Computer-Assisted analysis of subcellular localization signals and post-translational modifications of human prion proteins

In the present work, computational analyses were applied to study the subcellular localiza-tion and posttranslational modifications of hu-man prion proteins (PrPs). The tentative location of prion protein was determined to be in the nu-cleolus inside the nucleus by the following bio-informatics tools: Hum-PLoc, Euk-PLoc and Nuc-PLoc. Based on our results signal peptides with average of 22 base pairs in N-terminal were identified in human PrPs. This theoretical study demonstrates that PrP is post-translationally modified by: 1) attachment of two N-linked complex carbohydrate moieties (N181 and N197), 2) attachmet of glycosylphosphatidylinositol (GPI) at serine 230 and 3) formation of two di-sulfide bonds between “6–22” and “179–214” cysteines. Furthermore, ten protein kinase phosphorylation sites were predicted in human PrP. The above-noted phosphorylation was car-ried out by PKC and CK2. By using bioinfor-matics tools, we have shown that computation-ally human PrPs locate particularly into the nu-cleolus.

Selective N-terminal modification of peptides and proteins:Recent progresses and applications

Numerous strategies for linking desired chemical probes with target peptides and proteins have been developed and applied in the field of biological chemistry.Approaches for site-specific modification of native amino acid residues in test tubes and biological contexts represent novel biological tools for understanding the role of peptides and proteins.Selective N-terminal modification strategies have been broadly studied especially in the last 10 years,as N-terminal positions are typically solvent exposed and provide chemically distinct sites for many peptide and protein targets,making N terminus distinct from other functional groups.A growing number of chemical and enzymatic techniques have been developed to modify N-terminal amino acids,and those techniques have the potential in the fields of medicine,basic research and applied materials science.This review focuses on appraising modification methodologies with the potential for biological applications from the past 10 years.

Polydopamine-modified chitin conduits with sustained release of bioactive peptides enhance peripheral nerve regeneration in rats

The introduction of neurotrophic factors into injured peripheral nerve sites is beneficial to peripheral nerve regeneration.However,neurotrophic facto rs are rapidly degraded in vivo and obstruct axonal regeneration when used at a supraphysiological dose,which limits their clinical benefits.Bioactive mimetic peptides have been developed to be used in place of neurotrophic factors because they have a similar mode of action to the original growth fa ctors and can activate the equivalent receptors but have simplified sequences and structures.In this study,we created polydopamine-modified chitin conduits loaded with brain-derived neurotrophic factor mimetic peptides and vascular endothelial growth fa ctor mimetic peptides(Chi/PDA-Ps).We found that the Chi/PDA-Ps conduits were less cytotoxic in vitro than chitin conduits alone and provided sustained release of functional peptides.In this study,we evaluated the biocompatibility of the Chi/P DA-Ps conduits.Brain-derived neurotrophic factor mimetic peptide and vascular endothelial growth fa ctor mimetic peptide synergistically promoted prolife ration of Schwann cells and secretion of neurotrophic factors by Schwann cells and attachment and migration of endothelial cells in vitro.The Chi/P DA-Ps conduits were used to bridge a 2 mm gap between the nerve stumps in rat models of sciatic nerve injury.We found that the application of Chi/PDA-Ps conduits could improve the motor function of rats and reduce gastrocnemius atrophy.The electrophysiological results and the microstructure of regenerative nerves showed that the nerve conduction function and re myelination was further resto red.These findings suggest that the Chi/PDA-Ps conduits have great potential in peripheral nerve injury repair.

Transition-metal-catalyzed C-H functionalization for late-stage modification of peptides and proteins

Late-stage modification of peptides and proteins meets the increasing demand in biochemical and pharmaceutical communities. These modification strategies could provide functionalized nonproteinogenic analogues with enhanced biological activities or improved therapeutic capabilities compared to their natural counterparts. Recent years, transition-metal-promoted functionalization of ubiquitous C-H bonds has been emerged as a powerful and tunable tool in this area, both for backbone diversifications and labeling of specific moieties. These reactions were flexible and expedient in both academic and industrial laboratories, especially considering their atom and step-economy, good functional group compatibility, accurate site selectivity. This review surveys the progress achieved in the late-stage modification of peptides and proteins utilizing transition-metal-catalyzed C-H functionalization with C-C and C-X(F, Cl, O, N, B, etc.) bonds formation.

Molecular characteristics and structure–activity relationships of food-derived bioactive peptides

Peptides are functional active fragments of proteins which can provide nutrients needed for human growth and development,and they also have unique physiological activity characteristics relative to proteins.Bioactive peptides contain a great deal of development potential.More specifically,food-derived bioactive peptides have the advantages of a wide variety of sources,unique structures,high efficiency and safety,so they have broad development prospects.This review provides an overview of the current advances regarding the preparation,functional characteristics,and structure–activity relationships of food-derived bioactive peptides.Moreover,the prospects for the future development and application of food-derived bioactive peptides are discussed.This review may provide a better understanding of foodderived bioactive peptides,and some constructive inspirations for further research and applications in the food industry.

Progress in Chemical Synthesis of Peptides and Proteins

For the proteins that cannot be expressed exactly by cell expression technology(e.g., proteins with multiple posttranslational modifications or toxic proteins), chemical synthesis is an important substitute. Given the limited peptide length offered by solid-phase peptide synthesis invented by Professor Merrifield, peptide ligation plays a key role in long peptide or protein synthesis by ligating two small peptides to a long one. Moreover, high-molecularweight proteins must be synthesized using two or more peptide ligation steps, and sequential peptide ligation is such an efficient way. In this paper, we reviewed the development of chemical protein synthesis, including solid-phase peptide synthesis, chemical ligation, and sequential chemical ligation.

Antioxidant peptides encrypted in flaxseed proteome:An in silico assessment

Flaxseed proteins and antioxidant peptides(AP)encrypted in their sequences were analysed in silico with a range of bioinformatics tools to study their physicochemical properties,allergenicity,and toxicity.Nine proteases(digestive,plant and microbial sources)were assessed for their ability to release known APs from 23 mature flaxseed storage proteins using the BIOPEP database.The families of proteins identified were predominantly globulins,oleosins,and small amount of conlinin.Overall,253 APs were identified from these proteins.More peptides were released by enzymatic hydrolysis from the globulins than those from oleosins and conlinin.Compared with other enzymes studied,the plant proteases(papain,ficin,and bromelain)were found to be superior to releasing APs from the flaxseed proteins.Analysis of toxicity by ToxinPred showed that none of the peptides released was toxic.Most of the APs showed structural features that are important for antioxidation,including relatively low molecular weight(dipeptides and tripeptides only);amphipathic properties(hydrophobicity range of-0.5 to+0.5);relatively low Boman index(≤2);broad range of pI(3.7-10.8),and an abundance of antioxidant amino acid residues(e.g.glutamic acid and histidine).This study demonstrate the suitability of flaxseed proteins as a source of APs.

Mussel meat protein: Extraction, processing, nutrition value, and application in food products

The rapidly increasing global population has resulted in an increased demand for proteins in the human diet.The mussel meat protein is an affordable,abundant,sustainable,and environmentally friendly source of proteins suitable for human consumption.This review described the composition and characteristics of mussel meat proteins,as well as methods for sufficiently isolating and extracting these proteins.Several non-thermal processing technologies including high-pressure homogenization and ultrasonication are introduced for the mussel meat protein modification,and the impact of these processing treatments on the structure and functional properties of mussel meat proteins is also discussed.Moreover,the production of bioactive peptides with various kinds of biological activities from mussel meat proteins is highlighted.Finally,industrial and commercial applications of mussel meat proteins and their derived bioactive peptides in food formulations are discussed.The research findings demonstrated that homogenization and ultrasonication treatments could induce the change in protein conformation and significantly improve the functionality of mussel meat proteins.Enzymatic hydrolysis and fermentation are effective approaches to produce bioactive peptides with multiple biological activities from mussels.Mussel meat proteins exhibit considerable nutritional and functional values compared to other widely used protein sources.Therefore,mussel meat protein could be used as a sustainable alternative protein resource in food applications.

Potential antioxidant bioactive peptides from camel milk proteins

Camel milk is traditionally considered to have medicinal characteristics that it has potential health benefits and could help to treat several illnesses. Particularly, it is closest to human breast milk and has high levels of nutrients and bioactive components. The aim of this study was to explore the antioxidant peptides derived from protein fractions of camel milk. Camel milk proteins(CMP) were fractionated into camel casein protein(CCP) and camel whey protein(CWP), which were hydrolyzed with pepsin to produce peptic digests P-CCP and P-CWP, respectively. RP-HPLC was used for fractionation of the peptides from the P-CCP and P-CWP. The antioxidant activities were evaluated using superoxide anion generating system of xanthine oxidase(XOD)and 2,2-diphenyl-1-picrylhydrazyl(DPPH) scavenging assay. Active peptides were analyzed using matrixassisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS) whereas a number of antioxidant peptides, with masses ranging from 913 to 2,951 Da, derived mainly from alpha-casein, lactophorin and lactoferrin, were identified. When yeast cells are used as a system for modeling mitochondrial disease, the peptides in caseins and whey fractions significantly enhanced the tolerance of yeast cells against peroxide-induced oxidative stress. The results show that both caseins and whey proteins of camel milk possess bioactive peptides with significant radical-scavenging activities and thus herald a fascinating opportunity for their potential as nutraceuticals or therapeutic peptides for prevention and treatment of oxidative stress-associated diseases.

基于分子模拟技术筛选牛乳清蛋白源抗氧化肽

传统抗氧化肽的筛选往往需要经过酶解、纯化、分离、鉴定及体外试验验证,时间和物力花费较大。本研究以生物信息学为理论基础,利用计算机虚拟消化、相关活性预测与评价、药物代谢动力学及分子模拟技术对牛乳清蛋白源抗氧化肽进行依次筛选,对其可能发生的Keap1-Nrf2-ARE抗氧化途径进行分析、解释,分析其分子反应过程中构象的稳定性。应用PeptideRanker程序对生物活性进行预测评分,筛选得到137条评分大于0.4的肽段,同时采用ToxinPred、Innovagen、Expasy-compute及Pepdraw程序进一步分析肽段的理化性质,并通过药物代谢动力学ADMET、TOPKAT分析对肽段进行筛选,得到14条无毒、无致敏性、无致突变性、无致癌性及水溶性良好的多肽,然后将其与Keap1受体进行分子对接。将分子对接评分前4位的多肽与Keap1的最优结合复合物进行构象作用机制分析,最终通过复合物构象分子间作用力评价CDEF序列最具抗氧化活性的潜力,并将CDEF-Keap1受体复合物进行分子动力学模拟,结果显示动力学模拟过程中构象变化较稳定,因此CDEF抗氧化肽可在人体中较好地发挥抗氧化活性。与传统酶解方法相比,本方法可快速、高效筛选抗氧化肽,为天然食品源抗氧化肽的快速筛选提供了新思路。

Rationally designed bioactive milk-derived protein scaffolds enhanced new bone formation

Recently,a number of studies reported that casein was composed of various multifunctional bioactive peptides such as casein phosphopeptide andβ-casochemotide-1 that bind calcium ions and induce macrophage chemotaxis,which is crucial for bone homeostasis and bone fracture repair by cytokines secreted in the process.We hypothesized that the effects of the multifunctional biopeptides in casein would contribute to improving bone regeneration.Thus,we designed a tissue engineering platform that consisted of casein and polyvinyl alcohol,which was a physical-crosslinked scaffold(milk-derived protein;MDP),via simple freeze-thaw cycles and performed surface modification using 3,4-dihydroxy-L-phenylalanine(DOPA),a mussel adhesive protein,for immobilizing adhesive proteins and cytokines for recruiting cells in vivo(MDP-DOPA).Both the MDP and MDP-DOPA groups proved indirectly contribution of macrophages migration as RAW 264.7 cells were highly migrated toward materials by contained bioactive peptides.We implanted MDP and MDP-DOPA in a mouse calvarial defect orthotopic model and evaluated whether MDP-DOPA showed much faster mineral deposition and higher bone density than that of the no-treatment and MDP groups.The MDP-DOPA group showed the accumulation of host M2 macrophages and mesenchymal stem cells(MSCs)around the scaffold,whereas MDP presented mostly M1 macrophages in the early stage.

A comprehensive review on bioactive peptides derived from milk and milkproducts of minor dairy species

Milk from different species has been exploited for the isolation of various functional ingredients for decades.Irrespective of the source,milk is considered as a complete food,as it provides essential nutrients required by the human body.Proteins and their fractions are valuable sources of bioactive peptides that might exert a health beneficial role in the human body such as immune-modulation,antioxidant activity,ACE-inhibitory activity,anti-neoplastic,anti-microbial,etc.In milk,bioactive peptides may either be present in their natural form or released from their parental proteins due to enzymatic action.The increasing interest in bioactive peptides among researchers has lately augmented the exploration of minor dairy species such as sheep,goat,camel,mithun,mare,and donkey.Alternative to cow,milk from minor dairy species have also been proven to be healthier from infancy to older age owing to their higher digestibility and other nutritive components.Therefore,realizing the significance of milk from such species and incentivized interest towards the derivatization of bioactive peptides,the present review highlights the significant research achievements on bioactive peptides from milk and milk products of minor dairy species.

Electrophoresis(SDS-PAGE)as a Method for Screening Species of Passiflora Using Seed Proteins as Molecular Markers

Brazil is the center of diversity and dispersion of species of the genus Passiflora.Two species of passion fruit Passiflora tenuifila BRS VT(passiflora garlic)and another of Passiflora setacea BRS PC were used in this study.The plants were grown on the Embrapa Cerrados experimental farm.The fruits were washed,minimally cut and their constituents separated.Both species differed in the proportion of wet mass between husk,seed,and pulp.As expected,both species had a higher proportion of husks and seeds and a lower amount of pulp.A semi-purification procedure for the proteins present in the seeds and husks was described and the molecular aspects were analyzed by polyacrylamide gel electrophoresis(SDS-PAGE).The quantification of soluble proteins was performed using the Bradford method.The analysis of the soluble protein extracted from the seeds and husks showed that these values are up to 70 times higher for the seeds of P.tenuifila and 28.5 times for P.setacea,in the husks.It was verified that both species present a similar protein profile,observed by the intense and diverse bands found in the polyacrylamide gel,mainly in the range of 32 to 19 kDa.This work opens up an unexplored field of tracking bioactive proteins and/or peptides including forms of nanostructure systems that protect other bioactive molecules.The objective of this work was to present an analytical procedure to semi-purify seed proteins of two Passiflora species and use electrophoresis as an analysis tool for further screening of their protein profiles and selection of a molecular marker to differentiate them.

紫苏粕蛋白抗氧化活性肽的制备、分离纯化及序列鉴定

以紫苏粕粉为原料,使用碱性蛋白酶进行水解反应,制备抗氧化活性肽。采用超滤离心、凝胶过滤色谱和反相色谱等分离纯化手段对其富集。结果表明,相较其它3种蛋白酶,碱性蛋白酶Alcalase对紫苏粕蛋白的水解程度最高,约为(25.94±0.21)%;碱性蛋白酶作用紫苏蛋白后的酶解产物的抗氧化活性最好,对DPPH自由基清除率约为(91.01±0.73)%。酶解上清液经超滤离心分离后最小分子质量组分F1(小于3 ku)抗氧化活性最强,F1组分经Sephadex G-25凝胶过滤色谱分离后按照分子质量大小依次得到P1、P2、P33个组分,其中分子质量最小的P3组分抗氧化活性最高。P3组分经反相色谱分离所得4个组分中,最后被洗脱出来的P3-4组分疏水性最强且DPPH·自由基清除率最高,质量浓度为3μg/mL的P3-4溶液的DPPH·清除率为(58.8±0.78)%。通过LC-MS-MS鉴定,抗氧化活性肽P3-4组分为十二肽,其氨基酸序列为Lys-Leu-Lys-Asp-Ser-Phe-Glu-Arg-Gln-Gly-Met-Val,分子质量为1437.8 u。本研究结果为深入开发紫苏蛋白资源,研发紫苏抗氧化活性肽提供理论参考。

驼血蛋白中胰脂肪酶抑制肽的计算机模拟评估

本研究利用中性蛋白酶对驼血蛋白进行酶解,对其酶解工艺、胰脂肪酶(PDB code:1ETH)抑制活性、多肽分子特征以及分子结合模式进行研究,以期获得高活性的胰脂肪酶抑制肽。结果表明:中性蛋白酶在酶用量为5%、酶解pH为7.0、酶解温度为55℃、酶解时间为3 h的最佳工艺条件下酶解驼血蛋白所获多肽的胰脂肪酶抑制率为42.13%。使用LC-MS/MS共鉴定出533种肽段,肽兰克评分大于0.8的有8条肽段。其中ALERMFLGF、GQPAVPVRF、WDPSKVPPR三条肽段与胰脂肪酶结合显著。通过AMBER 19进行分子动力学模拟,结果显示,三条肽段均能与胰脂肪酶活性口袋残基结合,主要通过静电作用、氢键和疏水相互作用抑制其活性。其中GQPAVPVRF结合能最高且具有更多的结合位点而成为最具潜能的胰脂肪酶抑制肽。本研究为开发新型PL抑制剂肽提供了参考,为驼血蛋白的高价值利用提供了新途径。

超高压技术在蛋白质改性和活性肽制备中的应用研究进展

生物活性肽可以从植物、动物、海洋资源中获得,其具有多种生物活性,如抗高血压、抗氧化、抗血栓、降胆固醇、降血压、抗菌、免疫调节、细胞调节、结合矿物质等。提高蛋白质向多肽的转化率一直以来是多肽研究的难点之一,超高压技术是一种适合于规模化生产的新型非热加工物理技术,近年来被广泛地应用于蛋白质工程,超高压技术的应用改善了蛋白质的结构和功能特性,提高了生物活性肽提取、生产的效率。本文重点综述超高压技术在蛋白质改性和肽制备方面的应用,概述超高压制备生物活性肽的类型、来源和生物活性,以期为深入应用超高压技术辅助蛋白质改性和活性肽制备提供参考。

Recent advances in chemical protein synthesis:method developments and biological applications

The central dogma of modern biology underscores the pivotal roles proteins play in diverse biological processes,the study of which necessitates advanced methods to produce proteins with precision and versatility.Chemical protein synthesis,a powerful approach utilizing chemical reactions for the de novo construction of structurally accurate proteins,has emerged as a transformative tool for studying proteins and generating protein derivatives/mimics inaccessible by natural biological machinery,including post-translationally modified proteins,proteins comprised of unnatural amino acids,as well as mirror-image proteins.This review summarizes recent strides in synthetic method developments for chemical protein synthesis,including innovative techniques in solid-phase peptide synthesis,the challenges presented by difficult sequences in either synthesis or folding and the exploration of novel ligation reactions using both chemical and enzymatic methods.Furthermore,the review also delves into newly developed protocols for site-selective protein modifications and the generation of stapled or macrocyclized peptides/miniproteins,highlighting the power of chemical methods to make structurally diverse proteins.Recent applications of synthetic proteins in investigating post-translational modifications(phosphorylation,lipidation,glycosylation,ubiquitination,etc.),mirror-image biological processes and drug development are further discussed.Together,these topics provide a comprehensive overview of the current landscape of chemical protein synthesis.

An improved installation of 2-hydroxy-4-methoxybenzyl(iHmb)method for chemical protein synthesis

The 2-hydroxy-4-methoxybenzyl(Hmb)backbone modification can prevent amide bond-mediated sidereactions(e.g.,aspartimide formation,peptide aggregation)by installing the removable Hmb group into a peptide bond,thus improving the synthesis of long and challenging peptides and proteins.However,its use is largely precluded by the limited Hmb’s installation sites.In this report,an improved installation of Hmb(iHmb)method was developed to achieve the flexible installation and the convenient removal of Hmb.The iHmb method involves two critical steps:(1)oxidative diazotization of the readily installed 2-hydroxy-4-methoxy-5-amino-benzyl(Hmab)to give 2-hydroxy-4-methoxy-5-diazonium-benzyl(Hmdab)by combining soamyl nitrite(IAN)/HBF_(4),and(2)reductive elimination of Hmdab to give the desired Hmb by 1,2-ethanedithiol(EDT).The iHmb method enables the installation of Hmb at any primary amino acid including the highly sterically hindered amino acids(e.g.,valine and isoleucine).The practicality and utility of the iHmb method was demonstrated by one-shot solid-phase synthesis of a challenging aspartimide-prone peptide,the mirror-image version of a hydrophobic peptide and a long-chain peptide up to 76-residue.Furthermore,the iHmb method can be utilized to facilitate chemical protein ligation,as exemplified by the synthesis of the single-spanning membrane protein sarcolipin.The iHmb method expands the toolkit for peptide synthesis and ligation and facilitates the preparation of peptides/proteins.

乳蛋白对肠道菌群影响的研究进展

牛乳中蛋白完整的分子结构及众多的生物活性肽在促进肠道有益菌增殖,维持肠道菌群平衡中发挥着重要的作用。乳中的免疫球蛋白、溶菌酶等也具有抑制病原菌定殖黏附,保护肠道健康的功效。本文综述了乳中多种活性蛋白对肠道菌群的影响,为科学充分地利用乳蛋白预防和保护肠道健康提供理论依据与参考。

动物血液蛋白质及活性肽开发研究进展

动物血液作为畜禽屠宰及加工的主要副产物,富含蛋白质、微量元素、矿物质、激素、维生素、酶类等多种功能成分,营养价值较高。其中,蛋白质作为动物血液的主要营养成分,占动物全血含量的10%~20%,其氨基酸组成丰富,富含赖氨酸,是一种优质的动物蛋白资源,素有“液态肉”之称,具有广阔的开发应用前景。本文主要综述动物血液蛋白质的研究进展及开发应用现状,重点总结了近年来以动物血液蛋白为原料开发的具有特定生理功能的生物活性肽,如抗氧化肽、抗菌肽、免疫调节肽、抗癌肽、降血压肽、抗糖尿病肽等,对活性肽的结构、序列及活性功能进行系统阐述,并对动物血液蛋白实际应用中出现的问题以及未来发展趋势进行展望,旨在为动物血液蛋白的高附加值开发利用提供参考。