Cloning and characterization of BS-cadherin, a novel cadherin from the colonial urochordate Botryllus schlosseri

The genomic DNA for a novel member of the cadherin family (BS-cadherin) was cloned and characterized from the colonial marine invertebrate, Botryllus schlosseri. Using a differential display of mRNA by means of PCR, a small cDNA fragment of 380 nucleotides was found to be specifically expressed in a colony undergoing allogeneic rejection processes, as compared with naïve parts of the same genotype. This cDNA fragment was used as a probe to screen a genomic library of Botryllus schlosseri. A genomic fragment containing an ORF of 2718 nucleotides, with no introns, was isolated. The encoded protein exhibits a typical structure of cadherins; an extracellular domain with conserved repeated sequences (cadherin signatures), a single transmembrane domain and a conserved cytoplasmic tail region. The BS-cadherin amino-acid sequence shows 32–35% identity to mature classical cadherins type I, e.g., N-, P- and E-cadherin as well as mature classical cadherins type II, e.g., human cadherin-6, -8 and OB-cadherin. This cadherin represents a new cadherin gene family, evolutionarily distant to all other known classical cadherins.     

Isolation and characterization of a cDNA encoding a serine proteinase from the root-knot nematode Meloidogyne incognita

This report describes the first serine proteinase gene isolated from the sedentary nematode Meloidogyne incognita. Using degenerate primers, a 1372bp cDNA encoding a chymotrypsin-like serine proteinase (Mi-ser1) was amplified from total RNA of adult females by RT-PCR and 5' and 3' rapid amplification of cDNA ends. The deduced amino acid sequence of Mi-ser1 encoded a putative signal peptide and a prodomain of 22 and 33 amino acids, respectively, and a mature proteinase of 341 amino acids with a predicted molecular mass of 37,680Da. Sequence identity with the top serine proteinases matches from the databases ranged from 23 to 27%, including sequences from insects, mammals, and other nematodes. Southern blot analysis suggested that Mi-ser1 is encoded by a single or few gene copies. The pattern of developmental expression analyzed by Northern blot and RT-PCR indicated that Mi-ser1 was transcribed mainly in females. The domain architecture composed of a single chymotrypsin-like catalytic domain and the detection of a putative signal peptide suggested a digestive role for Mi-ser1.     

Eimeria tenella: cloning of a novel Eimeria tenella cDNA encoding a protein related to rhomboid family from F2 hybrid strain

A novel cDNA sequence with an open reading frame of 774 bp from Eimeria tenella F2 hybrid strain (ETRH01) was isolated from a lambda cDNA library with a monoclonal antibody against sporozoite. Analysis of the genomic sequence suggests that this is an intronless gene. The deduced protein sequence has 257 amino acids with a calculated molecular weight of 28.349 kDa and an isoelectric point of 8.56. Sequence analysis revealed seven transmembrane domains and a rhomboid domain within the protein. RT-PCR result indicates that this gene was expressed in all of the five E. tenella isolates analyzed. To further study the role of this novel gene in the life cycle of E. tenella, ETRH01 was successfully expressed using pET28b(+) expression system.     

Cypher, a striated muscle-restricted PDZ and LIM domain-containing protein, binds to alpha-actinin-2 and protein kinase C.

We have cloned and characterized a novel striated muscle-restricted protein (Cypher) that has two mRNA splice variants, designated Cypher1 and Cypher2. Both proteins contain an amino-terminal PDZ domain. Cypher1, but not Cypher2, contains three carboxyl-terminal LIM domains and an amino acid repeat sequence that exhibits homology to a repeat sequence found in the largest subunit of RNA polymerase II. cypher1 and cypher2 mRNAs exhibited identical expression patterns. Both are exclusively expressed in cardiac and striated muscle in embryonic and adult stages. By biochemical assays, we have demonstrated that Cypher1 and Cypher2 bind to alpha-actinin-2 via their PDZ domains. This interaction has been further confirmed by immunohistochemical studies that demonstrated co-localization of Cypher and alpha-actinin at the Z-lines of cardiac muscle. We have also found that Cypher1 binds to protein kinase C through its LIM domains. Phosphorylation of Cypher by protein kinase C has demonstrated the functional significance of this interaction. Together, our data suggest that Cypher1 may function as an adaptor in striated muscle to couple protein kinase C-mediated signaling, via its LIM domains, to the cytoskeleton (alpha-actinin-2) through its PDZ domain.     

The PDZ Domain of the LIM Protein Enigma Binds to β-Tropomyosin

PDZ and LIM domains are modular protein interaction motifs present in proteins with diverse functions. Enigma is representative of a family of proteins composed of a series of conserved PDZ and LIM domains. The LIM domains of Enigma and its most related family member, Enigma homology protein, bind to protein kinases, whereas the PDZ domains of Enigma and family member actin-associated LIM protein bind to actin filaments. Enigma localizes to actin filaments in fibroblasts via its PDZ domain, and actin-associated LIM protein binds to and colocalizes with the actin-binding protein alpha-actinin-2 at Z lines in skeletal muscle. We show that Enigma is present at the Z line in skeletal muscle and that the PDZ domain of Enigma binds to a skeletal muscle target, the actin-binding protein tropomyosin (skeletal beta-TM). The interaction between Enigma and skeletal beta-TM was specific for the PDZ domain of Enigma, was abolished by mutations in the PDZ domain, and required the PDZ-binding consensus sequence (Thr-Ser-Leu) at the extreme carboxyl terminus of skeletal beta-TM. Enigma interacted with isoforms of tropomyosin expressed in C2C12 myotubes and formed an immunoprecipitable complex with skeletal beta-TM in transfected cells. The association of Enigma with skeletal beta-TM suggests a role for Enigma as an adapter protein that directs LIM-binding proteins to actin filaments of muscle cells.     

ZRP-1, a zyxin-related protein, interacts with the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E.

Protein-protein interactions play an important role in the specificity of cellular signaling cascades. By using the yeast two-hybrid system, a specific interaction was identified between the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E and a novel protein, which was termed ZRP-1 to indicate its sequence similarity to the Zyxin protein family. The mRNA encoding this protein is distributed widely in human tissues and contains an open reading frame of 1428 base pairs, predicting a polypeptide of 476 amino acid residues. The deduced protein displays a proline-rich amino-terminal region and three double zinc finger LIM domains at its carboxyl terminus. The specific interaction of this novel protein with the second PDZ domain of hPTP1E was demonstrated both in vitro, using bacterially expressed proteins, and in vivo, by co-immunoprecipitation studies. Deletion analysis indicated that an intact carboxyl terminus is required for its interaction with the second PDZ domain of hPTP1E in the yeast two-hybrid system and suggested that other sequences, including the LIM domains, also participate in the interaction. The genomic organization of the ZRP-1 coding sequence is identical to that of the lipoma preferred partner gene, another Zyxin-related protein, suggesting that the two genes have evolved from a recent gene duplication event.     

LIM蛋白家族的研究进展

LIM蛋白是分子结构中含有一个或多个LIM结构域的蛋白质家族,该家族中的蛋白质通过其LIM结构域与某些结构蛋白,激酶,转录调控因子等多种蛋白质相互作用,对某些基因的表达,细胞分化与发育,细胞骨架形成等发挥重要调控作用。本文介绍LIM蛋白家族的分类与功能,LIM蛋白及其与其他蛋白之间的相互作用,以及LIM蛋白在心血管系统中的作用。     

Distinct LIM domains of Hic-5/ARA55 are required for nuclear matrix targeting and glucocorticoid receptor binding and coactivation

Hydrogen peroxide-inducible clone-5 (Hic-5), belongs to the group III LIM domain protein family and contains four carboxyl-terminal LIM domains (LIM1-LIM4). In addition to its role in focal adhesion signaling, Hic-5 acts in the nucleus as a coactivator for some steroid hormone receptors such as the glucocorticoid receptor (GR) and androgen receptor (AR). Based upon its effect on AR transactivation, Hic-5 has also been designated as ARA55. Here, we report mapping studies of Hic-5/ARA55 functional domains and establish that LIM3 and LIM4 are necessary for maximal effects on GR transactivation. However, results from yeast two-hybrid assays demonstrated that these two LIM domains together, while necessary, are not sufficient to interact with the tau2 transactivation domain of GR. LIM4 also functions as a nuclear matrix targeting sequence (NMTS) for Hic-5/ARA55, as it is both necessary and sufficient to target a heterologous protein to the nuclear matrix. Thus, as suggested from previous analysis of LIM domain-containing proteins, separate but highly related LIM domains serve distinct functions.     

A new multi-domain member of the cystatin superfamily expressed by Fasciola hepatica

Cystatins are cysteine protease inhibitors that are widespread in the plant and animal kingdoms. Cystatins are expressed by helminth parasites that may employ these proteins to regulate parasite cysteine protease activity and to modulate host immune responses. Here, we describe the cloning of a cDNA encoding a high molecular weight protein of Fasciola hepatica that contains two domains with significant identity to the cardinal cystatin signatures and four domains with degenerated cystatin signatures. This is the first report of a multi-domain cystatin in an invertebrate species. While cystatins are divided into three evolutionary related families, our phylogenetic analysis shows that all cystatin domains within this protein, like several other helminth cystatins, belong to the cystatin family 2. The DNA region encoding the domain 4 that is the best conserved at the level of its cystatin signatures was expressed in Drosophila cells and a recombinant protein was produced and purified. This protein was a potent inhibitor of the papain and of the major cysteine protease of F. hepatica, the cathepsin L1.     

Sequence similarities between chicken intestinal 110-kDa ATPase and myosin I-like enzymes

We report the partial amino acid sequence of chicken intestinal microvillar 110-kDa protein that, as a complex with calmodulin, has previously been shown to exhibit myosin-like ATPase and actin-binding activities. The sequence shows a high degree of similarity to the sequence of a novel vertebrate myosin I-like heavy chain encoded by a cDNA isolated from bovine intestine. This confirms that the bovine and chicken proteins are the first examples of Acanthamoeba myosin I-like proteins from higher eukaryotes. Comparison of available structural and functional data leads us to postulate that the myosin I family of proteins result from the fusion of a conserved myosin headlike motor domain, with variable COOH-terminal domains responsible for binding to specific intracellular structures.     

VHDL, a larval storage protein from the corn earworm, Helicoverpa zea, is a member of the vitellogenin gene family

The hemolymph of last instar larvae of the corn earworm, Helicoverpa zea contains a blue very high-density lipoprotein (VHDL) that is selectively taken up into fat body prior to pupation. Its amino-terminal sequence was determined by Edman degradation, and used to design a degenerate primer for PCR amplification. With 5' and 3' RACE techniques, the entire cDNA coding for VHDL was amplified and sequenced. Conceptual translation reveals a 173 kDa protein that contains a 15 amino acid signal sequence immediately before the experimentally determined N-terminus of the mature protein. The protein contains a typical lipoprotein N-terminal domain, and shows high sequence similarity to vitellogenins from Lepidoptera and other insect species. VHDL mRNA was not detectable in adult H. zea, and antibodies raised against VHDL did not react with adult hemolymph or yolk proteins. Therefore VHDL, although a member of the vitellogenin gene family, seems to be distinct from the vitellogenin expressed in adult females.     

小立碗藓冷驯化相关基因Pp-LIM only A的克隆与表达

植物经历冷驯化后抗冻能力会有所提高.利用cDNA-AFLP方法从经过0℃冷驯化处理的小立碗藓中筛选到差异表达的Pp-LIM only A基因片段.cDNA和基因序列比较分析表明此基因含有7个内含子和8个外显子,编码由345个氨基酸残基组成的蛋白质,其中只含有一个LIM结构域,与动物蛋白质PDZ/LIM家族有很高的同源性,推测是一种新的植物LIM蛋白.实时定量PCR分析显示其在冷驯化6 h后表达量即开始明显增加,并随着冷驯化时间的延长表达量大幅度提高.Pp-LIM only A蛋白可能通过LIM结构域对细胞骨架的作用而影响了细胞膜的稳定性,本研究对其在抗冻中的作用作了进一步讨论.     

Taenia solium: identification and preliminary characterization of a lipid binding protein with homology to the SEC14 catalytic domain

The objective of this work is to identify proteins of the human and porcine parasite, Taenia solium, which may be exploited for control of the parasite. Through screening a cDNA library of T. solium metacestodes, we have identified a novel Sec-14-like Taenia lipid-binding protein that may play an important role in membrane trafficking. The Sec14-like sequence is a single copy gene, encoding a putative polypeptide of 320 amino acids and 36.1 kDa (sec14Tsol protein). Secondary amino acid structural analysis suggested that the sec14Tsol protein might contain two distinct structural domains, an amino-terminal alpha-helix rich domain and a mixed alpha-helix/beta-stand carboxy-terminal zone, showing homology with the conserved SEC14 domain found in a great number of proteins that bind lipids, as the regulators of membrane trafficking between Golgi membrane bilayers. Significantly, therefore, in a phosphoinositide-binding assay, sec14Tsol purified recombinant protein specifically interacted with important lipid regulators of membrane trafficking, with a preference for PI(3)P(2), PI(3,4)P(2), PI(4,5)P(2) and phosphatidic acid. Moreover, the sec14Tsol protein was localized in the Golgi apparatus of transfected cells and in the spiral canal region of T. solium metacestode tegument. As sec14Tsol protein may play an important role in membrane trafficking, its demonstrated localisation in the intact parasite tegument suggests its involvement in the function of the tegument and thus perhaps interaction with the host.     

SCLIP: A Novel SCG10-Like Protein of the Stathmin Family Expressed in the Nervous System

Abstract: Stathmin is a cytosolic phosphoprotein previously described as a ubiquitous relay integrating various signaling pathways. It is the generic element of a protein family in mammals as in Xenopus , including SCG10, a neuron-specific, growth-associated protein, and RB3/XB3, related to the expression of differentiated functions of mature cells of the nervous system. In an extensive search for other members of the stathmin family, we identified cDNAs coding for two novel stathmin-related proteins: (a) a cDNA from a rat striatum cDNA library codes for RB3", a splice variant of RB3, with an additional basic domain in its N-terminal region; and (b) another cDNA identified through a systematic search in EST databases codes for a novel protein, SCLIP, for "SCG10-like protein," displaying 70% identity with SCG10 and sharing the same domain organization, with an N-terminal domain likely involved in membrane attachment and a C-terminal stathmin-like domain. Northern blot analysis as well as in situ hybridization on 14-day rat embryos showed that SCLIP mRNA is expressed only in neural structures. SCLIP mRNA is expressed at comparable levels in neonatal and adult rat brain, suggesting a potential role not only in the acquisition, but also in the expression of differentiated neuronal functions.