人和大鼠自噬相关新基因WIPI- 3 的电子克隆和序列分析

目的：人和大鼠WIPI-3基因的电子克隆和序列分析。方法：综合运用基因电子拼接和比较基因组分析等生物信息学手段进行新基因的电子克隆和注释。通过拼接CR593190、NM 019613和BC00097 cDNA序列获得人WIPI-3 cDNA全长序列,通过拼接EST序列CB727439、CB737031、BF557312、BG663387和预测的CDS获得大鼠WIPI3基因的cDNA序列。结果：成功克隆了人和大鼠自噬相关新基因WIPI-3的cDNA,上述两个新基因序列均得到了人类基因组序列和EST序列的双重支持,另外经RT-PCR验证电子克隆的基因序列也是正确的,而且序列均已被GenBank收录,其编号分别为：AM182326和NM-001039587。其中,人WIPI-3基因修正了目前基因库中注释的WIPI-3序列,而大鼠基因则是国际上首次克隆,井被确定为参考序列。结论：基因电子拼接结合种属同源基因比较分析,可大大提高电子克隆的精确性,这种方法可有效用于新基因的克隆和注释。     

Ras-like Small GTPases Form a Large Family of Proteins in the Marine Sponge <Emphasis Type="Italic">Suberites domuncula</Emphasis>

Sponges (Porifera) are the simplest and the most ancient metazoan animals, which branched off first from the common ancestor of all multicellular animals. We have inspected ∼13,000 partial cDNA sequences (ESTs) from the marine sponge Suberites domuncula and have identified full or partial cDNA sequences coding for ∼50 different Ras-like small GTPases. Forty-four sponge proteins from the Ras family are described here: 6 proteins from the Ras subfamily, 5 from Rho, 6 from Arf, 1 Ran, and 26 Rabs or Rab-like proteins. No isoforms of these proteins were detected; the closest related proteins are two Rho proteins with 74% identity. Small GTPases from sponge display a higher degree of sequence conservation with orthologues from vertebrates (53%–93% identity) than with those from either Caenorhabditis elegans or Drosophila melanogaster. The real number of small GTPases in this sponge is certainly much higher than 50, because the actual S. domuncula database of ∼13,000 ESTs contains at most 3000 nonredundant cDNA sequences. The number of genes for Ras-like small GTPases in yeast, C. elegans, D. melanogaster, and humans is 30, 56, 90, and 174, respectively. Both model invertebrates have only 29 Rabs or Rab-like proteins, compared with 26 already found in sponge, and are missing at least 1 Rab (Rab24) present in S. domuncula and mammals. Our results indicate that duplications and diversifications of genes encoding Ras-like small GTPases, especially the Rab subfamily of small GTPases, happened very early in the evolution of Metazoa. Reviewing Editor: Dr. Martin Kreitman Vera Gamulin passed away on 12 October 2006. We feel privileged to have had the opportunity to have worked with her.     

Proteome analysis of wheat lemma

We report here for the first time on the construction of proteomes from wheat lemma at the anthesis stage. After transfer of lemma proteins to polyvinylidene difluoride membranes, seventy larger spots were subjected to peptide sequence analysis; the amino acid sequences could be described for forty-eight of these proteins. The result suggested that wheat proteins were less N-terminally blocked compared to rice proteins, which are known to have a much higher ratio of N-terminal blocks. We further analyzed the internal sequences of eight blocked proteins by the Cleveland peptide mapping method. Out of these total 56 amino acid sequences, forty-one could be assigned to the corresponding expressed sequence tags (ESTs). The expression profile of lemma proteins was generally similar to that of leaf, and the majority of identified proteins were related to cellular metabolisms. We analyzed the internal sequences of one protein spot present in lemma, which was not present in leaf.     

Isolation, characterization, and mapping of the mouse and human WDR8 genes, members of a novel WD-repeat gene family

The Trp-Asp (WD) motif has been shown to exist in a number of proteins. Genes containing repeats of the WD motif compose a large gene family associated with a variety of cellular functions and can be divided into a number of functional subfamilies. By means of the differential display method using ttw, a mouse model for the early stage of ectopic ossification, we have identified a novel mouse gene, Wdr8 (WD repeat domain 8), which contains two WD repeats, together with its human orthologue. The human and mouse WDR8 genes encode 460 and 462 amino acids, respectively, with 89% identity, and are expressed in almost all tissues, including bone and cartilage, and in bone-forming cells, including osteoblasts and chondrocytes. Wdr8 expression in cartilage was differentially displayed by stimuli for ectopic ossification in ttw and was observed strongly only at a transition period from hypertrophic to mineralizing stages in ATDC5, a chondrogenic cell line that exhibits endochondral ossification, suggesting a potential role for Wdr8 in the process of ossification. The WDR8 protein is highly conserved among a variety of species, but is distinctly different from other WD-repeat proteins, indicating that it represents a novel subfamily of the WD-repeat gene family.     

Identification, structure, and differential expression of members of a BURP domain containing protein family in soybean.

Expressed sequence tags (ESTs) exhibiting homology to a BURP domain containing gene family were identified from the Glycine max (L.) Merr. EST database. These ESTs were assembled into 16 contigs of variable sizes and lengths. Consistent with the structure of known BURP domain containing proteins, the translation products exhibit a modular structure consisting of a C-terminal BURP domain, an N-terminal signal sequence, and a variable internal region. The soybean family members exhibit 35-98% similarity in a -100-amino-acid C-terminal region, and a phylogenetic tree constructed using this region shows that some soybean family members group together in closely related pairs, triplets, and quartets, whereas others remain as singletons. The structure of these groups suggests that multiple gene duplication events occurred during the evolutionary history of this family. The depth and diversity of G. max EST libraries allowed tissue-specific expression patterns of the putative soybean BURPs to be examined. Consistent with known BURP proteins, the newly identified soybean BURPs have diverse expression patterns. Furthermore, putative paralogs can have both spatially and quantitatively distinct expression patterns. We discuss the functional and evolutionary implications of these findings, as well as the utility of EST-based analyses for identifying and characterizing gene families.     

The structure of Ski8p, a protein regulating mRNA degradation: Implications for WD protein structure

Ski8p is a 44-kD protein that primarily functions in the regulation of exosome-mediated, 3'--> 5' degradation of damaged mRNA. It does so by forming a complex with two partner proteins, Ski2p and Ski3p, which complete a complex that is capable of recruiting and activating the exosome/Ski7p complex that functions in RNA degradation. Ski8p also functions in meiotic recombination in complex with Spo11 in yeast. It is one of the many hundreds of primarily eukaryotic proteins containing tandem copies of WD repeats (also known as WD40 or beta-transducin repeats), which are short ~40 amino acid motifs, often terminating in a Trp-Asp dipeptide. Genomic analyses have demonstrated that WD repeats are found in 1%-2% of proteins in a typical eukaryote, but are extremely rare in prokaryotes. Almost all structurally characterized WD-repeat proteins are composed of seven such repeats and fold into seven-bladed beta propellers. Ski8p was thought to contain five WD repeats on the basis of primary sequence analysis implying a five-bladed propeller. The 1.9 A crystal structure unexpectedly exhibits a seven-bladed propeller fold with seven structurally authentic WD repeats. Structure-based sequence alignments show additional sequence diversity in the two undetected repeats. This demonstrates that many WD repeats have not yet been identified in sequences and also raises the possibility that the seven-bladed propeller may be the predominant fold for this family of proteins.     

The characterisation of novel secreted Ly-6 proteins from rat urine by the combined use of two-dimensional gel electrophoresis, microbore high performance liquid chromatography and expressed sequence tag data

A proteomic study of rat urine was undertaken using two-dimensional gel electrophoresis, microbore high performance liquid chromatography, mass spectrometry and N-terminal sequencing. Five known urinary proteins were identified but two novel peptide fragments matched a large number of rat expressed sequence tags (ESTs) from a liver library. By combining protein chemical and nucleotide data, two 101-residue open reading frames with 90% amino acid identity were determined, rat urinary protein 1 (RUP-1) and RUP-2. The data established signal peptide removal and provided evidence for N-glycosylation. A third related sequence, rat spleen protein (RSP-1) was confirmed from EST searches. These three proteins have been submitted to SWISS-PROT as P81827, P81828 and Q9QXN2, respectively. A fourth novel homologue was found in porcine and bovine ESTs from embryo libraries. Alignment with known homologues showed conserved cysteine positions characteristic of a secreted subfamily of Ly-6 proteins. In two cases, antineoplastic urinary protein and caltrin, these homologues have unverified functional annotations. The RUP sequences showed high scoring matches to three unrelated rat mRNAs subsequently established to be chimeric. Two of these share extended sectional identity to RUP-1 but the third may represent another novel Ly-6 homologue. These chimeras have caused serious annotation errors in secondary databases.     

Predicted structures of two proteins involved in human diseases

Structures of 79 proteins involved in human diseases were predicted by sequence alignments with structural templates. The predicted structures for ALDP and CSA, proteins responsible for adrenoleukodystrophy and the Cockayne syndrome, respectively, were analyzed to elucidate the molecular basis of disease mutations. In particular we positioned residue P484 of ALDP in the homodimer interface. This positioning is consistent with a recent experimental finding that the mutation P484R significantly decreases the self-interaction of ALDP and suggests that the disease mechanism of this mutation lies in the impaired ALDP dimerization. We identified two new WD repeats in CSA and suggest that one of these forms part of the interaction surface with other proteins.     

Identification of candidate antigen in Whipple's disease using a serological proteomic approach

Whipple's disease (WD) is a chronic multisystemic infection, caused by Tropheryma whipplei, a Gram-positive rod. Recently, a reliable method has been developed for cultivating T. whipplei in vitro. This together with the availability of complete genome sequence of T. whipplei prompted us to initiate proteome analysis of T. whipplei. The objective of the present study was to identify candidate proteins for serological diagnosis of WD. Immunoreactivities of sera collected from 18 patients with WD were compared with those of 24 control subjects who did not have WD. For this, we used 2-DE, immunoblotting, and MS. In total, we identified 23 candidate antigenic proteins. These included a subset of six proteins, each of which was found significantly more frequently in cases as compared to their controls. The remaining 17 proteins were found exclusively in cases. The methods we used in the current study enabled us to identify candidate antigens that, in our view, might be useful for serological diagnosis of WD.     

Leucine-Rich Repeat Kinase 2 Binds to Neuronal Vesicles through Protein Interactions Mediated by Its C-Terminal WD40 Domain

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson''s disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function.     

Reticulon-like proteins in Arabidopsis thaliana: structural organization and ER localization

Reticulons are proteins that have been found predominantly associated with the endoplasmic reticulum in yeast and mammalian cells. While their functions are still poorly understood, recent findings suggest that they participate in the shaping of the tubular endoplamic reticulum (ER). Although reticulon-like proteins have been identified in plants, very little is known about their cellular localization and functions. Here, we characterized the reticulon-like protein family of Arabidopsis thaliana. Three subfamilies can be distinguished on the basis of structural organization and sequence homology. We investigated the subcellular localization of two members of the largest subfamily, i.e. AtRTNLB2 and AtRTNLB4, using fluorescent protein tags. The results demonstrate for the first time that plant reticulon-like proteins are associated with the ER. Both AtRTNLB proteins are located in the tubular ER but AtRTNLB4 is also found in the lamellar ER cisternae, and in ER tubules in close association with the chloroplasts. Similarity in protein structure and subcellular localization between AtRTNLB2 and mammalian reticulons suggests that they could assume similar basic functions inside the cell.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Cmr1 protein preferentially binds to UV-damaged DNA <Emphasis Type="Italic">in vitro</Emphasis>

DNA metabolic processes such as DNA replication, recombination, and repair are fundamentally important for the maintenance of genome integrity and cell viability. Although a large number of proteins involved in these pathways have been extensively studied, many proteins still remain to be identified. In this study, we isolated DNA-binding proteins from Saccharomyces cerevisiae using DNA-cellulose columns. By analyzing the proteins using mass spectrometry, an uncharacterized protein, Cmr1/YDL156W, was identified. Cmr1 showed sequence homology to human Damaged-DNA binding protein 2 in its C-terminal WD40 repeats. Consistent with this finding, the purified recombinant Cmr1 protein was found to be intrinsically associated with DNA-binding activity and exhibited higher affinity to UV-damaged DNA substrates. Chromatin isolation experiments revealed that Cmr1 localized in both the chromatin and supernatant fractions, and the level of Cmr1 in the chromatin fraction increased when yeast cells were irradiated with UV. These results suggest that Cmr1 may be involved in DNA-damage responses in yeast.     

Subfamily I Treponema pallidum repeat protein family: sequence variation and immunity

A 12-membered Treponema pallidum repeat (Tpr) protein family has been identified in T. pallidum subsp. pallidum, the causative agent of syphilis. The subfamily I Tpr proteins (C, D, F, and I) possess conserved sequence at the N- and C-termini and central regions that differentiate the members. These proteins may be important in the immune response during syphilis infection and in protective immunity. Strong antibody responses have been observed toward some of the subfamily I Tpr proteins during infection with different syphilis isolates. Some sequence variation has also been identified in one subfamily I Tpr member, TprD, among T. pallidum subsp. pallidum isolates. In this study, we examined sequences in the remaining subfamily I Tpr proteins among strains. Both TprF and TprI were conserved among T. pallidum subsp. pallidum isolates.While some heterogeneity was identified in TprC. We further examined the immune response and protective capacity of TprF protein in this paper. We demonstrate that the N-terminal conserved region of the subfamily I Tpr proteins elicits strong antibody and T-cell responses during infection, and immunization with this region attenuates syphilitic lesion development upon infectious challenge.