Prediction of transmembrane helical segments in transmembrane proteins based on wavelet transform

Tmnsmembrane（TM） protein plays an important role in the life activity of the cells, and the prediction of transmembrane helical segments （TMHs） is an important subject in the bioinformatics research. Thus far, several prediction methods have been reported, but there are some deficiencies in prediction accuracy and adaptability in these methods. In this paper, a method based on discrete wavelet transform （DWT） was developed to predict the TMHs. Two sets of test data sets containing total 60 protein sequences were utilized to access the effect of the method. Compared with the prediction results of TMHMM2.0 and MEMSAT, the obtained results indicate that the presented method has high prediction accuracy.

Association of Lysosome Associated Protein Transmembrane 4 Beta Gene Polymorphism with the Risk of Pancreatic Cancer

Objective： Lysosome associated protein transmembrane 4 beta （LAPTM4B） was originally identified as a gene in human hepatocellular carcinoma （HCC）. It was successfully cloned by fluorescence differential display, rapid amplification of cDNA ends （RACE） and reverse transcription polymerase chain reaction （RT-PCR）. Previous study showed that the novel gene played an important role in the occurrence, development, migration and prognosis of tumors. Pancreatic cancer is an aggressive malignancy with the majority of patients dying within one year after diagnosis. This study tries to find out the relationship between lysosome associated protein transmembrane 4 beta gene polymorphism and the susceptibility of pancreatic cancer. Methods： A case-control study was conducted in China, including 58 pancreatic cancer cases and 156 healthy controls. Human genomic DNA was used as the template, polymerase chain reaction （PCR） was used to detect the distribution of LAPTM4B genotype. Analyses Odds ratio （OR） and corresponding 95% confidence interval （95%CI） with logistic regression were performed. Results： Two alleles of LAPTM4B generated three kinds of genotypes in population, ＊1/1, ＊1/2, and ＊2/2. The genotype frequency of ＊1/1, ＊1/2 and ＊2/2 in the pancreatic cancer group were 41.4%, 44.8% and 13.8% respectively, which were not significantly different from those of healthy group （47.4%, 42.9%, 9.6%） （P=0.773, P=0.291）. Also the ＊2 allele frequency of LAPTM4B among pancreatic cancer had no significantly difference with the controls （P=0.354）. When compared to the ＊1 allele, the people with ＊2 allele had no increased risk of pancreatic cancer. Conclusion： The gene polymorphism of LAPTM4B may not influence the susceptibility of pancreatic cancer.

Molecular cloning and characterization of human age-related NADH oxidase (arNOX) proteins as members of the TM9 superfamily of transmembrane proteins

Age-related NADH oxidase (arNOX = ENOX3) proteins are superoxide-generating cell surface oxidases that increase in activity with age beginning at about 30 y. A soluble and truncated exfoliated form of the activity is present in blood and other body fluids. The activity was purified to apparent homogeneity from human urine and resolved by 2-D gel electrophoresis into a series of 24 to 32 kDa components of low isoelectric point. The purified proteins were resistant both to N-terminal sequencing and trypsin cleavage. Cleavage with pepsin revealed peptides corresponding to the TM9 family of transmembrane proteins. Peptide antisera raised to all five members of the human TM9 family sequentially blocked the arNOX activity of human saliva and sera. The soluble truncated N-terminus of the human homolog TM9SF4 was expressed in bacteria. The recombinant protein was characterized biochemically and exhibited ar-NOX activity. The findings identify five arNOX isoforms each of which correspond to one of the five known TM9 family members. The exfoliated soluble arNOX forms are derived from the 24 to 32 kDa N-termini exposed to the cell’s exterior at the cell surface. Each of the shed forms contain putative functional motifs characteristic of ECTO-NOX (ENOX) proteins despite only minimal sequence identity. Our findings identify arNOX as having functional characteristics of ENOX proteins and the TM9 superfamily of proteins as the genetic origins of the five known arNOX isoforms present in human sera, plasma and other body fluids1.

Potential role of transmembrane 9 superfamily member 1 as a biomarker in urothelial cancer

Bladder cancer is a urological tumor with high rates of recurrence despite recent advances in novel therapies.Many proteins involved in the molecular mechanisms are currently an enigma,especially the transmembrane 9 superfamily member 1 which has an unclear function.Wei et al published the function and mechanism of this protein,and showed that it could participate in the proliferation,migration and invasion of tumor cells in bladder cancer,therefore treatments directed against this protein may be beneficial in avoiding this condition.

Prediction of hydrophobic regions effectively in transmembrane proteins using digital filter

The hydrophobic effect is the major factor that drives a protein molecule towards folding and to a great degree the stability of protein structures. Therefore the knowledge of hydrophobic regions and its prediction is of great help in understanding the structure and function of the protein. Hence determination of membrane buried region is a computationally intensive task in bioinformatics. Several prediction methods have been reported but there are some deficiencies in prediction accuracy and adaptability of these methods. Of these proteins that are found embedded in cellular membranes, called as membrane proteins, are of particular importance because they form targets for over 60% of drugs on the market. 20-30% of all the proteins in any organism are membrane proteins. Thus transmembrane protein plays important role in the life activity of the cells. Hence prediction of membrane buried segments in transmembrane proteins is of particular importance. In this paper we have proposed signal processing algorithms based on digital filter for prediction of hydrophobic regions in the transmembrane proteins and found improved prediction efficiency than the existing methods. Hydrophobic regions are extracted by assigning physico-chemical parameter such as hydrophobicity and hydration energy index to each amino acid residue and the resulting numerical representation of the protein is subjected to digital low pass filter. The proposed method is validated on transmembrane proteins using Orientation of Proteins in Membranes (OPM) dataset with various prediction measures and found better prediction accuracy than the existing methods.

Favorable and unfavorable amino acid residues in water-soluble and transmembrane proteins

We analyzed the amino acid residues present in the water-soluble and transmembrane proteins of 6 thermophilic and 6 mesophilic species of the domains Archaea and Eubacteria, and characterized them as favorable or unfavorable. The characterization was performed by comparing the observed number of each amino acid residue to the expected number calculated from the percentage of nucleotides present in each gene. Amino acids that were more or less abundant than expected were considered as favorable or unfavorable, respectively. Comparisons of amino acid compositions indicated that the water-soluble proteins were rich in charged residues such as Glu, Asp, Lys, and His, whereas hydrophobic residues such as Trp, Phe, and Leu were abundant in transmembrane proteins. Interestingly, our results found that although the Trp residue was abundant in transmembrane proteins, it was not defined as favorable by our calculations, indicating that increased numbers of a particular amino acid does not necessary indicate it is a favorable residue. Amino acids with high G + C content such as Ala, Gly, and Pro were frequently observed as favorable in species with low G + C content. Comparatively, amino acids with low G + C content such as Phe, Tyr, Lys, Ile, and Met were frequently observed as favorable in species with high G + C content. These are the examples to increase the supply of amino acids than expected. Amino acids with neutral G + C content, i.e., Glu and Asp were favorable in water-soluble proteins from all species analyzed, and Cys was unfavorable both in water-soluble and transmembrane proteins. These results indicate that amino acid compositions are essentially determined by the nucleotide sequence of the genes, and the amino acid content is altered by a deviation from expectation.

Cloning and characterization of a novel gene encoding a putative seven-span transmembrane protein localized in endoplasmic reticulum

Objective: To clone and analyze the structure of a novel gene, named EST 1 (endoplasmic reticulum localized seven span transmembrane protein 1) and to analyze the expression pattern and intracellular location of EST 1. Methods: The cDNA library was screened to isolate novel cDNA fragment. The structure of novel gene was analysed by computer software. Expression of EST 1 was analyzed by dot blot and Northern blotting. Intracellular localization was observed after EST 1 enhanced green fluorescence protein (EGFP) fusion gene was transfected into mammalian cells. Results: The full length cDNA of mouse EST 1 was 1 802 bp, with a 1 293 bp open reading frame encoding 431 amino acids. It was predicated that protein encoded by EST 1 contained a signal peptide sequence at the N terminus, seven putative transmembrane domains, and an ER retaining signal at the C terminus. EST 1 EGFP fusion protein showed an ER like intracellular distribution in mammalian cells. Expression pattern analysis showed that EST 1 is expressed in all tissues examined. Conclusion: EST 1 is encoding a putative seven span transmembrane protein localized in endoplasmic reticulum. EST 1 was expressed in all tissues examined, suggesting an essential function of EST 1 in cells.

MicroRNA-363-3p inhibits colorectal cancer progression by targeting interferon-induced transmembrane protein 1

BACKGROUND The molecular mechanisms of colorectal cancer development and progression are far from being elucidated.AIM To investigate the role of microRNA-363-3p(miR-363-3p)in the progression of colorectal cancer.METHODS Real-time polymerase chain reaction was performed to detect miRNA expression in human colorectal cancer tissues and paired normal colorectal tissues.PITA 6 was utilized to predict the targets of miR-363-3p.Dual-luciferase reporter system was used to validate the target of miR-363-3p.Plate colony formation assay and wound-healing assay were performed to evaluate cancer cells’clonogenic survival ability and migration ability,respectively.Cell proliferation was examined by cell counting kit-8 assay.Immunohistochemical staining was used to determine the expression level of interferon-induced transmembrane protein 1(IFITM1)in colorectal cancer tissues and adjacent tissues.The TCGA and GTEx databases were used to compare the expression levels of IFITM1 mRNA in colorectal cancer tissues and normal colorectal tissues and analyze the correlation between the expression levels of IFITM1 mRNA and overall survival and disease-free survival of patients.A colorectal cancer cell line with a deficiency of IFITM1 was constructed,and the regulation effect of IFITM1 on the clonogenic growth of colorectal cancer cells was clarified.RESULTS MiR-363-3p was decreased in colorectal cancer tissues compared to normal colorectal tissues.IFITM1 was characterized as a direct target of miR-363-3p.Overexpression of miR-363-3p led to decreased clonogenic survival,proliferation,and migration of colorectal cancer cells,which could be reversed by forced IFITM1 expression.CONCLUSION MiR-363-3p can constrain clonogenic survival,proliferation,and migration of colorectal cancer cells via targeting IFITM1.

Cell Surface Transmembrane Protein Database for Detecting Potential Targets of Antibody Drug in Application of Cancer Therapies

Background： Antibody drug conjugated （ADC） is one kind of very important method of therapy to cancer diseases. In this research, the authors introduce BLAST and some other important algorithms to create transmembrane protein databases. These databases are acquired from well-known databases such as NCBI or Swiss-Prot as template, and then collect all possible transmembrane protein by using BLAST or physical character. After collect these databases, the authors will aim at each nucleotide sequences to design the probes of oligonucleotide microarray, which can detect the high express transmembrane proteins very efficiently. Finally, the authors can accelerate the anti-cancer drug discovery by using these databases. Result： This study constructed a web service, the Transmembrane Protein Database, to researchers that are interested in or need to oligonucleotide microarray probe design for detecting potential targets of antibody drug. With user friendly web based windows containing each necessary selections, users can easily choose the parameters and get the suitable probe design suggestions. Conclusion： Transmembrane protein database is very important and powerful in detecting cancers or other human disease. By using this database, the authors offer a good strategy in transmembrane protein research as well.

Emerging role of cystic fibrosis transmembrane conductance regulator- an epithelial chloride channel in gastrointestinal cancers

Cystic fibrosis transmembrane conductance regulator(CFTR), a glycoprotein with 1480 amino acids, has been well established as a chloride channel mainly expressed in the epithelial cells of various tissues and organs such as lungs, sweat glands, gastrointestinal system, and reproductive organs. Although defective CFTR leads to cystic fibrosis, a common genetic disorder in the Caucasian population, there is accumulating evidence that suggests a novel role of CFTR in various cancers, especially in gastroenterological cancers, such as pancreatic cancer and colon cancer. In this review, we summarize the emerging findings that link CFTR with various cancers, with focus on the association between CFTR defects and gastrointestinal cancers as well as the underlying mechanisms. Further study of CFTR in cancer biology may help pave a new way for the diagnosis and treatment of gastrointestinal cancers.

THE COMBINATION PREDICTION OF TRANSMEMBRANE REGIONS BASED ON DEMPSTER-SHAFER THEORY OF EVIDENCE

Transmembrane proteins are some special and important proteins in cells. Because of their importance and specificity, the prediction of the transmembrane regions has very important theoretical and practical significance. At present, the prediction methods are mainly based on the physicochemical property and statistic analysis of amino acids. However, these methods are suitable for some environments but inapplicable for other environments. In this paper, the multi-sources information fusion theory has been introduced to predict the transmembrane regions. The proposed method is test on a data set of transmembrane proteins. The results show that the proposed method has the ability of predicting the transmembrane regions as a good performance and powerful tool.

N-乙酰氨基葡萄糖转移酶Ⅴ过表达对人肝癌细胞粘着斑复合物介导的信号转导作用

为了探讨过表达N 乙酰氨基葡萄糖转移酶Ⅴ (GnT Ⅴ )后 772 1细胞侵袭、迁移等行为改变的机制 ,检测了GnT Ⅴ 772 1及pcDNA3 772 1两组细胞中与恶性表型密切相关的粘着斑激酶 (focalad hesionkinase ,FAK)、PTEN蛋白、蛋白激酶B(PKB)等重要信号分子的表达水平 ,同时测定了 2组细胞非贴壁依赖生长的能力 .利用Western印迹方法检测FAK、PTEN、PKB的表达或磷酸化水平 .利用poly hema使细胞非贴壁生长 ,2组细胞悬浮无血清培养 2 0h ,采用流式细胞仪方法检测细胞的失巢凋亡 (anoikis) .研究发现 ,转染GnT Ⅴ后的肝癌细胞的FAK表达无明显变化 ,FAK的酪氨酸磷酸化水平增高 70 %;而PTEN的表达下降了 4 9%;PKB的磷酸化增加 2 0 0 %;pcDNA3 772 1细胞已有明显凋亡 ,而转染GnT Ⅴ的 772 1细胞未发生凋亡 .结果提示 ,转染GnT Ⅴ后的肝癌细胞迁移力增强 ,可能与其FAK的磷酸化程度升高 ,激酶活力增强有关 ;而能逃逸失巢凋亡是因为PTEN的表达下降 ,PTEN蛋白的磷酸酶活性降低 ,细胞Akt PKB磷酸化水平保持在较高水平 .

变形链球菌AgⅠ/Ⅱ蛋白编码可变区(Ⅴ+)基因表达质粒的构建

目的 构建变形链球菌 (S .mutans血清型f、s .mutnas血清型c)表面蛋白编码Ⅴ +基因重组质粒pCVc、pCVf和重组表达质粒pSRVc、pSRVf。 方法 用PCR方法从sr基因中扩增目的基因片段与质粒 pCR2 .1连接 ,再将Ⅴ +区基因片段转移到高效表达质粒pET2 1a(+) ,酶切电泳检测。结果 PCR扩增产物为 1.16kb的DNA带 ,测序结果与sr基因序列Ⅴ +区一致。酶切电泳证实Ⅴ +区基因片段整合到 pET2 1a(+)的适当部位 ,构建重组表达质粒 pSRVc、pSRVf。 结论 本试验成功地构建了携带Ⅴ +基因片段的表达质粒 pSRVc、pSRVf。

抑制肝癌细胞N-乙酰氨基葡萄糖转移酶Ⅴ表达导致细胞未折叠蛋白质反应

目的探讨N乙酰氨基葡萄糖转移酶Ⅴ(GnTⅤ)对细胞基因表达和功能的影响。方法用WesternBlot验证GnTⅤ的反义cDNA稳转的细胞株AsGnTⅤ7721(AsGnTⅤSMMC7721)中GnTⅤ表达,用基因芯片技术比较AsGnTⅤ7721与7721(SMMC7721)细胞的基因表达差异,同时用RTPCR和WesternBlot检测AsGnTⅤ7721细胞中未折叠蛋白质反应信号通路关键分子Bip和XBP1的表达变化。结果基因芯片检测到的AsGnTⅤ7721细胞中的基因表达变化表明,细胞中产生未折叠蛋白质反应。同时AsGnTⅤ7721细胞中未折叠蛋白质反应信号途径中的关键分子Bip的mRNA和蛋白表达升高,XBP1mRNA被剪接。结论GnTⅤ表达受阻能导致细胞产生未折叠蛋白质反应。

FⅤ Leiden突变与缺血性卒中

F Ⅴ Leiden突变可导致活化蛋白C抵抗现象,是引起白种人静脉血栓栓塞的最常见遗传因素。但对这一突变与缺血性卒中的关系尚存有争议。文章综述了F Ⅴ Leiden突变的分子机制及近年来在缺血性卒中方面的研究情况。

β-1,4-半乳糖基转移酶Ⅱ、Ⅴ表达的亚细胞结构定位研究

为了研究β-1,4-半乳糖基转移酶 和 (β-1,4-Gal T- and )蛋白表达的亚细胞结构定位 ,本实验构建了β-1,4-Gal T- 和 融合绿色荧光蛋白 (GFP)表达质粒 ,分别将构建的质粒转染到 PC12细胞和肝癌 772 1细胞中 ,在荧光显微镜下观察β-1,4-Gal T- 和 在其中表达的亚细胞结构定位。发现 ,β-1,4-Gal T- 和 主要表达在这两种细胞的细胞核旁的 Golgi复合体 ,说明它们主要分布在 Golgi复合体上。提示它们可能是在

复合杂合突变致遗传性凝血因子Ⅴ缺陷症家系的表型与基因型分析

目的:通过检测复合杂合突变导致遗传性凝血因子Ⅴ(FⅤ)缺陷家系的表型和基因突变分析,探讨其分子发病机制。方法:检测先证者及其家系成员(共3代10人)血浆凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、纤维蛋白原(FIB)、FⅤ促凝活性(FⅤ∶C)、FⅤ抗原(FⅤ∶Ag)及其他相关凝血指标以明确诊断。采用DNA直接测序分析F5基因的所有外显子、侧翼、5'和3'非翻译区及家系成员相应的突变位点区域,发现突变位点用反向测序证实。使用ClustalX-2.1-win软件分析突变氨基酸的保守性,PROVEAN和MutationTaster在线生物信息学软件预测突变对蛋白质功能的影响,Swiss-PdbViewer软件在突变位点上进行蛋白质模型和氨基酸相互作用分析。结果:先证者PT和APTT较正常对照健康体检者显著延长,分别为34.2 vs 13.2 s和119.3 vs 36.0 s;FⅤ∶C和FⅤ∶Ag极度降低,分别为3%和6%。先证者的第二子、第三子、女儿和孙子的PT和APTT略有延长,FⅤ∶C和FⅤ∶Ag均有不同程度的降低,其他家庭成员的相关凝血参数均在正常范围内。先证者6号外显子上存在c.911G>A杂合错义突变,导致p.Gly276Glu;16号外显子上存在c.5343C>G杂合错义突变,导致p.Ser1781Arg。其第二子、第三子和孙子均携带p.Gly276Glu的杂合子,其女儿携带p.Ser1781Arg的杂合子,其他家庭成员均为野生型。保守分析结果表明,Gly276和Ser1781在同源物种中高度保守。2种生物信息学软件的预测结果相同,PROVEAN(得分-6.214和-12.79)表明,该复合杂合突变是一种有害突变;MutationTaster(得分0.976和0.999)提示,这些突变可能引起相应疾病。p.Gly276Glu蛋白质模型分析显示,Glu侧链延长,分子量变大,这将增加它与周围氨基酸之间的空间位阻,影响FⅤ蛋白的正常局部折叠,最终导致蛋白质活性和含量降低。由于尚无16号外显子FⅤ的X射线3D结构文件,本研究无法对p.Ser1781Arg突变蛋白进行空间结构分析。结论:在本研究中鉴定的新型复合杂合突变(p.Gly276Glu和p.Ser1781Arg)是该家系FⅤ水平下降的主要原因,其中p.Ser1781Arg国内外鲜见报道。

脑梗死患者凝血因子Ⅴ基因3种多态性和APC-R、LA的检测

目的 :研究脑梗死患者狼疮抗凝物质 (LA)、活化蛋白C抵抗 (APC R)和凝血因子 Ⅴ 基因 3种多态性的关系。方法 :测定脑梗死患者和正常对照血浆中LA、APC R ,并用限制性内切酶片段多态性方法测定FⅤ Gl691→A、G10 91→C、A10 90→G等 3种基因多态性的发生情况。结果 :脑梗死患者LA阳性占 45 5 6% ,APC R阳性占 3 3 3 % ,但 3种基因多态性均未见阳性。结论 :脑梗死患者LA阳性率明显高于正常对照。LA可能是引起脑梗死患者脑血栓形成的重要原因之一。脑梗死患者存在APC R ,但可能和凝血因子 Ⅴ 基因 3种多态性无关。

Membrane Proteins as Potential Colon Cancer Biomarkers: Verification of 4 Candidates from a Secretome Dataset

Colorectal cancer (CRC) is an important health issue in Taiwan. There were over ten thousand newly diagnosed CRC patients each year. The outcome of late stage CRC still remains to be improved, and tumor markers are expected to improve CRC detection and management. From a colorectal cancer cell secretome database, we chose four proteins as candidates for clinical verification, including tumor-associated calcium signal transducer 2 (TROP2, TACSTD2), transmembrane 9 superfamily member 2 (TM9SF2), and tetraspanin-6 (TSPAN6), and tumor necrosis factor receptor superfamily member 16 (NGFR). Different groups of 30 CRC patients’ tissue samples collected from Chang Gung Memorial Hospital were analyzed by immunohistochemistry (IHC) for the four proteins, and the results were scored by pathologist. For all the four candidate proteins, marked differences of IHC score existed between tumor and adjacent non-tumor counterpart. However, there were only trends between higher protein expression levels and worse outcome. Three proteins (TROP2, TM9SF2 and NGFR) had trends between higher tissue expression and tumor stage or lymph node metastasis. Our study revealed that tissue expression of four proteins (TROP2, TM9SF2, TSPAN6, and NGFR) was markedly different between tumor and adjacent non-tumor counterparts. Overexpression of all these four proteins showed some trends with poorer survival.

鲍曼不动杆菌OmpA蛋白的生物学特性分析

目的提取临床分离的鲍曼不动杆菌外膜蛋白A(OmpA)的基因,并进行生物学预测和分析。方法查询鲍曼不动杆菌OmpA基因序列,设计特异性PCR引物,以提取的鲍曼不动杆菌基因组DNA为模板,PCR扩增OmpA片段。回收目的片段,采用生物信息学软件分析OmpA蛋白的生物学特性。结果12株鲍曼不动杆菌多位点序列分型(MLST)序列分析有6个ST分型,分别是ST 208、ST 229、ST 191、ST 195、ST540、ST 1145,鲍曼不动杆菌OmpA基因序列全长为1070 bp的,单核苷酸多态性(SNP)位点较少,预测蛋白为跨膜蛋白,具有6种三级结构,均由β-桶状结构组成的保守结构域。结论临床分离的不同鲍曼不动杆菌株OmpA蛋白为结构相对保守的跨膜蛋白,具有维持细胞的形状和稳定性,参与细菌耐药机制及免疫原性作用。