ERK8, a new member of the mitogen-activated protein kinase family

The ERKs are a subfamily of the MAPKs that have been implicated in cell growth and differentiation. By using the rat ERK7 cDNA to screen a human multiple tissue cDNA library, we identified a new member of the ERK family, ERK8, that shares 69% amino acid sequence identity with ERK7. Northern analysis demonstrates that ERK8 is present in a number of tissues with maximal expression in the lung and kidney. Fluorescence in situ hybridization localized the ERK8 gene to chromosome 8, band q24.3. Expression of ERK8 in COS cells and bacteria indicates that, in contrast to constitutively active ERK7, ERK8 has minimal basal kinase activity and a unique substrate profile. ERK8, which contains two SH3-binding motifs in its C-terminal region, associates with the c-Src SH3 domain in vitro and co-immunoprecipitates with c-Src in vivo. Co-transfection with either v-Src or a constitutively active c-Src increases ERK8 activation indicating that ERK8 can be activated downstream of c-Src. ERK8 is also activated following serum stimulation, and the extent of this activation is reduced by pretreatment with the specific Src family inhibitor PP2. The ERK8 activation by serum or Src was not affected by the MEK inhibitor U0126 indicating that activation of ERK8 does not require MEK1, MEK2, or MEK5. Although most closely related to ERK7, the relatively low sequence identity, minimal basal activity, and different substrate profile identify ERK8 as a distinct member of the MAPK family that is activated by an Src-dependent signaling pathway.     

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.     

Purification and characterization of a new member of the S-100 protein family from human placenta.

A novel Ca(2+)-binding protein which is termed S-100P was purified from human placenta with a hydrophobic column followed by an anion exchange column and reverse phase high performance liquid chromatography (HPLC). Molecular mass of the protein was 10 kDa according to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Using immunoblotting technique, anti-human calcyclin antibodies did not bind to the S-100P. Isoelectric point of S-100P was pI = 4.6. S-100P did not formed disulfide-linked dimer. Calcium binding ability was proved by UV difference spectrometry, urea/alkaline gel electrophoresis, and 45Ca overlay technique. A ninety amino acid sequence of S-100P was determined. It is 49% identical with human S-100 beta, 38% with human calcyclin, and 37% with human cystic fibrosis antigen.     

Identification and characterization of thrombospondin-4, a new member of the thrombospondin gene family

A new member of the thrombospondin gene family, designated thrombospondin-4, has been identified in the Xenopus laevis genome. The predicted amino acid sequence indicates that the protein is similar to the other members of this gene family in the structure of the type 3 repeats and the COOH-terminal domain. Thrombospondin-4 contains four type 2 repeats and lacks the type 1 repeats that are found in thrombospondin-1 and 2. The amino-terminal domain of thrombospondin-4 has no significant homology with the other members of the thrombospondin gene family or with other proteins in the database. RNAse protection analysis establishes that the initial expression of Xenopus thrombospondin-4 is observed during neurulation. Levels of mRNA expression increase twofold during tailbud stages but decrease by the feeding tadpole stage. The size of the thrombospondin-4 message is 3.3 Kb and 3.4 Kb in the frog and human, respectively. Northern blot analysis of human tissues reveals high levels of thrombospondin-4 expression in heart and skeletal muscle, low levels in brain, lung and pancreas and undetectable levels in the placenta, liver and kidney. These data establish the existence of a new member of the thrombospondin gene family that may participate in the genesis and function of cardiac and skeletal muscle.     

Identification and characterization of a new member of the prolactin family, placental lactogen-I variant

This report describes the identification and characterization of a new member of the placental prolactin (PRL) family, termed placental lactogen-I variant (PL-Iv). PL-Iv was isolated from medium conditioned by late gestation placental explants. Rat PL-Iv was found to be closely related to rat PL-I. Amino-terminal sequence analysis indicated that PL-Iv shared approximately 88% sequence identity with the amino terminus of PL-I. PL-Iv proteins cross-reacted with antiserum to recombinant mouse PL-I and PL-Iv mRNA hybridized with a PL-I cDNA. Multiple PL-I and PL-Iv species were present in placental cytosol. Despite the structural similarities between PL-I and PL-Iv, distinct differences were also evident. Antibodies generated to the amino-terminal 19 amino acids of PL-Iv specifically recognized PL-Iv, while failing to recognize PL-I. Secreted PL-Iv had an affinity for concanavalin A, whereas secreted PL-I lacked affinity for the lectin. PL-I was predominantly secreted as a 36-40-kDa species and PL-Iv was predominantly secreted as a 33-kDa species. Furthermore, PL-I and PL-Iv were synthesized at different times during gestation and by different cell types. PL-I was synthesized by trophoblast giant cells during the first half of gestation, while PL-Iv was predominantly synthesized by spongiotrophoblast cells during the later stages of gestation. PL-Iv was shown to stimulate the proliferation of rat Nb2 lymphoma cells, an in vitro measure of lactogenic activity. In summary, PL-Iv shares structural similarities with PL-I; however, it shows other structural differences in addition to unique cell- and temporal-specific patterns of expression in the rat chorioallantoic placenta.     

Identification and characterization of RsmE, the founding member of a new RNA base methyltransferase family

A variety of RNA methyltransferases act during ribosomal RNA maturation to modify nucleotides in a site-specific manner. However, of the 10 base-methylated nucleotides present in the small ribosomal subunit of Escherichia coli, only three enzymes responsible for modification of four bases are known. Here, we show that the protein encoded by yggJ, a member of the uncharacterized DUF558 protein family of predicted alpha/beta (trefoil) knot methyltransferases is responsible for methylation at U1498 in 16S rRNA. The gene is well-conserved across bacteria and plants, and likely performs the same function in other organisms. A yggJ deletion strain lacks the methyl group at U1498 as well as the specific methyltransferase activity. Moreover, purified recombinant YggJ specifically methylates m3U1498 in vitro. The deletion strain was unaffected in exponential growth in rich or minimal media at multiple temperatures, but it was defective when grown in competition with isogenic wild-type cells. Based on these data, we conclude that yggJ is the founding member of a family of RNA base methyltransferases, and propose that it be renamed rsmE.     

Identification and characterization of nonmuscle myosin II-C, a new member of the myosin II family

A previously unrecognized nonmuscle myosin II heavy chain (NMHC II), which constitutes a distinct branch of the nonmuscle/smooth muscle myosin II family, has recently been revealed in genome data bases. We characterized the biochemical properties and expression patterns of this myosin. Using nucleotide probes and affinity-purified antibodies, we found that the distribution of NMHC II-C mRNA and protein (MYH14) is widespread in human and mouse organs but is quantitatively and qualitatively distinct from NMHC II-A and II-B. In contrast to NMHC II-A and II-B, the mRNA level in human fetal tissues is substantially lower than in adult tissues. Immunofluorescence microscopy showed distinct patterns of expression for all three NMHC isoforms. NMHC II-C contains an alternatively spliced exon of 24 nucleotides in loop I at a location analogous to where a spliced exon appears in NMHC II-B and in the smooth muscle myosin heavy chain. However, unlike neuron-specific expression of the NMHC II-B insert, the NMHC II-C inserted isoform has widespread tissue distribution. Baculovirus expression of noninserted and inserted NMHC II-C heavy meromyosin (HMM II-C/HMM II-C1) resulted in significant quantities of expressed protein (mg of protein) for HMM II-C1 but not for HMM II-C. Functional characterization of HMM II-C1 by actin-activated MgATPase activity demonstrated a V(max) of 0.55 + 0.18 s(-1), which was half-maximally activated at an actin concentration of 16.5 + 7.2 microm. HMM II-C1 translocated actin filaments at a rate of 0.05 + 0.011 microm/s in the absence of tropomyosin and at 0.072 + 0.019 microm/s in the presence of tropomyosin in an in vitro motility assay.     

Cloning and characterization of a 22 kDa protein from rat adipocytes: a new member of the reticulon family

In the course of our examination of proteins associated with the GLUT4-containing vesicles of rat adipocytes we have identified a new 22 kDa member of the family of endoplasmic reticulum (ER) proteins known as reticulons. The protein, which we refer to as vp20, was purified from a preparation of GLUT4-containing vesicles of rat adipocytes, and tryptic peptides were micro-sequenced. From this information a cDNA encoding a single open reading frame for a protein of 22 kDa was cloned. This protein is homologous to known members of the reticulon protein family. vp20 has two hydrophobic stretches of about 35 amino acids that could be membrane spanning domains and an ER retention motif at its carboxy-terminus. vp20 was most abundant in the high density microsome fraction of adipocytes, which is the fraction most enriched in ER. Only a small fraction of vp20 was present in the GLUT4 vesicle population, and that fraction appears to be due to ER vesicles that were non-specifically bound to the adsorbent. Analysis of tissue distribution of vp20 in rats revealed that it is concentrated in muscle, fat and the brain.     

Cloning and characterization of a Saccharomyces cerevisiae gene encoding a new member of the ubiquitin-conjugating protein family

Ubiquitin-conjugating enzymes (E2s), which participate in the post-translational conjugation of ubiquitin to proteins, are encoded by a multigene family in the yeast Saccharomyces cerevisiae. E2s function in a variety of cellular activities including intracellular proteolysis, DNA repair, sporulation, and cell cycle traverse. Here, we report the cloning and characterization of a new member of the yeast UBC gene family, UBC8. UBC8 encodes a 206-amino acid protein containing a highly acidic carboxyl terminus. The primary structure of the protein is similar to that of all other known E2s, with the highest homology being to the E2 (23 kDa) of wheat germ. Haploid strains in which the UBC8 gene is disrupted are viable, and the disruption does not produce any obvious phenotype. The UBC8 protein, produced in Escherichia coli, forms thiol ester adducts with ubiquitin and, apparently, diubiquitin, but does not transfer ubiquitin to histones.     

Conkunitzin-S1 is the first member of a new Kunitz-type neurotoxin family. Structural and functional characterization

Conkunitzin-S1 (Conk-S1) is a 60-residue neurotoxin from the venom of the cone snail Conus striatus that interacts with voltage-gated potassium channels. Conk-S1 shares sequence homology with Kunitz-type proteins but contains only two out of the three highly conserved cysteine bridges, which are typically found in these small, basic protein modules. In this study the three-dimensional structure of Conk-S1 has been solved by multidimensional NMR spectroscopy. The solution structure of recombinant Conk-S1 shows that a Kunitz fold is present, even though one of the highly conserved disulfide cross-links is missing. Introduction of a third, homologous disulfide bond into Conk-S1 results in a functional toxin with similar affinity for Shaker potassium channels. The affinity of Conk-S1 can be enhanced by a pore mutation within the Shaker channel pore indicating an interaction of Conk-S1 with the vestibule of potassium channels.     

Identification and functional characterization of the BAG protein family in Arabidopsis thaliana

The genes that control mammalian programmed cell death are conserved across wide evolutionary distances. Although plant cells can undergo apoptosis-like cell death, plant homologs of mammalian regulators of apoptosis have, in general, not been found. This is in part due to the lack of primary sequence conservation between animal and putative plant regulators of apoptosis. Thus, alternative approaches beyond sequence similarities are required to find functional plant homologs of apoptosis regulators. Here, we present the results of using advanced bioinformatic tools to uncover the Arabidopsis family of BAG proteins. The mammalian BAG (Bcl-2-associated athanogene) proteins are a family of chaperone regulators that modulate a number of diverse processes ranging from proliferation to growth arrest and cell death. Such proteins are distinguished by a conserved BAG domain that directly interacts with Hsp70 and Hsc70 proteins to regulate their activity. Our searches of the Arabidopsis thaliana genome sequence revealed seven homologs of the BAG protein family. We further show that plant BAG family members are also multifunctional and remarkably similar to their animal counterparts, as they regulate apoptosis-like processes ranging from pathogen attack to abiotic stress and development.     

Isolation and characterization of a cDNA encoding a novel member of the human regenerating protein family: Reg IV

Human Reg and Reg-related genes constitute a multi-gene family belonging to the calcium (C-type) dependent lectin superfamily. Regenerating gene family members are expressed in the proximal gastrointestinal (GI) tract and ectopically at other sites in the setting of tissue injury. By high-throughput sequence analysis of a large inflammatory bowel disease library, two cDNAs have been isolated which encode a novel member of this multigene family. Based on primary sequence homology, tissue expression profiles, and shared exon-intron junction genomic organization, we assign this gene to the regenerating gene family. Specific protein structural differences suggest that the current three regenerating gene subtypes should be expanded to four. We demonstrate that Reg IV has a highly restricted tissue expression pattern, with prominent expression in the gastrointestinal tract. Reg IV mRNA expression is significantly up-regulated by mucosal injury from active Crohn's disease or ulcerative colitis.     

Cloning and functional characterization of ACAD-9, a novel member of human acyl-CoA dehydrogenase family

Acyl-CoA dehydrogenases (ACADs) are a family of mitochondrial enzymes catalyzing the initial rate-limiting step in the beta-oxidation of fatty acyl-CoA. The reaction provides main source of energy for human heart and skeletal muscle. Eight human ACADs have been described. Deficiency of these enzymes, especially very long-chain acyl-CoA dehydrogenase (VLCAD), usually leads to severe human organic diseases, such as sudden death in infancy, infantile cardiomyopathy (CM), hypoketotic hypoglycemia, or hepatic dysfunction. By large-scale random sequencing, we identified a novel homolog of ACADs from human dendritic cell (DC) cDNA library. It contains an open reading frame (ORF) of 1866bp, which encodes a 621 amino acid protein. It shares approximately 47% amino acid identity and 65% similarity with human VLCAD. So, the novel molecule is named as acyl-CoA dehydrogenase-9 (ACAD-9), the ninth member of ACADs. The new gene consists of 18 exons and 17 introns, and is mapped to chromosome 3q26. It contains the two signatures shared by all members of the ACADs. ACAD-9 mRNA is ubiquitously expressed in most normal human tissues and cancer cell lines with high level of expression in heart, skeletal muscles, brain, kidney, and liver. Enzymatic assay proved that the recombinant ACAD-9 protein has the dehydrogenase activity on palmitoyl-coenzyme A (C16:0) and stearoyl-coenzyme A (C18:0). Our results indicate that ACAD-9 is a novel member of ACADs.     

Cloning and characterization of a new member of the T-box gene family

The T-box is a strongly conserved protein domain, 174 to 186 amino acids in length, that binds DNA. Many genes from many species have been shown to encode T-box domain-containing proteins. Here we report the cloning and characterization of a novel T-box gene, TBX21. The human cDNA contains an open reading frame encoding a 535-amino-acid protein with a predicted molecular mass of 58.3 kDa. Except for the T-box sequence, database searches revealed no significant homology to any known sequences at the nucleotide or protein level. In addition to the human cDNA sequence, we report the cDNA sequence of the murine homologue, the structure and organization of the murine Tbx21 gene, and its localization to mouse chromosome 11. TBX21 expression was detected in peripheral blood lymphocytes, spleen, lung, and natural killer cells.