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UniProt(https://www.uniprot.org/)是国际知名蛋白质数据库,主要包括UniProtKB知识库、UniParc归档库和UniRef参考序列集三部分。UniProtKB知识库是UniProt的核心,除蛋白质序列数据外,还包括大量注释信息。UniProtKB知识库分Swiss-Prot和TrEMBL两个子库。Swiss-Prot子库中50多万条序列均由人工审阅和注释,而TrEMBL子库中1.4亿多条序列是由核酸序列数据库EMBL中的蛋白质编码序列翻译所得,并由计算机根据一定规则进行注释。UniParc归档库将存放于不同数据库中的同一个蛋白质归并到一个记录中以避免冗余,并赋予序列唯一性特定标识符。UniRef参考序列集按相似性程度将UniProtKB和UniParc中的序列分为UniRef100、UniRef90和UniRef50三个数据集。UniProt网站为用户提供了高效实用的高级检索系统和大量帮助文档。UniProt数据库每4周发布新版的同时也发布统计报表,用户可通过统计报表了解该数据库的数据量及更新情况、数据类别和物种分布等基本信息,查看常规注释信息、序列特征注释信息和数据库交叉链接等统计数据。UniProt是目前国际上序列数据最完整、注释信息最丰富的非冗余蛋白质序列数据库,自本世纪初创建以来,为生命科学领域提供了宝贵资源。 相似文献
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Arginine-rich cell-penetrating peptides are short cationic peptides capable of traversing the plasma membranes of eukaryotic cells. While successful intracellular delivery of many biologically active macromolecules has been accomplished using these peptides, their mechanisms of cell entry are still under investigation. Recent dialogue has centered on a debate over the roles that direct translocation and endocytotic pathways play in internalization of cell-penetrating peptides. In this paper, we review the evidence for the broad range of proposed mechanisms, and show that each distinct process requires negative Gaussian membrane curvature as a necessary condition. Generation of negative Gaussian curvature by cell-penetrating peptides is directly related to their arginine content. We illustrate these concepts using HIV TAT as an example. 相似文献
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Pancreatic-type ribonucleases can exert toxic activity by catalyzing the degradation of cellular RNA. Their ability to enter cells is essential for their cytotoxicity. Here, we determine the mechanism by which bovine pancreatic ribonuclease (RNase A) enters human cells. Inhibiting clathrin-dependent endocytosis with dynasore or chlorpromazine decreases RNase A-uptake by ~70%. Limited colocalization between RNase A and transferrin indicates that RNase A is not routed through recycling endosomes. Instead, vesicular staining of RNase A overlaps substantially with that of nona-arginine and the cationic peptide corresponding to residues 47-57 of the HIV-1 TAT protein. At low concentrations (<5 μM), internalization of RNase A and these cell-penetrating peptides (CPPs) is inhibited by chlorpromazine as well as the macropinocytosis inhibitors cytochalasin D and 5-(N-ethyl-N-isopropyl)amiloride to a similar extent, indicative of common endocytic mechanism. At high concentrations, CPPs adopt a nonendocytic mechanism of cellular entry that is not shared by RNase A. Collectively, these data suggest that RNase A is internalized via a multipathway mechanism that involves both clathrin-coated vesicles and macropinosomes. The parallel between the uptake of RNase A and CPPs validates reference to RNase A as a "cell-penetrating protein". 相似文献
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Cascales L Henriques ST Kerr MC Huang YH Sweet MJ Daly NL Craik DJ 《The Journal of biological chemistry》2011,286(42):36932-36943
Cell-penetrating peptides can translocate across the plasma membrane of living cells and thus are potentially useful agents in drug delivery applications. Disulfide-rich cyclic peptides also have promise in drug design because of their exceptional stability, but to date only one cyclic peptide has been reported to penetrate cells, the Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). MCoTI-II belongs to the cyclotide family of plant-derived cyclic peptides that are characterized by a cyclic cystine knot motif. Previous studies in fixed cells showed that MCoTI-II could penetrate cells but kalata B1, a prototypic cyclotide from a separate subfamily of cyclotides, was bound to the plasma membrane and did not translocate into cells. Here, we show by live cell imaging that both MCoTI-II and kalata B1 can enter cells. Kalata B1 has the same cyclic cystine knot structural motif as MCoTI-II but differs significantly in sequence, and the mechanism by which these two peptides enter cells also differs. MCoTI-II appears to enter via macropinocytosis, presumably mediated by interaction of positively charged residues with phosphoinositides in the cell membrane, whereas kalata B1 interacts directly with the membrane by targeting phosphatidylethanolamine phospholipids, probably leading to membrane bending and vesicle formation. We also show that another plant-derived cyclic peptide, SFTI-1, can penetrate cells. SFTI-1 includes just 14 amino acids and, with the exception of its cyclic backbone, is structurally very different from the cyclotides, which are twice the size. Intriguingly, SFTI-1 does not interact with any of the phospholipids tested, and its mechanism of penetration appears to be distinct from MCoTI-II and kalata B1. The ability of diverse disulfide-rich cyclic peptides to penetrate cells enhances their potential in drug design, and we propose a new classification for them, i.e. cyclic cell-penetrating peptides. 相似文献
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We report a first test of the hypothesis that the mechanism of antimicrobial, cytolytic, and amphipathic cell-penetrating peptides in model membranes is determined by the thermodynamics of insertion of the peptide into the lipid bilayer from the surface-associated state. Three peptides were designed with minimal mutations relative to the sequence of TP10W, the Y3W variant of transportan 10, which is a helical, amphipathic cell-penetrating peptide previously studied. Binding to 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membranes and release of dye from those vesicles were assessed by stopped-flow fluorescence, and the secondary structure of the peptides on the membrane was determined by circular dichroism. The Gibbs energy of binding determined experimentally was in excellent agreement with that calculated using the Wimley-White interfacial hydrophobicity scale, taking into account the helical content of the membrane-associated peptide. Release of dye from POPC vesicles remained graded, as predicted by the hypothesis. More significantly, as the Gibbs energy of insertion into the bilayer became more unfavorable, which was estimated using the Wimley-White octanol hydrophobicity scale, dye release became slower, in quantitative agreement with the prediction. 相似文献
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Science China Life Sciences - 相似文献
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The uptake of different cell-penetrating peptides (CPPs) in two yeast species, Saccharomyces cerevisiae and Candida albicans, was studied using fluorescence HPLC-analyses of cell content. Comparison of the ability of penetratin, pVEC and (KFF)(3)K to traverse the yeast cell envelope shows that the cellular uptake of the peptides varies widely. Moreover, the intracellular degradation of the CPPs studied varies from complete stability to complete degradation. We show that intracellular degradation into membrane impermeable products can significantly contribute to the fluorescence signal. pVEC displayed highest internalizing capacity, and considering its stability in both yeast species, it is an attractive candidate for further studies. 相似文献
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Narumi T Komoriya M Hashimoto C Wu H Nomura W Suzuki S Tanaka T Chiba J Yamamoto N Murakami T Tamamura H 《Bioorganic & medicinal chemistry》2012,20(4):1468-1474
Compounds which inhibit the HIV-1 replication cycle have been found amongst fragment peptides derived from an HIV-1 matrix (MA) protein. Overlapping peptide libraries covering the whole sequence of MA were designed and constructed with the addition of an octa-arginyl group to increase their cell membrane permeability. Imaging experiments with fluorescent-labeled peptides demonstrated these peptides with an octa-arginyl group can penetrate cell membranes. The fusion of an octa-arginyl group was proven to be an efficient way to find active peptides in cells such as HIV-inhibitory peptides. 相似文献
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Studying the uptake of cell-penetrating peptides 总被引:1,自引:0,他引:1
More than a decade ago, it was discovered that cationic peptides could traverse the cellular plasma membrane without specific transporter proteins or membrane damage. Subsequently, it was found that these peptides, known as cell-penetrating peptides (CPPs), were also capable of delivering cargos into cells, hence the great potential of these vectors was acknowledged. Today, many different research groups are working with CPPs, which necessitates efforts to develop unified assays enabling the comparison of data. Here we contribute three protocols for evaluation of CPPs which, if used in conjunction, provide complementary data about the amount and mechanism of uptake (fluorometric analysis and confocal microscopy, respectively), as well as the extent of degradation (HPLC analysis of cell lysates). All three protocols are based on the use of fluorescently labeled peptides and can be performed on the same workday. 相似文献
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Although cell-penetrating peptides (CPPs), also denoted protein transduction domains (PTDs), have been widely used for intracellular delivery of large and hydrophilic molecules, the mechanism of uptake is still poorly understood. In a recent live cell study of the uptake of penetratin and tryptophan-containing analogues of Tat(48-60) and oligoarginine, denoted TatP59W, TatLysP59W and R(7)W, respectively, it was found that both endocytotic and non-endocytotic uptake pathways are involved [Thoren et al., Biochem. Biophys. Res. Commun. 307 (2003) 100-107]. Non-endocytotic uptake was only observed for the arginine-rich peptides TatP59W and R(7)W. In this paper, the interactions of penetratin, R(7)W, TatP59W and TatLysP59W with phospholipid vesicles are compared in the search for an understanding of the mechanisms for cellular uptake. While R(7)W, TatP59W and TatLysP59W are found to promote vesicle fusion, indicated by mixing of membrane components, penetratin merely induces vesicle aggregation. Studies of the leakage from dye-loaded vesicles indicate that none of the peptides forms membrane pores and that vesicle fusion is not accompanied by leakage of the aqueous contents of the vesicles. These observations are important for a proper interpretation of future experiments on the interactions of these peptides with model membranes. We suggest that the discovered variations in propensity to destabilize phospholipid bilayers between the peptides investigated, in some cases sufficient to induce fusion, may be related to their different cellular uptake properties. 相似文献
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RAPD分子标记简介 总被引:44,自引:0,他引:44
RAPD分子标记简介邹喻苹(中国科学院植物研究所系统与进化植物学开放研究实验室,北京100093)1引言地球上的人口急剧增长,突破50亿。而人类藉以生存的环境却日益恶化。人类衣、食、住、行所依赖的生物资源—“物种”正在以地质史上的记载的最大速度灭绝或... 相似文献
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Cells are protected from the surrounding environment by plasma membrane which is impenetrable for most hydrophilic molecules. In the last 10 years cell-penetrating peptides (CPPs) have been discovered and developed. CPPs enter mammalian cells and carry cargo molecules over the plasma membrane with a molecular weight several times their own. Known transformation methods for plant cells have relatively low efficiency and require improvement. The possibility to use CPPs as potential delivery vectors for internalisation in plant cells has been studied in the present work. We analyse and compare the uptake of the fluorescein-labeled CPPs, transportan, TP10, penetratin and pVEC in Bowes human melanoma cells and Nicotiana tabacum cultivar (cv.) SR-1 protoplasts (plant cells without cell wall). We study the internalisation efficiency of CPPs with fluorescence microscopy, spectrofluorometry and fluorescence-activated cell sorter (FACS). All methods indicate, for the first time, that these CPPs can internalise into N. tabacum cv. SR-1 protoplasts. Transportan has the highest uptake efficacy among the studied peptides, both in mammalian cells and plant protoplast. The internalisation of CPPs by plant protoplasts may open up a new effective method for transfection in plants. 相似文献