells. In this study, we purified a 33.5 kDa Ubi-L adduct from D.10 cell lysates by sequential chromatography on DEAE, anti-(Ubi-L) Ig-conjugated Sepharose, and hydroxylapatite. MALDI-TOF-MS fingerprinting revealed that this Ubi-L adduct consists of an 8.5 kDa Ubi-L and a Bcl2-like protein, murine orthologue of a previously cloned human BCL-G gene product with pro-apoptotic function. Murine Bcl-G mRNA was highly expressed in testis and significantly in spleen. In addition, the level of Bcl-G mRNA expression was increased in concanavalin A- and interferon gamma-activated D.10 cells. The 33.5 kDa Ubi-L adduct was expressed in spleen but not in testis, even though Bcl-G protein was highly expressed in this tissue. The antisense oligonucleotide to Bcl-G significantly decreased the level of the Ubi-L adduct formation in concanavalin A-activated D.10 cells and the proliferative response of the D.10 cells. These results suggest that the post-translational modification of Bcl-G by Ubi-L might be implicated in T-cell activation.