综述与专论：血浆低分子量蛋白与多肽的研究进展

血浆是临床上使用频率最高的体液样本,具有采样无创、使用便捷以及内含物丰富等特点,是挖掘疾病相关潜在生物标志物的重要来源。血浆中低分子量蛋白与多肽(low molecular weight proteins and peptides,LMWPs)包括细胞因子、生长因子、多肽类激素和蛋白质降解产物等,其以分子质量较小、序列可表征、来源广泛等优势,得到了研究人员的青睐。近年来,随着高分辨率、高灵敏度质谱仪与计算科学的不断发展,新技术新方法的融入,LMWPs相关研究也有了新的发展。本文结合当前本领域的研究热点,介绍了血浆LMWPs的分类,包括完整小蛋白、蛋白质降解物、神经肽、免疫多肽、载体蛋白结合的LMWPs和小开放阅读框编码的多肽,同时总结了血浆LMWPs在富集方法、研究策略、功能研究以及疾病相关生物标志物方面的研究进展,并对血浆LMWPs面临的挑战和未来的研究方向进行了讨论。     

多肽药物的发展现状

随着生物技术与多肽合成技术的日臻成熟,越来越多的多肽药物被开发并应用于临床。因适应证广、安全性高且疗效显著,多肽药物目前已广泛应用于肿瘤、肝炎、糖尿病、艾滋病等疾病的预防、诊断和治疗,具有广阔的开发前景。简介多肽药物的来源与特点及制备方法,重点综述国内外多肽药物的研发概况、国外近年获准上市的主要多肽药物和我国自主研发并进入临床研究的主要多肽药物,旨在为该类新药的研发提供参考。     

抗肿瘤小分子多肽的研究进展

抗肿瘤小分子多肽具有分子量小、低毒性、高活性、易于穿透肿瘤细胞等特点,一些抗肿瘤小分子多肽已进入临床研究,成为肿瘤药物研发的新热点。本文从抗肿瘤小分子多肽的不同来源途径、结构改造及生物活性等方面,对其近年来的研究进展作简要概述。     

草菇子实体多肽提取工艺及其抗氧化活性

为了优化草菇子实体多肽的提取工艺和探究其抗氧化活性,以草菇子实体为原料,采用酶解法提取草菇子实体多肽,通过单因素试验得出最佳的酶解工艺,并使用Box-Behnken设计试验组合。结果表明：草菇子实体提取多肽的最佳工艺为料液比1:52 (g/mL)、加酶量7 200 U/g、酶解温度43 ℃,此工艺条件下的多肽得率为67.76%。从1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除能力、铁离子还原能力、超氧阴离子自由基清除能力和羟自由基清除能力4个方面研究其体外抗氧化能力,结果表明,草菇子实体多肽对DPPH自由基清除率为74.11%,超氧阴离子自由基和羟自由基清除率分别在69.64%和91.83%达到稳定,草菇子实体多肽还具有一定的还原力,说明草菇子实体多肽可以作为优质抗氧化肽的良好来源。该研究为草菇多肽的高效制备和抗氧化肽等高附加值产品的研发提供理论依据。     

海绵来源真菌天然产物的研究进展

随着海洋药物及其药物先导化合物研究的不断深入,海绵来源微生物,因其种类繁多、代谢产物复杂多样,引发了科研工作者的兴趣。许多化合结构新颖、具有强烈生物活性的化合物从海绵来源真菌中发现,包括多肽、生物碱、聚酮类、萜类等。本文仅就近几年来所报道的海绵来源真菌新天然产物的结构类型及其生物活性做简要介绍。     

新生牛肝脏生长因子的纯化及其理化特性与生物活性

新生牛肝细胞生长因子是一种热稳定、蛋白酶及酸化(pH<1.5)敏感的蛋白质或多肽。研究结果表明,它可促进肝来源的细胞系的DNA合成,但不能刺激非肝来源细胞系的DNA合成。它还可提高某些化合物(CCl_4,D-Gal)引起的急性肝衰竭小鼠的存活率。     

微生物纤溶酶的多样性及应用前景

微生物是溶栓药物的重要来源.本文综述了细菌、放线茵和真菌产生的纤溶酶的种类、性质及药理作用等进展,整理了基因工程技术在筛选产酶菌株及提高分泌物产量方面的研究近况,并对纤溶酶作为溶栓剂的应用前景进行了展望.     

抗氧化多肽研究及其应用前景

活性氧是一类含未配对电子的含氧类物质,是机体进行有氧呼吸的副产物,可以独立存在并具有强氧化性。人和动物体内过多的活性氧蓄积会引发氧化还原失衡,导致氧化应激,造成核酸和蛋白质结构损伤,诱发系列疾病。目前主要通过使用抗氧化剂来清除活性氧,可分为人工合成抗氧化剂和天然抗氧化剂。与具有潜在基因毒性的合成抗氧化剂不同,属于天然抗氧化剂的抗氧化多肽具有来源广泛、易吸收、活性强的特点,能有效清除活性氧,减缓氧化损伤,预防和缓解多种慢性疾病。现梳理了近年来有关抗氧化多肽的研究,总结了已发现的抗氧化多肽的种类及其特性,着重分析了抗氧化多肽构效关系及其活性影响因素,并简要讨论了抗氧化多肽的应用前景及其面临的挑战。     

MALDI-TOF/TOF和LC-MS/MS液质联用分析大鼠脑脉络丛的多肽组

脉络丛(choroid plexus,CP)位于血液与脑脊液(cerebrospinal fluid,CSF)之间,不仅是CSF的重要来源,而且是构成血液-脑脊液屏障(blood-cerebrospinal fluid barrier,BCB)的组织基础.CP参与脑组织中一些血源性多肽的输送以及自身多肽合成的生理过程,在维持脑微环境动态平衡和调节中枢神经系统的正常功能方面起到非常重要的作用.本研究分别运用MALDI-TOF/TOF和LC-MS/MS液质联用系统分析了成年SD大鼠血液-脑脊液屏障(即脉络丛组织)中的多肽组.共鉴定到163个多肽(P0.001),这些多肽为69种蛋白质的降解肽段,其中ATP合酶(ATP synthase),细胞色素c(cytochrome c),血红蛋白(hemoglobin),NADH-辅酶Q氧化还原酶(NADH-ubiquinone oxidoreductase),β珠蛋白(beta-globin)这5种蛋白质的肽段数占总肽段数的50%以上,并且部分多肽序列相似度高,类似其前体蛋白的逐步降解片段,而这些前体蛋白质的分子量多数在10kD至20kD之间.上述研究结果为SD大鼠脉络丛组织的生理功能研究及组织多肽组学的研究方法提供了有价值的科学资料.     

乳清蛋白及其生物活性肽血压调节功能研究进展

乳清蛋白既是优质蛋白来源，也是抗高血压生物活性肽的理想来源，开发具有调节血压功效的蛋白多肽类产品对未来临床高血压防治将发挥重要作用。文章综述了乳清蛋白生物活性肽血压调节功能的基本机制和研究现状，并对其未来发展趋势和应用前景进行了讨论。     

虫蛹活性多肽的制备、分离和生物活性研究进展

我国拥有悠久的蜂、蚕养殖历史,其虫蛹富含各种营养物质,其中蛋白质的含量最高。现代营养学研究发现,通过酶解等生物手段从虫蛹蛋白中分离出的生物活性肽,具有多种生物活性,如抗氧化、抗肿瘤、降血压和增强免疫等,同时可有效地提高虫蛹的利用率。基于此,就近年来国内外对虫蛹多肽的制备、分离纯化的方法以及生物活性的研究进展作一综述,以期为虫蛹多肽的深入研究与开发提供参考。     

Interaction of β-sheet folds with a gold surface

The adsorption of proteins on inorganic surfaces is of fundamental biological importance. Further, biomedical and nanotechnological applications increasingly use interfaces between inorganic material and polypeptides. Yet, the underlying adsorption mechanism of polypeptides on surfaces is not well understood and experimentally difficult to analyze. Therefore, we investigate here the interactions of polypeptides with a gold(111) surface using computational molecular dynamics (MD) simulations with a polarizable gold model in explicit water. Our focus in this paper is the investigation of the interaction of polypeptides with β-sheet folds. First, we concentrate on a β-sheet forming model peptide. Second, we investigate the interactions of two domains with high β-sheet content of the biologically important extracellular matrix protein fibronectin (FN). We find that adsorption occurs in a stepwise mechanism both for the model peptide and the protein. The positively charged amino acid Arg facilitates the initial contact formation between protein and gold surface. Our results suggest that an effective gold-binding surface patch is overall uncharged, but contains Arg for contact initiation. The polypeptides do not unfold on the gold surface within the simulation time. However, for the two FN domains, the relative domain-domain orientation changes. The observation of a very fast and strong adsorption indicates that in a biological matrix, no bare gold surfaces will be present. Hence, the bioactivity of gold surfaces (like bare gold nanoparticles) will critically depend on the history of particle administration and the proteins present during initial contact between gold and biological material. Further, gold particles may act as seeds for protein aggregation. Structural re-organization and protein aggregation are potentially of immunological importance.     

Pore-forming polypeptides of the pathogenic protozoon Naegleria fowleri

The free-living amoeboflagellate and potential human pathogen Naegleria fowleri causes the often fatal disease primary amoebic meningoencephalitis. The molecular repertoire responsible for the cytolytic and tissue-destructive activity of this amoeboid protozoon is largely unknown. We isolated two pore-forming polypeptides from extracts of highly virulent trophozoites of N. fowleri by measuring their membrane-permeabilizing activity. N-terminal sequencing and subsequent molecular cloning yielded the complete primary structures and revealed that the two polypeptides are isoforms. Both polypeptides share similar structural properties with antimicrobial and cytolytic polypeptides of the protozoon Entamoeba histolytica (amoebapores) and of cytotoxic natural killer (NK) and T cells of human (granulysin) and pig (NK-lysin), all characterized by a structure of amphipathic alpha-helices and an invariant framework of cysteine residues involved in disulfide bonds. In contrast to the aforementioned proteins, the Naegleria polypeptides both are processed from large precursor molecules containing additional isoforms of substantial sequence divergence. Moreover, biochemical characterization of the isolated polypeptides in combination with mass determination showed that they are N-glycosylated and variably processed at the C terminus. The biological activity of the purified polypeptides of Naegleria was examined toward human cells and bacteria, and it was found that these factors, named naegleriapores, are active against both types of target cells, which is in good agreement with their proposed biological role as a broad-spectrum effector molecule.     

Post-translational modification of genetically encoded polypeptide libraries

The genetic encoding of polypeptides with biological display systems enables the facile generation and screening of very large combinatorial libraries of molecules. By post-translationally modifying the encoded polypeptides, chemically and structurally more diverse molecules beyond linear amino acid polymers can be generated. The first post-translational modification applied to encoded polypeptides, the oxidation of cysteine residues to form disulfide bridges, is a natural one and was used to cyclise short peptides soon after the invention of phage display. Recently a range of non-natural chemical strategies for the post-translational modification of encoded polypeptide repertoires were applied to generate optical biosensors, semisynthetic polypeptides, peptide-drug conjugates, redox-insensitive monocyclic peptides or multicyclic peptides, and these strategies are reviewed in this article.     

Cationic polypeptides are required for antibacterial activity of human airway fluid

In a search for direct evidence leading to the biological relevance of airway secretions in innate host defense, we characterized the antibacterial function of cationic polypeptides within minimally manipulated nasal fluid. In this study, we show that cationic antimicrobial polypeptides are responsible for most of the bactericidal activity of whole nasal fluid. The removal of cationic polypeptides using a cation-exchange resin ablated the activity of nasal fluid against Escherichia coli, Listeria monocytogenes, and Pseudomonas aeruginosa. By using a novel proteomic approach, we identified a dozen cationic peptides and proteins within nasal fluid, all of which either are known antimicrobial polypeptides or have other proposed roles in host defense. Of the three most abundant cationic polypeptides in nasal fluid, lysozyme was more effective than either lactoferrin or secretory leukoprotease inhibitor in restoring the antibacterial activity of the cationic polypeptide-depleted fluid against a mucoid cystic fibrosis isolate of P. aeruginosa.     

Cyclic peptides from Oldenlandia affinis DC. Molecular and biological properties

A new isolation procedure for Kalata polypeptides from the tropical plant Oldenlandia affinis DC is described. Fractions were screened by thin-layer chromatography, and Van Urk positive fractions were tested for oxytocic activity in estrogenized rat uteri. By using this procedure, we were able to isolate and characterize three macrocyclic polypeptides with uterine activity. Their amino acid sequence and biological effects have been analyzed, and their NMR spectra were compared with those of the earlier ones. All three peptides showed hemolytic activity on human blood, and were tested for antibiotic effect against E. coli, Staphylococcus aureus, and Hemophilus influenzae.     

Biological synthesis of circular polypeptides

Here, we review the use of different biochemical approaches for biological synthesis of circular or backbone-cyclized proteins and peptides. These methods allow the production of circular polypeptides either in vitro or in vivo using standard recombinant DNA expression techniques. Protein circularization can significantly impact protein engineering and research in protein folding. Basic polymer theory predicts that circularization should lead to a net thermodynamic stabilization of a folded protein by reducing the entropy associated with the unfolded state. Protein cyclization also provides a valuable tool for exploring the effects of topology on protein folding kinetics. Furthermore, the biological production of cyclic polypeptides makes possible the production of cyclic polypeptide libraries. The generation of such libraries, which was previously restricted to the domain of synthetic chemists, now offers biologists access to highly diverse and stable molecular libraries for probing protein structure and function.     

The Acrolepiopsis assectella silk cocoon: kairomonal function and chemical characterisation

Two soluble sericin-like polypeptides, B1 and B2, from leek moth (Acrolepiopsis assectella) cocoons trigger host-acceptance behaviour in the parasitoid, Diadromus pulchellus (Proc. Roy. Soc. London B 269 (2002) 1879). We found that these polypeptides were particularly cysteine-rich and lost their ability to trigger host-acceptance behaviour after being denatured and purified. This suggests that inter-disulphide bonds and the secondary structure of B1 and B2 are important for their biological activity. We also isolated six insoluble polypeptides (or polypeptides of low solubility) from A. assectella cocoons. At least four of these polypeptides triggered host-acceptance behaviour. The strongest responses were observed with P22, a light-chain fibroin or a seroin-peptide, and P100, a sericin-like polypeptide that is probably more strongly associated with the silk core than are B1 and B2. In conclusion, several polypeptides from different parts of the A. assectella silk-cocoon (the insoluble core and coating of the silk thread) are able to elicit host-acceptance behaviour in D. pulchellus females. These polypeptides belong to different silk protein families and are used as kairomones by this specialist parasitoid.     

大鼠α-酰胺酶在变铅青链霉菌中的克隆及表达研究

α－酰胺酶（α－ａｍｉｄａｓｅ,α－ＡＥ）催化神经和内分泌系统中活性多肽的Ｃ－端酰胺化,多多肽的生物活性至关重要。以大鼠心房组织的总ＲＮＡ为模板,采用ＲＴ－ＰＣＲ技术扩增获得编码α－酰胺酶的ｃＤＮＡ,并进行了克隆和测序。为了使α－酰胺酶能在链霉菌中分泌表达,将其ｃＤＮＡ与链霉菌酷氮酶酶（ｍｅｌＣ１）信号的编码序列融合得到融合ｍｅｌ／ＡＥ,将ｍｅｌ／ＡＥ插入链霉菌质粒ｐＩＪ６８０,获得重组质粒ｐＩＪ     

How do proteins fold?

This article emphasizes the importance of getting students to understand the ways in which polypeptides fold to form protein molecules with complex higher-ordered structures. Modern views on how this folding occurs in vitro and in the cell are summarized and set within an appropriate biological context.