丝氨酸蛋白酶超家族分子结构进化研究

采用刚体结构比较法进行蛋白质的结构比较,根据结构比较分数构建分子进化树, 研究丝氨酸蛋白酶超家族分子的进化规律。对分子进化树进行了一些初步分析,得到了一些有意义的结果。根据蛋白质的进化,可以比较精确的确定某物种的进化地位,对于物种的分类具有重要意义。通过对超家族分子进化的研究可以了解蛋白质超家族不同蛋白质之间的亲缘关系和蛋白质之间的进化差异,对于蛋白质工程分子设计提供帮助,对蛋白质结构预测具有一定意义     

Molecular and Functional Characterization of the Four-Transmembrane Molecule L6 in Epidermal Keratinocytes

In normal human epidermal keratinocytes (NHEK) proteolytic detachment from the substrate induces a complex activation cascade including expression of new proteins, morphological alterations, and the onset of migration for epidermal regeneration. By subtractive cloning we have shown that L6, a four-transmembrane protein, is newly expressed after proteolytic keratinocyte detachment. In this study, we have generated a novel anti-L6 antibody (clone HD-pKe#104-1.1) and investigated L6 expression regulation in vitro and in vivo as well as L6 function in keratinocyte migration. Dispase-mediated detachment induced L6 expression in NHEK at the mRNA and protein level. Immunohistology of skin biopsies displayed a strong expression of L6 in follicular epidermis and epidermolytic lesions of autoimmune bullous dermatoses (bullous pemphigoid, pemphigus vulgaris), but not in normal interfollicular epidermis. In contrast to normal keratinocytes, HaCaT cells showed constitutive L6 expression, indicating a constitutively active phenotype. After artificial wounding of confluent HaCaT cultures, anti-L6 antibody strongly impaired cell migration velocity and migratory reepithelization of the defect, indicating L6 involvement in keratinocyte migration. These findings suggest that L6 is an important activation-dependent regulator of keratinocyte function and epidermal tissue regeneration.     

模式匹配分析方法检索免疫球蛋白样功能区

运用计算机进行核酸和蛋白质的序列分析是分子生物学研究的一个较新发展,这项技术已越来越多地用于研究大量积累的序列数据。蛋白质功能区是蛋白质分子中能独立折叠成具有一定结构并执行特定功能的结构域,所有具有同一类功能区的分子统称为一个蛋白质的超族(protein superfamily)。本文通过对免疫球蛋白(Ig)超族及其功能区序列所进行的分析,建立了一种根据功能区之保守片段残基组成的模式匹配分析检索蛋白质功能区的方法,它先根据多序列的对准比较确定某一类功能区之保守片段,再对已知的保守片段各位置上氨基酸残基组成进行统计分析,然后根据与统计数值相匹配的方法,计算待检序列残基组成的统计学意义,由此确定功能区的存在。该方法的优点在于它不仅可以检出已知的具有某一类功能区的分子,而且还可能发现新的具有该功能区的分子,从而推测后者的功能。     

What Was the Set of Ubiquitin and Ubiquitin-Like Conjugating Enzymes in the Eukaryote Common Ancestor?

Ubiquitin (Ub)-conjugating enzymes (E2) are key enzymes in ubiquitination or Ub-like modifications of proteins. We searched for all proteins belonging to the E2 enzyme super-family in seven species (Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Arabidopsis thaliana) to identify families and to reconstruct each family’s phylogeny. Our phylogenetic analysis of 207 genes led us to define 17 E2 families, with 37 E2 genes, in the human genome. The subdivision of E2 into four classes did not correspond to the phylogenetic tree. The sequence signature HPN (histidine–proline–asparagine), followed by a tryptophan residue at 16 (up to 29) amino acids, was highly conserved. When present, the active cysteine was found 7 to 8 amino acids from the C-terminal end of HPN. The secondary structures were characterized by a canonical alpha/beta fold. Only family 10 deviated from the common organization because the proteins were devoid of enzymatic activity. Family 7 had an insertion between beta strands 1 and 2; families 3, 5 and 14 had an insertion between the active cysteine and the conserved tryptophan. The three-dimensional data of these proteins highlight a strong structural conservation of the core domain. Our analysis shows that the primitive eukaryote ancestor possessed a diversified set of E2 enzymes, thus emphasizing the importance of the Ub pathway. This comprehensive overview of E2 enzymes emphasizes the diversity and evolution of this superfamily and helps clarify the nomenclature and true orthologies. A better understanding of the functions of these enzymes is necessary to decipher several human diseases. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expansion of the APC superfamily of secondary carriers

The amino acid‐polyamine‐organoCation (APC) superfamily is the second largest superfamily of secondary carriers currently known. In this study, we establish homology between previously recognized APC superfamily members and proteins of seven new families. These families include the PAAP (Putative Amino Acid Permease), LIVCS (Branched Chain Amino Acid:Cation Symporter), NRAMP (Natural Resistance‐Associated Macrophage Protein), CstA (Carbon starvation A protein), KUP (K⁺ Uptake Permease), BenE (Benzoate:H⁺ Virginia Symporter), and AE (Anion Exchanger). The topology of the well‐characterized human Anion Exchanger 1 (AE1) conforms to a UraA‐like topology of 14 TMSs (12 α‐helical TMSs and 2 mixed coil/helical TMSs). All functionally characterized members of the APC superfamily use cation symport for substrate accumulation except for some members of the AE family which frequently use anion:anion exchange. We show how the different topologies fit into the framework of the common LeuT‐like fold, defined earlier (Proteins. 2014 Feb;82(2):336‐46), and determine that some of the new members contain previously undocumented topological variations. All new entries contain the two 5 or 7 TMS APC superfamily repeat units, sometimes with extra TMSs at the ends, the variations being greatest within the CstA family. New, functionally characterized members transport amino acids, peptides, and inorganic anions or cations. Except for anions, these are typical substrates of established APC superfamily members. Active site TMSs are rich in glycyl residues in variable but conserved constellations. This work expands the APC superfamily and our understanding of its topological variations. Proteins 2014; 82:2797–2811. © 2014 Wiley Periodicals, Inc.     

A common protocadherin tail: Multiple protocadherins share the same sequence in their cytoplasmic domains and are expressed in different regions of brain

To study the diversity of protocadherins, a rat brain cDNA library was screened using a cDNA for the cytoplasmic domain of human protocadherin Pcdh2 as a probe. The resultant clones contained three different types. One type corresponds to rat Pcdh2; the other two types are distinct from Pcdh2 but contain the same sequence in their cytoplasmic domains and part of the 3′ flanking sequence. To clarify the structure of the proteins defined by the new clones, a putative entire coding sequence corresponding to one of the clones was determined. The overall structure is essentially the same as Pcdh2, indicating that the proteins defined by this clone, and probably by other clones, belong to the protocadherin family. Two PCR experiments and an RNase protection assay showed the existence of the corresponding mRNAs in rat brain preparations. Human and mouse cDNA clones with the same sequence properties were also isolated. Taken together, these results indicate that the clones are not cloning artifacts and that corresponding mRNAs are actually expressed in brains of various species. The results of in situ hybridization showed that the mRNAs corresponding to these clones were expressed in different regions in brain. Since protocadherins encoded by these mRNAs are likely to have different specificity in their interaction and share a common activity at their cytoplasmic domains, these protocadherins may provide a molecular basis, in part, to support the complex cell-cell interaction in brain.     

Structure of Xanthomonas axonopodis pv. citri YaeQ reveals a new compact protein fold built around a variation of the PD-(D/E)XK nuclease motif

The YaeQ family of proteins are found in many Gram-negative and a few Gram-positive bacteria. We have determined the first structure of a member of the YaeQ family by X-ray crystallography. Comparisons with other structures indicate that YaeQ represents a new compact protein fold built around a variation of the PD-(D/E)XK nuclease motif found in type II endonucleases and enzymes involved in DNA replication, repair, and recombination. We show that catalytically important residues in the PD-(D/E)XK nuclease superfamily are spatially conserved in YaeQ and other highly conserved YaeQ residues may be poised to interact with nucleic acid structures.     

Rediscovered parasitism of Andrena savignyi Spinola (Hymenoptera,Andrenidae) by Stylops (Strepsiptera,Stylopidae) and revised taxonomic status of the parasite

Parasitism of Andrena (Suandrena) savignyi Spinola (Hymenoptera: Andrenidae) by Stylops Kirby (Strepsiptera: Stylopidae) has been recorded only once, and from an individual collected in Egypt almost a century ago, with the parasite described as Stylops savignyi Hofeneder. The recent rediscovery of this Stylops from an individual of Andrena savignyi permits a reinterpretation of the species and its affinities among other Stylops. The bee was collected at flowers of Zilla spinosa (Turra) Prantl. (Brassicaceae) in Amariah, Riyadh, Kingdom of Saudi Arabia. Based on DNA barcode sequences from material sampled across Africa, Asia, and Europe, it is apparent that Stylops savignyi is conspecific with Stylops nassonowi Pierce, and we accordingly synonymize this name (syn. n.), with the latter representing the senior and valid name for the species. A differential diagnosis is provided for Stylops nassonowi and the morphology of the female is described, as well as the first instars.     

Contribution to the knowledge of Galumnoidea (Acari,Oribatida) of Cuba

An annotated checklist of identified oribatid mites of the superfamily Galumnoidea collected from Cuba, including ten species from four genera and two families, is provided. Galumna flabellifera Hammer, 1958, Pergalumna bifissurata Hammer, 1972, Pergalumna bryani (Jacot, 1934), Pergalumna decorata Balogh & Mahunka, 1977 and Galumnopsis secunda Sellnick, 1923 are recorded for the first time in the Cuban fauna. A new species of Pergalumna, Pergalumna cubaensis sp. n., is described; it is morphologically similar to Pergalumna decorata Balogh & Mahunka, 1977, but differs from the latter by the larger body size, heavily granulated prodorsum and well-developed interlamellar setae. The adult of Allogalumna cubana Balogh & Mahunka, 1979 is redescribed.     

Impaired growth of pancreatic exocrine cells in transgenic mice expressing human activin betaE subunit

Activins, TGF-beta superfamily members, have multiple functions in a variety of cells and tissues. Recently, additional activin beta subunit genes, betaC and betaE, have been identified. To explore the role of activin E, we created transgenic mice overexpressing human activin betaE subunit. There were pronounced differences in the pancreata of the transgenic animals as compared with their wild-type counterparts. Pancreatic weight, expressed relative to total body weight, was significantly reduced. Histologically, adipose replacement of acini in the exocrine pancreas was observed. There was a significant decrease in the number of PCNA-positive cells in the acinar cells, indicating reduced proliferation in the exocrine pancreas of the transgenic mice. However, quantitative pancreatic morphometry showed that the total number and mass of the islets of the transgenic mice were comparable with those of the nontransgenic control mice. Our findings suggest a role for activin E in regulating the proliferation of pancreatic exocrine cells.