Identification and characterization of MtMTP1, a Zn transporter of CDF family,in the Medicago truncatula

Zn is an essential micronutrient in plants, and the mechanisms of Zn homeostasis are under intensive study. In this report, we have identified MtMTP1, a Zn transporter of the CDF family in the legume model plant Medicago truncatula. The ORF of the MtMTP1 cDNA encodes a protein consisting of 407 amino acid residues with a predicted molecular mass of 45 kDa. Like other metal tolerance proteins (MTPs) in plants, heterologous expression of MtMTP1 can complement the Zn-susceptible zrc1 cot1 yeast double mutant. The expression pattern was studied by quantitative fluorescent PCR. The expression of MtMTP1 was detected in all vegetative organs with the highest level of expression observed in leaves. With Zn supplementation its expression in roots was reduced while its expression in stems was increased in the first 2 days. No obvious changes were detected in leaves. Inoculation with Rhizobium meliloti down-regulated its expression in roots.     

Deneddylation修饰酶NEDP1的作用机制及研究进展

神经前体表达发育下调蛋白8(neural precursor cell-expressed developmentally downregulated 8,Nedd8)是一种类泛素蛋白,其参与蛋白质修饰的作用机制与泛素高度相似,即通过与底物的赖氨酸残基共价结合,从而对底物进行Neddylation修饰。Neddylation修饰可调控多种重要的生命活动,如细胞周期和免疫应答等。Nedd8蛋白酶1(Nedd8 protease 1, NEDP1)是隶属于小类泛素修饰物特异性蛋白酶家族(small ubiquitin-like modifier specific protease family, Ulp/Senp family)家族的半胱氨酸蛋白酶,可以特异性去除底物与Nedd8的共价结合,例如,NEDP1能够去除p53、鼠双微体蛋白(murine double min-utes 2 protein, Mdm2)、smad泛素化调节因子1(smad ubiquitylation regulatory factor 1, Smurf1)等多个蛋白质的Neddylation修饰,进而调节Neddylation修饰蛋白的生物学功能。临床研究表明,NEDP1与肿瘤的发生发展密切相关,包括肺癌、结肠癌、胶质母细胞瘤等,另外,NEDP1还参与成骨发育异常、神经退行性疾病、血管炎症等病变过程。本文对NEDP1蛋白的结构,调控Neddylation通路的作用模式和NEDP1作用底物的进展进行总结,以期为肿瘤等相关疾病的诊断和治疗提供参考。     

Identification and initial characterization of four novel members of the interleukin-1 family

Interleukin-1 (IL-1), fibroblast growth factors (FGFs), and their homologues are secreted factors that share a common beta-barrel structure and act on target cells by binding to cell surface receptors with immunoglobulin-like folds in their extracellular domain. While numerous members of the FGF family have been discovered, the IL-1 family has remained small and outnumbered by IL-1 receptor homologues. From expressed sequence tag data base searches, we have now identified four additional IL-1 homologues, IL-1H1, IL-1H2, IL-1H3, and IL-1H4. Like most other IL-1/FGFs, these proteins do not contain a hydrophobic leader sequence. IL-1H4 has a propeptide sequence, while IL-1H1, IL-1H2, and IL-1H3 encode only the mature protein. Circular dichroism spectra and thermal stability analysis suggest that IL-1H1 folds similarly to IL-1ra. The novel homologues are not widely expressed in mammals. IL-1H1 is constitutively expressed only in placenta and the squamous epithelium of the esophagus. However, IL-1H1 could be induced in vitro in keratinocytes by interferon-gamma and tumor necrosis factor-alpha and in vivo via a contact hypersensitivity reaction or herpes simplex virus infection. This suggests that IL-1H1 may be involved in pathogenesis of immune mediated disease processes. The addition of four novel IL-1 homologues suggests that the IL-1 family is significantly larger than previously thought.     

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 a sphingolipid delta 4-desaturase family

Sphingolipids desaturated at the Delta4-position are important signaling molecules in many eukaryotic organisms, including mammals. In a bioinformatics approach, we now identified a new family of protein sequences from animals, plants, and fungi and characterized these sequences biochemically by expression in Saccharomyces cerevisiae. This resulted in the identification of the enzyme sphingolipid Delta4-desaturase (dihydroceramide desaturase) from Homo sapiens, Mus musculus, Drosophila melanogaster, and Candida albicans, in addition to a bifunctional sphingolipid Delta4-desaturase/C-4-hydroxylase from M. musculus. Among the sequences investigated are the Homo sapiens membrane lipid desaturase, the M. musculus degenerative spermatocyte, and the Drosophila melanogaster degenerative spermatocyte proteins. During spermatogenesis, but not oogenesis of des mutant flies, both cell cycle and spermatid differentiation are specifically blocked at the entry into the first meiotic division, leading to male sterility. This mutant phenotype can be restored to wild-type by complementation with a functional copy of the des gene (Endo, K., Akiyama, T., Kobayashi S., and Okada, M. (1996) Mol. Gen. Genet. 253, 157-165). These results suggest that Delta4-desaturated sphingolipids provide an early signal necessary to trigger the entry into both meiotic and spermatid differentiation pathways during Drosophila spermatogenesis.     

Identification and characterization of a novel member of olfactomedin-related protein family, hGC-1, expressed during myeloid lineage development

We have cloned a novel hematopoietic granulocyte colony-stimulating factor (G-CSF)-induced olfactomedin-related glycoprotein, termed hGC-1 (human G-CSF-stimulated clone-1). mRNA differential display was used in conjunction with a modified two-phase liquid culture system. Cultures were enriched for early precursors of erythroid, myeloid, and megakaryocytic lineages, which were isolated after induction with erythropoietin, G-CSF, and thrombopoietin, respectively. RNA from the enriched cells was subjected to differential display analysis to identify lineage-specific expressed genes. One clone specifically induced by G-CSF, hGC-1, was characterized. The 2861 bp cDNA clone of hGC-1 contained an open reading frame of 1530 nucleotides, translating into a protein of 510 amino acids with a signal peptide and six N-linked glycosylation motifs. The protein sequence of hGC-1 showed it to be a glycoprotein of the olfactomedin family, which includes olfactomedin, TIGR, Noelin-2 and latrophilin-1. Olfactomedin-like genes show characteristic tissue-restricted patterns of expression; the specific tissues expressing these genes differ among the family members. hGC-1 was strongly expressed in the prostate, small intestine, and colon, moderately expressed in the bone marrow and stomach, and not detectable in other tissues. In vitro translation and ex vivo expression showed hGC-1 to be an N-linked glycoprotein. The hGC-1 gene locus mapped to chromosome 13q14.3. Together, our findings indicate that hGC-1 is primarily expressed as an extracellular olfactomedin-related glycoprotein during normal myeloid-specific lineage differentiation, suggesting the possibility of a matrix-related function for hGC-1 in differentiation.     

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.     

Identification, characterization and cloning of myr 1, a mammalian myosin-I

We have identified, characterized and cloned a novel mammalian myosin-I motor-molecule, called myr 1 (myosin-I from rat). Myr 1 exists in three alternative splice forms: myr 1a, myr 1b, and myr 1c. These splice forms differ in their numbers of putative calmodulin/light chain binding sites. Myr 1a-c were selectively released by ATP, bound in a nucleotide-dependent manner to F-actin and exhibited amino acid sequences characteristic of myosin-I motor domains. In addition to the motor domain, they contained a regulatory domain with up to six putative calmodulin/light chain binding sites and a tail domain. The tail domain exhibited 47% amino acid sequence identity to the brush border myosin-I tail domain, demonstrating that myr 1 is related to the only other mammalian myosin-I motor molecule that has been characterized so far. In contrast to brush border myosin-I which is expressed in mature enterocytes, myr 1 splice forms were differentially expressed in all tested tissues. Therefore, myr 1 is the first mammalian myosin-I motor molecule with a widespread tissue distribution in neonatal and adult tissues. The myr 1a splice form was preferentially expressed in neuronal tissues. Its expression was developmentally regulated during rat forebrain ontogeny and subcellular fractionation revealed an enrichment in purified growth cone particles, data consistent with a role for myr 1a in neuronal development.     

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.     

Identification and characterization of a family of Rab11-interacting proteins

Rab11a is a small GTP-binding protein enriched in the pericentriolar plasma membrane recycling systems. We hypothesized that Rab11a-binding proteins exist as downstream effectors of its action. Here we define a family of four Rab11-interacting proteins: Rab11-Family Interacting Protein 1 (Rab11-FIP1), Rab11-Family Interacting Protein 2 (Rab11-FIP2), Rab11-Family Interacting Protein 3 (Rab11-FIP3), and pp75/Rip11. All four interacting proteins associated with wild type Rab11a and dominant active Rab11a (Rab11aS20V) as well as Rab11b and Rab25. Rab11-FIP2 also interacted with dominant negative Rab11a (Rab11aS25N) and the tail of myosin Vb. The binding of Rab11-FIP1, Rab11-FIP2, and Rab11-FIP3 to Rab11a was dependent upon a conserved carboxyl-terminal amphipathic alpha-helix. Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 colocalized with Rab11a in plasma membrane recycling systems in both non-polarized HeLa cells and polarized Madin-Darby canine kidney cells. GFP-Rab11-FIP3 also colocalized with Rab11a in HeLa cells. Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 also coenriched with Rab11a and H(+)K(+)-ATPase on parietal cell tubulovesicles, and Rab11-FIP1 and Rab11-FIP2 translocated with Rab11a and the H(+)K(+)-ATPase upon stimulating parietal cells with histamine. The results suggest that the function of Rab11a in plasma membrane recycling systems is dependent upon a compendium of protein effectors.     

Identification and characterization of YME1L1, a novel paraplegin-related gene

A gene responsible for an autosomal recessive form of hereditary spastic paraplegia (SPG7) was recently identified. This gene encodes paraplegin, a mitochondrial protein highly homologous to the yeast mitochondrial AAA proteases Afg3p, Rca1p, and Yme1p, which have both proteolytic and chaperone-like activities at the inner mitochondrial membrane. By screening the expressed sequence tag database, we identified and characterized a novel human gene, YME1L1 (YME1L1-like1, HGMW-approved symbol). This gene encodes a predicted protein of 716 amino acids highly similar to all mitochondrial AAA proteases and in particular to yeast Yme1p. Expression and immunofluorescence studies revealed that YME1L1 and paraplegin share a similar expression pattern and the same subcellular localization in the mitochondrial compartment. YME1L1 may represent a candidate gene for other forms of hereditary spastic paraplegia and possibly for other neurodegenerative disorders.