ADAMTS9, a novel member of the ADAM-TS/ metallospondin gene family

ADAM-TS/metallospondin genes encode a new family of proteins with structural homology to the ADAM metalloprotease-disintegrin family. However, unlike other ADAMs, these proteins contain thrombospondin type 1 (TSP1) repeats at the carboxy-terminal end and are secreted proteins instead of being membrane bound. Members of the ADAM-TS family have been implicated in the cleavage of proteoglycans, the control of organ shape during development, and the inhibition of angiogenesis. We have cloned a new member of the ADAM-TS/metallospondin family designated here as ADAMTS9. This protein has a metalloprotease domain, a disintegrin-like domain, one internal TSP1 motif, and three carboxy-terminal TSP1-like submotifs. In contrast to other ADAM-TS family members, ADAMTS9 is expressed in all fetal tissues examined as well as some adult tissues. Using FISH and radiation hybrid analysis, we have localized ADAMTS9 to chromosome 3p14.2-p14.3, an area known to be lost in hereditary renal tumors.     

Radixin is a novel member of the band 4.1 family

Radixin is an actin barbed-end capping protein which is highly concentrated in the undercoat of the cell-to-cell adherens junction and the cleavage furrow in the interphase and mitotic phase, respectively (Tsukita, Sa., Y. Hieda, and Sh. Tsukita. 1989 a.J. Cell Biol. 108:2369-2382; Sato, N., S. Yonemura, T. Obinata, Sa. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 113:321-330). To further understand the structure and functions of the radixin molecule, we isolated and sequenced the cDNA clones encoding mouse radixin. Direct peptide sequencing of radixin and immunological analysis with antiserum to a fusion protein were performed to confirm that the protein encoded by these clones is identical to radixin. The composite cDNA is 4,241 nucleotides long and codes for a 583-amino acid polypeptide with a calculated molecular mass of 68.5 kD. Sequence analysis has demonstrated that mouse radixin shares 75.3% identity with human ezrin, which was reported to be a member of the band 4.1 family. We then isolated the cDNA encoding mouse ezrin. Sequence analysis and Northern blot analysis revealed that radixin and ezrin are similar but distinct (74.9% identity), leading us to conclude that radixin is a novel member of the band 4.1 family. In erythrocytes the band 4.1 protein acts as a key protein in the association of short actin filaments with a plasma membrane protein (glycophorin), together with spectrin. Therefore, the sequence similarity between radixin and band 4.1 protein described in this study favors the idea that radixin plays a crucial role in the association of the barbed ends of actin filaments with the plasma membrane in the cell-to-cell adherens junction and the cleavage furrow.     

A novel member of a zinc transporter family is defective in acrodermatitis enteropathica

The rare inherited condition acrodermatitis enteropathica (AE) results from a defect in the absorption of dietary zinc. Recently, we used homozygosity mapping in consanguineous Middle Eastern kindreds to localize the AE gene to an approximately 3.5-cM region on 8q24. In this article, we identify a gene, SLC39A4, located in the candidate region and, in patients with AE, document mutations that likely lead to the disease. The gene encodes a histidine-rich protein, which we refer to as "hZIP4," which is a member of a large family of transmembrane proteins, some of which are known to serve as zinc-uptake proteins. We show that Slc39A4 is abundantly expressed in mouse enterocytes and that the protein resides in the apical membrane of these cells. These findings suggest that the hZIP4 transporter is responsible for intestinal absorption of zinc.     

Protein-tyrosine phosphatase-sigma is a novel member of the functional family of alpha-latrotoxin receptors

Receptor-like protein-tyrosine phosphatase sigma (PTPvarsigma) is essential for neuronal development and function. Here we report that PTPvarsigma is a target of alpha-latrotoxin, a strong stimulator of neuronal exocytosis. alpha-Latrotoxin binds to the cell adhesion-like extracellular region of PTPvarsigma. This binding results in the stimulation of exocytosis. The toxin-binding site is located in the C-terminal part of the PTPvarsigma ectodomain and includes two fibronectin type III repeats. The intracellular catalytic domains of PTPvarsigma are not required for the alpha-latrotoxin binding and secretory response triggered by the toxin in chromaffin cells. These features of PTPvarsigma resemble two other previously described alpha-latrotoxin receptors, neurexin and CIRL. Thus, alpha-latrotoxin represents an unusual example of the neurotoxin that has three independent, equally potent, and yet structurally distinct targets. The known structural and functional characteristics of PTPvarsigma, neurexin, and CIRL suggest that they define a functional family of neuronal membrane receptors with complementary or converging roles in presynaptic function via a mechanism that involves cell-to-cell and cell-to-matrix interaction.     

ADAMTS: a novel family of extracellular matrix proteases

ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) is a novel family of extracellular proteases found in both mammals and invertebrates. Members of the family may be distinguished from the ADAM (a disintegrin and metalloprotease) family members based on the multiple copies of thrombospondin 1-like repeats they carry. With at least nine members in mammals alone, the ADAMTS family members are predicted by their structural domains to be extracellular matrix (ECM) proteins with a wide range of activities and functions distinct from members of the ADAM family that are largely anchored on the cell surface. ADAMTS2 is a procollagen N-proteinase, and the mutations of its gene are responsible for Human Ehlers-Danlos syndrome type VII C and bovine dermatosparaxis. ADAMTS4 and ADAMTS5 are aggrecanases implicated in the degradation of cartilage aggrecan in arthritic diseases. Other members of the ADAMTS family have also been implicated in roles during embryonic development and angiogenesis. Current and future studies on this emerging group of ECM proteases may provide important insights into developmental or pathological processes involving ECM remodeling.     

A novel ubiquitously expressed alpha-latrotoxin receptor is a member of the CIRL family of G-protein-coupled receptors

Poisoning with alpha-latrotoxin, a neurotoxic protein from black widow spider venom, results in a robust increase of spontaneous synaptic transmission and subsequent degeneration of affected nerve terminals. The neurotoxic action of alpha-latrotoxin involves extracellular binding to its high affinity receptors as a first step. One of these proteins, CIRL, is a neuronal G-protein-coupled receptor implicated in the regulation of secretion. We now demonstrate that CIRL has two close homologs with a similar domain structure and high degree of overall identity. These novel receptors, which we propose to name CIRL-2 and CIRL-3, together with CIRL (CIRL-1) belong to a recently identified subfamily of large orphan receptors with structural features typical of both G-protein-coupled receptors and cell adhesion proteins. Northern blotting experiments indicate that CIRL-2 is expressed ubiquitously with highest concentrations found in placenta, kidney, spleen, ovary, heart, and lung, whereas CIRL-3 is expressed predominantly in brain similarly to CIRL-1. It appears that CIRL-2 can also bind alpha-latrotoxin, although its affinity to the toxin is about 14 times less than that of CIRL-1. When overexpressed in chromaffin cells, CIRL-2 increases their sensitivity to alpha-latrotoxin stimulation but also inhibits Ca2+-regulated secretion. Thus, CIRL-2 is a functionally competent receptor of alpha-latrotoxin. Our findings suggest that although the nervous system is the primary target of low doses of alpha-latrotoxin, cells of other tissues are also susceptible to the toxic effects of alpha-latrotoxin because of the presence of CIRL-2, a low affinity receptor of the toxin.     

Lecithin retinol acyltransferase is a founder member of a novel family of enzymes

Lecithin retinol acyltransferase (LRAT) catalyzes the reversible esterification of vitamin A using lecithin as the acyl donor. LRAT is the founder member of a new class of enzymes, which include class II tumor suppressors, proteins essential for development, and putative proteases. All of these proteins possess Cys and His residues homologous to C161 and H60 of LRAT. These two residues are shown here to be essential for LRAT activity and are part of a catalytic dyad reminiscent of that found in thiol proteases. However, the local primary sequence contexts of C161 and H60 of LRAT and family are not at all homologous to those found in the approximately 20 thiol protease families. Moreover, LRAT shows pKs of 8.3 and 10.8, compared to approximately 4.0 and 8.5 observed in the thiol proteases. LRAT also contains Gln177 and Asp67 residues, which are largely conserved in the homologues. However, neither of these residues is essential for catalysis. Thiol proteases often contain catalytically essential Asp or Gln residues. It is concluded that LRAT is the founder member of a new class of Cys-His enzymes with diverse functions.     

新型金属蛋白酶ADAMTS家族的研究进展

含Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶ADAMTSs(a disintegrin and metallo-proteinase with thrombospondin motifs)是一类新的Zn2 依赖的金属蛋白酶家族,广泛存在于哺乳动物和无脊椎动物体内.从1997年发现第一个ADAMTSs家族成员以来,迄今共有19个成员被发现,在保持凝血系统的稳态、器官生成、炎症、生育等方面有重要作用.尽管其中大部分酶的功能尚不清楚,但已有研究显示该家族成员与多种疾病密切相关.ADAMTSs与基质金属蛋白酶MMPs、解聚蛋白样金属蛋白酶ADAMs同属金属蛋白酶家族,但在结构组成、组织细胞分布、底物作用的特异性、酶活性的调节等方面有明显差别.本文综述了其在结构功能及与疾病关系的研究进展.     

A stationary-phase gene in Saccharomyces cerevisiae is a member of a novel, highly conserved gene family.

The regulation of cellular growth and proliferation in response to environmental cues is critical for development and the maintenance of viability in all organisms. In unicellular organisms, such as the budding yeast Saccharomyces cerevisiae, growth and proliferation are regulated by nutrient availability. We have described changes in the pattern of protein synthesis during the growth of S. cerevisiae cells to stationary phase (E. K. Fuge, E. L. Braun, and M. Werner-Washburne, J. Bacteriol. 176:5802-5813, 1994) and noted a protein, which we designated Snz1p (p35), that shows increased synthesis after entry into stationary phase. We report here the identification of the SNZ1 gene, which encodes this protein. We detected increased SNZ1 mRNA accumulation almost 2 days after glucose exhaustion, significantly later than that of mRNAs encoded by other postexponential genes. SNZ1-related sequences were detected in phylogenetically diverse organisms by sequence comparisons and low-stringency hybridization. Multiple SNZ1-related sequences were detected in some organisms, including S. cerevisiae. Snz1p was found to be among the most evolutionarily conserved proteins currently identified, indicating that we have identified a novel, highly conserved protein involved in growth arrest in S. cerevisiae. The broad phylogenetic distribution, the regulation of the SNZ1 mRNA and protein in S. cerevisiae, and identification of a Snz protein modified during sporulation in the gram-positive bacterium Bacillus subtilis support the hypothesis that Snz proteins are part of an ancient response that occurs during nutrient limitation and growth arrest.     

A polysomal ribonuclease involved in the destabilization of albumin mRNA is a novel member of the peroxidase gene family.

We have purified an approximately 60 kDa endoribonuclease from Xenopus liver polysomes with properties expected for a messenger RNase involved in the estrogen-regulated destabilization of serum protein mRNAs (Dompenciel et al., 1995, J Biol Chem 270:6108-6118). The present report describes the cloning of this protein and its identification as a novel member of the peroxidase gene family. This novel enzyme, named polysomal RNase 1, or PMR-1 has 57% sequence identity with myeloperoxidase, and like that protein, appears to be processed from a larger precursor. Unlike myeloperoxidase, however, PMR-1 lacks N-linked oligosaccharide, heme, and peroxidase activity. Western blot and immunoprecipitation experiments using epitope-specific antibodies to the derived protein sequence confirm the identity of the cloned cDNA to the protein originally isolated from polysomes. The 80 kDa pre-PMR-1 expressed in a recombinant baculovirus was not processed to the 60 kDa form in Sf9 cells and lacks RNase activity. However, the baculovirus-expressed mature 60-kDa form of the enzyme has RNase activity. The recombinant protein is an endonuclease that shows selectivity for albumin versus ferritin mRNA. While it does not cleave at consensus APyrUGA elements, recombinant PMR-1 generates the same minor cleavage products from albumin mRNA as PMR-1 purified from liver. Finally, we show estrogen induces only a small increase in the amount of PMR-1. This result is consistent with earlier data suggesting estrogen activates mRNA decay through a posttranslational pathway.     

Human Bop is a novel BH3-only member of the Bcl-2 protein family

One group of Bcl-2 protein family, which shares only the BH3 domain (BH3-only), is critically involved in the regulation of programmed cell death. Herein we demonstrated a novel human BH3-only protein (designated as Bop) which could induce apoptosis in a BH3 domain-dependent manner. Further analysis indicated that Bop mainly localized to mitochondria and used its BH3 domain to contact the loop regions of voltage dependent anion channel 1 (VDAC1) in the outer mitochondrial membrane. In addition, purified Bop protein induced the loss of mitochondrial transmembrane potential (ΔΨm) and the release of cytochrome c. Furthermore, Bop used its BH3 domain to contact pro-survival Bcl-2 family members (Bcl-2, Bcl-X_L, Mcl-1, A1 and Bcl-w), which could inhibit Bop-induced apoptosis. Bop would be constrained by pro-survival Bcl-2 proteins in resting cells, because Bop became released from phosphorylated Bcl-2 induced by microtubule-interfering agent like vincristine (VCR). Indeed, knockdown experiments indicated that Bop was partially required for VCR induced cell death. Finally, Bop might need to function through Bak and Bax, likely by releasing Bak from Bcl-X_L sequestration. In conclusion, Bop may be a novel BH3-only factor that can engage with the regulatory network of Bcl-2 family members to process intrinsic apoptotic signaling.     

A chloroplastic RNA-binding protein is a new member of the PPR family

P67, a new protein binding to a specific RNA probe, was purified from radish seedlings [Echeverria, M. and Lahmy, S. (1995) Nucleic Acids Res. 23, 4963–4970]. Amino acid sequence information obtained from P67 microsequencing allowed the isolation of genes encoding P67 in radish and Arabidopsis thaliana. Immunolocalisation experiments in transfected protoplasts demonstrated that this protein is addressed to the chloroplast. The RNA-binding activity of recombinant P67 was found to be similar to that of the native protein. A significant similarity with the maize protein CRP1 [Fisk, D.G., Walker, M.B. and Barkan, A. (1999) EMBO J. 18, 2621–2630] suggests that P67 belongs to the PPR family and could be involved in chloroplast RNA processing.     

The Golgi-associated hook3 protein is a member of a novel family of microtubule-binding proteins

Microtubules are central to the spatial organization of diverse membrane-trafficking systems. Here, we report that Hook proteins constitute a novel family of cytosolic coiled coil proteins that bind to organelles and to microtubules. The conserved NH(2)-terminal domains of Hook proteins mediate attachment to microtubules, whereas the more divergent COOH-terminal domains mediate the binding to organelles. Human Hook3 bound to Golgi membranes in vitro and was enriched in the cis-Golgi in vivo. Unlike other cis-Golgi-associated proteins, however, a large fraction of Hook3 maintained its juxtanuclear localization after Brefeldin A treatment, indicating a Golgi-independent mechanism for Hook3 localization. Because overexpression of Hook3 caused fragmentation of the Golgi complex, we propose that Hook3 participates in defining the architecture and localization of the mammalian Golgi complex.     

A sperm cytoskeletal protein TSA70 is a novel phosphorylated member of cenexin/odf2 family

Using post-vasectomy monoclonal antibody we recently identified a testis specific sperm auto-antigen called TSA70 which is post-meiotically expressed and plays a role in sperm motility and capacitation-acrosome reaction. In the present study, we report its cytoskeletal nature based on its resistance to various high ionic salt solutions. TSA70 is developmentally regulated and appears postpubertally. The two protein spots identified by 2D WB namely TSA1-pI=5.821, MW=77.050 and TSA3-pI=6.173, MW=75.519 showed sequence homology to Cenexin/odf2 indicating that two are isoforms of the same protein. The immunoreactivity of TSA70 with anti-Cenexin antibody substantiates its homology with Cenexin/odf2. In silico analysis revealed the presence of two leucine zippers in TSA70 and also predicted potential phosphorylation sites at serine, threonine, and tyrosine residues. The phosphorylated status of TSA70 was further confirmed by immunoblot analysis. The differential cellular expression suggests that TSA70 is a novel member of Cenexin/odf2 family that exhibits functional divergence.     

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.     

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.