组氨酸磷酸化修饰的鉴定方法及应用

蛋白质磷酸化是广受关注的翻译后修饰类型之一,组氨酸磷酸化作为一种非常见的磷酸化修饰,最早被发现在细菌和低等真核生物信号传导的级联反应中起关键作用。近年来研究显示,其在肿瘤发生发展过程中也可能扮演了重要角色。由于磷酸化组氨酸的化学不稳定性、低丰度、亚化学计量性质、缺乏特异性的富集试剂,导致研究手段缺乏,限制了人们对磷酸化组氨酸修饰底物蛋白质的认识。随着磷酸化组氨酸抗体的设计以及富集和质谱等鉴定方法的发展,更多的磷酸化组氨酸修饰底物被鉴定,从而加速了对磷酸化组氨酸生物学功能的认识。本文介绍了磷酸化组氨酸的化学性质、主要生物学功能,并综述了磷酸化修饰底物富集和鉴定技术等方面的最新进展,同时也要看到组氨酸磷酸化修饰组学研究仍然存在巨大的技术挑战。     

Effects of passive integrated transponder tags on short-term feeding patterns in European perch (Perca fluviatilis)

We tested the feeding behaviour of small European perch (Perca fluviatilis) in a laboratory study during the first 24 h after handling and 23 mm passive integrated transponder (PIT) tag implantation. Feeding commenced almost immediately following tagging and overall feeding patterns were unaffected by tagging. However, untagged perch had more feeding events than PIT-tagged individuals. This discrepancy could be attributed to post-tagging effects or/and reduced room for food due to the presence of the tag in the body cavity.     

Nasal Discs and the Vital Rates of Lesser Scaup

Nasal discs have been used to identify ducks in studies of survival and reproduction. To date, there has not been a comprehensive assessment of nasal-disc effects on the vital rates of wild ducks. We applied nasal discs to 603 juvenile and 784 adult lesser scaup (Aythya affinis) females from a population breeding in southwest Montana, USA, and released 1,399 juvenile and 71 adult females wearing only metal leg bands between June 2005 and September 2016. Using resighting, recapture, and hunter-recovery data collected from those individuals, we estimated survival and recovery probability with multistate capture-recapture models in Program MARK. We also assessed if recovery distance from our study site and pre-breeding and brood-rearing body condition were diminished for females wearing nasal discs. Model-averaged survival probabilities were 0.231 ± 0.035 (SE) for juveniles and 0.482 ± 0.019 for adults released with nasal discs. Survival was 1.8–3.4 times higher for females released with metal leg bands when compared to those released with nasal discs; survival of these juveniles was 0.433 ± 0.049 and 0.693 ± 0.039 for adults. We did not find evidence for recovery probability or recovery distance varying between females that wore nasal discs and those that did not. During the pre-breeding and brood-rearing seasons, we did not find females wearing nasal discs to be in lower body condition when compared to unmarked females. Our comprehensive assessment of nasal discs on wild lesser scaup suggests that survival probabilities estimated from nasal-marked study populations should be cautiously interpreted as minimum estimates. © 2021 The Wildlife Society.     

The Journey from Two-Step to Multi-Step Phosphorelay Signaling Systems

BackgroundThe two-component signaling (TCS) system is an important signal transduction machinery in prokaryotes and eukaryotes, excluding animals, that uses a protein phosphorylation mechanism for signal transmission.ConclusionProkaryotes have a primitive type of TCS machinery, which mainly comprises a membrane-bound sensory histidine kinase (HK) and its cognate cytoplasmic response regulator (RR). Hence, it is sometimes referred to as two-step phosphorelay (TSP). Eukaryotes have more sophisticated signaling machinery, with an extra component - a histidine-containing phosphotransfer (HPT) protein that shuttles between HK and RR to communicate signal baggage. As a result, the TSP has evolved from a two-step phosphorelay (His–Asp) in simple prokaryotes to a multi-step phosphorelay (MSP) cascade (His–Asp–His–Asp) in complex eukaryotic organisms, such as plants, to mediate the signaling network. This molecular evolution is also reflected in the form of considerable structural modifications in the domain architecture of the individual components of the TCS system. In this review, we present TCS system''s evolutionary journey from the primitive TSP to advanced MSP type across the genera. This information will be highly useful in designing the future strategies of crop improvement based on the individual members of the TCS machinery.     

蒙古黄芪病程相关蛋白AmPR-10于大肠杆菌不同载体的可溶性表达策略研究

蒙古黄芪病程相关蛋白（Astragalus membranaceus pathogenesis-related protein-10, AmPR-10）具有核酸酶活性,对黄芪生长发育及抗病机制具有重要意义。但从天然黄芪中提取蛋白质的传统方法成本较高,蛋白得率较少。研究首次利用大肠杆菌表达体系对AmPR-10进行可溶性外源表达,构建了3种重组子：①以pET28a为载体构建pET28a-AmPR-10;②以pET30a为载体构建pET30a-AmPR-10;③以pET30a为载体、大肠杆菌分子伴侣skp修饰构建pET30a-skp-AmPR-10。通过SDS-PAGE分析,目的蛋白可溶性表达量比较结果是：pET30a-skp-AmPR-10 > pET30a-AmPR-10 > pET28a-AmPR-10。由蛋白质三级结构模拟分析发现,载体pET-30a上的标签S-tag通过影响AmPR-10局部α螺旋构象而提高目标蛋白的可溶性表达,分子伴侣skp通过提高融合蛋白整体α螺旋比例进一步提升目标蛋白可溶性表达量。研究解决了目前从天然黄芪中提取蛋白操作复杂、提取量小的问题。同时,经测定,目的蛋白具有核酸酶活性,为外源AmPR-10相关活性研究提供了依据。     

Introduction of histidine units using benzotriazolide activation

N^α‐Boc‐N^im‐(4‐toluenesulfonyl‐l ‐histidylbenzotriazole) enables convenient acylation of N‐, O‐, S‐, and C‐nucleophiles with no detectable racemization. We report efficient syntheses of novel histidine‐containing di‐, tri‐, and tetra‐peptides and models for the preparation of potentially biologically active histidine N‐, O‐, S‐, and C‐conjugates. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Expression,purification, and characterization of N-terminal His-tagged proteins with mutations in zinc finger 3 of zinc finger protein ZNF191(243–368)

Abstract

Zinc finger protein ZNF191(243–368), the zinc finger region of ZNF191, is potentially associated with cell proliferation in hepatocellular carninoma. A His-tag expression system was used to express and purify proteins with mutations in the zinc finger 3 of ZNF191(243–368) for analysis of protein properties, structure, and functions. The purification of the His-tag fusion proteins was simpler and faster than that of the ZNF191(243–368) inclusion bodies. The properties and structures of the His-tag fusion mutant proteins were investigated using spectrographic techniques and DNA hydrolysis experiment. The His₆-tag system could be used to express ZNF191(243–368). The presence of the His₆-tag at the N-terminus of ZNF191(243–368) did not evidently affect its properties and structure. However, the site-directed mutations in zinc finger 3 affected the structure of the protein. The DNA hydrolase activity of His₆-ZF-F3/H4 suggested that four histidines in zinc finger 3 might form a structure similar to that of the active center in a hydrolase. This work reports that continuous histidines need to form a certain structure for specific functions, and provides new insights into the design of an artificial nuclease.     

High-throughput proteomics using Fourier transform ion cyclotron resonance mass spectrometry

The advent of high-throughput proteomic technologies for global detection and quantitation of proteins creates new opportunities and challenges for those seeking to gain greater understanding of the cellular machinery. Here, recent advances in high-resolution capillary liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry are reviewed along with its potential application to high-throughput proteomics. These technological advances combined with quantitative stable isotope labeling methodologies provide powerful tools for expanding our understanding of biology at the system level.     

Isobaric protein and peptide quantification: perspectives and issues

An important challenge for proteomics is the ability to compare protein levels across biological samples. Since their introduction, isotopic and isobaric peptide labeling have played an important role in relative quantitative comparisons of proteomes. One important drawback of most of the isotopic-labeling techniques is an increase in sample complexity. This problem was successfully addressed with the construction of isobaric labeling strategies, such as isobaric tag for relative and absolute quantification (iTRAQ), tandem mass tagging, the cleavable isobaric affinity tag, dimethylated leucines and isobaric peptide termini labeling. Furthermore, numerous applications for multiplexing using iTRAQ and tandem mass tagging have been reported.     

High-throughput proteomics of breast carcinoma cells: a focus on FTICR-MS

Discovery of better biomarkers for diagnosis, prognosis and therapy-response prediction is the most critical task of a scientific quest aimed at developing novel, tailormade therapies for patients with cancer. Consequently, a proteome-wide analysis, in addition to genomic studies, is an absolute requirement for a complete functional understanding of tumor biology. Ultra-sensitive, high-performance Fourier-transform ion-cyclotron resonance (FTICR) mass spectrometry (MS) currently holds an important role in fulfilling the demands of biomarker discovery. In this review, we describe the applicability of FTICR-MS for breast cancer proteomics, particularly for the analysis of complex protein mixtures obtained from a limited number of cells typically available from clinical specimens.