Purification and Chemical Analysis of Microbial Cell Flocculant Produced by Aspergillus sojae AJ7002

A bio-flocculant was isolated from the culture broth of Asp. sojae AJ 7002. It was partially purified by acetone or ethanol precipitation, by ion-exchange and gel chromatography, and by dialysis. The isolated polymer possessed chemical characteristics of a poly-hexosamine and a protein. Glucosamine and galactosamine were not acetylated. The flocculant contained 2-ketogluconic acid, but sulfur or phosphorus was not detected. This flocculant was thermo-stable and its activity varied with pH. It was suggested that the hexosamine moiety in the polymer played a major role in bio-flocculation, assisted by protein portion in enlargement of the molecular weight of the flocculant, and by 2-ketogluconic acid in endowing it with amphoteric character.     

Vital for Viruses: Intrinsically Disordered Proteins

Viruses infect all kingdoms of life; their genomes vary from DNA to RNA and in size from 2kB to 1 MB or more. Viruses frequently employ disordered proteins, that is, protein products of virus genes that do not themselves fold into independent three-dimensional structures, but rather, constitute a versatile molecular toolkit to accomplish a range of functions necessary for viral infection, assembly, and proliferation. Interestingly, disordered proteins have been discovered in almost all viruses so far studied, whether the viral genome consists of DNA or RNA, and whatever the configuration of the viral capsid or other outer covering. In this review, I present a wide-ranging set of stories illustrating the range of functions of IDPs in viruses. The field is rapidly expanding, and I have not tried to include everything. What is included is meant to be a survey of the variety of tasks that viruses accomplish using disordered proteins.     

Microbial strain improvement for enhanced polygalacturonase production by Aspergillus sojae

Strain improvement is a powerful tool in commercial development of microbial fermentation processes. Strains of Aspergillus sojae which were previously identified as polygalacturonase producers were subjected to the cost-effective mutagenesis and selection method, the so-called random screening. Physical (ultraviolet irradiation at 254 nm) and chemical mutagens (N-methyl-N′-nitro-N-nitrosoguanidine) were used in the development and implementation of a classical mutation and selection strategy for the improved production of pectic acid-degrading enzymes. Three mutation cycles of both mutagenic treatments and also the combination of them were performed to generate mutants descending from A. sojae ATCC 20235 and mutants of A. sojae CBS 100928. Pectinolytic enzyme production of the mutants was compared to their wild types in submerged and solid-state fermentation. Comparing both strains, higher pectinase activity was obtained by A. sojae ATCC 20235 and mutants thereof. The highest polygalacturonase activity (1,087.2?±?151.9 U/g) in solid-state culture was obtained by mutant M3, which was 1.7 times increased in comparison to the wild strain, A. sojae ATCC 20235. Additional, further mutation of mutant M3 for two more cycles of treatment by UV irradiation generated mutant DH56 with the highest polygalacturonase activity (98.8?±?8.7 U/mL) in submerged culture. This corresponded to 2.4-fold enhanced polygalacturonase production in comparison to the wild strain. The results of this study indicated the development of a classical mutation and selection strategy as a promising tool to improve pectinolytic enzyme production by both fungal strains.     

Computational Screening of Phase-separating Proteins

Phase separation is an important mechanism that mediates the compartmentalization of proteins in cells. Proteins that can undergo phase separation in cells share certain typical sequence features, like intrinsically disordered regions(IDRs) and multiple modular domains. Sequencebased analysis tools are commonly used in the screening of these proteins. However, current phase separation predictors are mostly designed for IDR-containing proteins, thus inevitably overlook the phase-separating proteins with relatively low IDR content. Features other than amino acid sequence could provide crucial information for identifying possible phase-separating proteins: protein–protein interaction(PPI) networks show multivalent interactions that underlie phase separation process; post-translational modifications(PTMs) are crucial in the regulation of phase separation behavior; spherical structures revealed in immunofluorescence(IF) images indicate condensed droplets formed by phase-separating proteins, distinguishing these proteins from non-phaseseparating proteins. Here, we summarize the sequence-based tools for predicting phaseseparating proteins and highlight the importance of incorporating PPIs, PTMs, and IF images into phase separation prediction in future studies.     

A Real-Time SUMO-Binding Assay for the Analysis of the SUMO-SIM Protein Interaction Network

SUMO-interacting motifs (SIMs) play a central role in the fate of SUMO-modified proteins. Here we report a real-time SUMO-binding assay. It can be applied to the identification of SIMs and to screening for the identification of novel SUMO-binding proteins. Using this assay, we investigated the SIMs in SETDB1 and MCAF1 to gain insight into the assembly of SETDB1-MCAF1-mediated gene silencing.     

Flocculant Production by Paecilomyces sp. Taxonomic Studies and Culture Conditions for Production

Screening of microorganisms producing a new kind of flocculating substance was carried out. A fungus that produced a large amount of a strong microbial cell flocculant was isolated from soil. The fungus was classified as a Paecilomyces species. The substance produced showed very interesting and unique flocculation characteristics. It could efficiently flocculate all tested suspended solids in aqueous solution, and flocculation was practically not affected by ionic strength, pH or temperature.

The influence of components of the culture medium upon the flocculant production by Paecilomyces sp. 1-1 was studied. Addition of hydrolysates of casein such as casamino acid and polypeptone stimulated the production of the flocculant. A high concentration of calcium ions also significantly increased the mycelial growth and the production of the flocculant. Under the optimum cultural conditions, a 500-fold dilution of the culture filtrate could efficiently flocculate and sediment E. coli cells.     

细胞自噬中关键蛋白乙酰化修饰与相关疾病诊治的研究进展

自噬是一种在进化上保守的溶酶体依赖的降解途径.近十多年来,自噬过程的分子机制研究得到了长足的发展.自噬过程中关键蛋白复合物的乙酰化修饰发挥了十分重要的作用.为此,该文阐述了细胞自噬过程中主要蛋白复合物的乙酰化修饰作用进展,并对蛋白质乙酰化修饰与肿瘤、神经退行性疾病等的关系作一总结.总之,自噬过程中蛋白乙酰化修饰已经成为...     

邻近标记在蛋白质组学中的发展及应用

人体内各种复杂的生命活动离不开蛋白质之间的相互作用。这种相互作用具有瞬时性和结合力弱等特点,并受到多种动态调节,特别是蛋白质翻译后修饰（post-translation modifications, PTM）。传统的亲和质谱检测方法存在蛋白纯化的局限性,在高效检测到动态变化方面存在不足。邻近标记是一种能够给与靶蛋白质瞬时靠近,或者互作（邻近）的蛋白质加上生物素的技术,它与质谱检测技术的联合使用能检测细胞过程中弱的、瞬时的蛋白质相互作用,有效解决上述问题。本文综述了基于生物素的邻近标记方法的发展现状,从依赖于融合序列的生物素标记开始,依次介绍有关生物素连接酶、过氧化物酶及其进化后的2代标记方法等经典生物素标记的方法和原理,比较各个方法间的差异和优缺点;也列举了一些近年来新出现的标记方法,如将生物素连接酶进行拆分、鉴定蛋白质在不同复合物中功能的方法、抗体靶向的标记方法,以及其他来源的生物素连接酶突变体,例如枯草芽孢杆菌（Bacillus subtilis）的C端氨基酸突变的生物素连接酶,能够应用在苍蝇和蠕虫中的生物素连接酶突变体。本文对这些方法进行归纳总结,旨在为初步接触该领域的科研工作者提供参考,同时也希望能够提供一些新的思路,推动蛋白质相互作用组学的发展。     

邻近标记在蛋白质组学中的发展及应用

人体内各种复杂的生命活动离不开蛋白质之间的相互作用。这种相互作用具有瞬时性和结合力弱等特点,并受到多种动态调节,特别是蛋白质翻译后修饰（post-translation modifications, PTM）。传统的亲和质谱检测方法存在蛋白纯化的局限性,在高效检测到动态变化方面存在不足。邻近标记是一种能够给与靶蛋白质瞬时靠近,或者互作（邻近）的蛋白质加上生物素的技术,它与质谱检测技术的联合使用能检测细胞过程中弱的、瞬时的蛋白质相互作用,有效解决上述问题。本文综述了基于生物素的邻近标记方法的发展现状,从依赖于融合序列的生物素标记开始,依次介绍有关生物素连接酶、过氧化物酶及其进化后的2代标记方法等经典生物素标记的方法和原理,比较各个方法间的差异和优缺点;也列举了一些近年来新出现的标记方法,如将生物素连接酶进行拆分、鉴定蛋白质在不同复合物中功能的方法、抗体靶向的标记方法,以及其他来源的生物素连接酶突变体,例如枯草芽孢杆菌（Bacillus subtilis）的C端氨基酸突变的生物素连接酶,能够应用在苍蝇和蠕虫中的生物素连接酶突变体。本文对这些方法进行归纳总结,旨在为初步接触该领域的科研工作者提供参考,同时也希望能够提供一些新的思路,推动蛋白质相互作用组学的发展。     

Plant Cell Wall Proteomics： Mass Spectrometry Data,a Trove for Research on Protein Structure/Function Relationships

Proteomics allows the large-scale study of protein expression either in whole organisms or in purified organelles. In particular, mass spectrometry （MS） analysis of gel-separated proteins produces data not only for protein identification, but for protein structure, location, and processing as well. An in-depth analysis was performed on MS data from etiolated hypocotyl cell wall proteomics ofArabidopsis thaliana. These analyses show that highly homologous members of multigene families can be differentiated. Two lectins presenting 93% amino acid identity were identified using peptide mass fingerprinting. Although the identification of structural proteins such as extensins or hydroxyproline/proline-rich proteins （H/PRPs） is arduous, different types of MS spectra were exploited to identify and characterize an H/PRP. Maturation events in a couple of cell wall proteins （CWPs） were analyzed using site mapping. N-glycosylation of CWPs as well as the hydroxylation or oxidation of amino acids were also explored, adding information to improve our understanding of CWP structure/function relationships. A bioinformatic tool was developed to locate by means of MS the N-terminus of mature secreted proteins and N-glycosylation.     

黑曲霉发酵产橙皮苷酶的工艺优化

目的:对黑曲霉WP124发酵产橙皮苷酶的工艺条件进行优化,旨在为利用酶法改造橙皮苷打下基础.方法:采用摇瓶培养,对培养基的成分和培养条件进行了优化.结果:黑曲霉WP124发酵产橙皮苷酶的最佳培养基组成是:蔗糖30g/L,酵母膏20g/L,磷酸二氢钾3g/L,桔皮粉50g/L;最佳培养条件是:起始pH为5.5,培养温度为30℃,摇瓶转速是180r/min.在上述条件下,经过72h的培养,橙皮苷酶酶活达到1398U/mL.结论:该菌株发酵过程具有较好的工业化应用的前景.     

Current trends in computational inference from mass spectrometry-based proteomics

Mass spectrometry offers a high-throughput approach to quantifying the proteome associated with a biological sample and hence has become the primary approach of proteomic analyses. Computation is tightly coupled to this advanced technological platform as a required component of not only peptide and protein identification, but quantification and functional inference, such as protein modifications and interactions. Proteomics faces several key computational challenges such as identification of proteins and peptides from tandem mass spectra as well as their quantitation. In addition, the application of proteomics to systems biology requires understanding the functional proteome, including how the dynamics of the cell change in response to protein modifications and complex interactions between biomolecules. This review presents an overview of recently developed methods and their impact on these core computational challenges currently facing proteomics.     

Amino acid-coded tagging approaches in quantitative proteomics

To improve the efficiency, accuracy, reproducibility, throughput and proteome coverage of mass spectrometry-based quantitative approaches, both in vitro and in vivo tagging of particular amino acid residues of cellular proteins have been introduced to assist mass spectrometry for global-scale comparative studies of differentially expressed proteins/modifications between different biologically relevant cell states or cells at different pathological states. The basic features of these methods introduce pair-wise isotope signals of each individual peptide containing a particular type of tagged amino acid (amino acid-coded mass tagging) that originated from different cell states. In this review, the applications of major amino acid-coded mass tagging-based quantitative proteomics approaches, including isotope-coded affinity tag, isobaric tags for relative and absolute quantification (iTRAQ?) and stable isotope labeling by amino acids in cell culture are summarized in the context of their respective strengths/weakness in identifying those differentially expressed or post-translational modified proteins regulated by particular cellular stress on a genomic scale in a high-throughput manner. Importantly, these gel-free, in-spectra quantitative mechanisms have been further explored to identify/characterize large-scale protein–protein interactions involving various functional pathways. Taken together, the information about quantitative proteome changes, including multiple regulated proteins and their interconnected relationships, will provide an important insight into the molecular mechanisms, where novel targets for diagnosis and therapeutic intervention will be identified.     

去泛素化酶活性的调控

泛素化是一种非常重要的蛋白质翻译后修饰方式,在细胞生命活动的各个方面发挥作用。泛素化修饰是可逆的过程,去泛素化酶通过催化去除底物蛋白质上的泛素从而逆转该过程。去泛素化酶是一类数量众多的蛋白水解酶家族,近年来不断有新的去泛素化酶被发现和报道。鉴于其在细胞功能中的重要作用,去泛素化酶活性受到严格的调控。目前的研究表明,影响去泛素化酶活性的因素很多。本文主要从转录水平的调控、翻译后修饰、蛋白质定位和蛋白质相互作用等调控方式进行论述,以期为研究和利用去泛素化酶治疗疾病提供新思路。     

嗜肺军团菌效应蛋白质介导的新型泛素化过程

泛素化是真核细胞特有的蛋白质翻译后修饰方式,调节真核细胞内多种重要生理过程,例如蛋白质稳态、细胞周期、免疫反应、DNA修复以及囊泡转运等。鉴于泛素化对于生命活动的重要性,病原菌在与宿主细胞的长期进化过程中衍生出一系列针对宿主泛素化过程的效应蛋白质,调控宿主体内泛素化过程,从而构建有利于病原菌自身生长繁殖的内环境。嗜肺军团菌是一种革兰氏阴性菌,是军团菌肺炎的致病菌,能够引起发热和肺部感染,重型病死率高达15%~30%。Dot/Icm Ⅳ型分泌系统是嗜肺军团菌侵染过程中最主要的毒力系统。在侵染宿主细胞的过程中,嗜肺军团菌利用该分泌系统,分泌超过330种效应蛋白质,协助细菌在宿主胞内生存、增殖和逃逸。多种嗜肺军团菌效应蛋白质通过直接或者间接的方式对宿主泛素化过程进行调控。近年的研究发现,多种效应蛋白质可以介导不同于真核生物经典泛素化的新型泛素化过程。本文介绍了嗜肺军团菌效应蛋白质介导的新型泛素化过程的最新研究进展,为理解泛素化过程在嗜肺军团菌致病过程中的重要作用提供参考依据。     

Crystal structure of SUMO-3-modified thymine-DNA glycosylase

Modification of cellular proteins by the small ubiquitin-like modifier SUMO is important in regulating various cellular events. Many different nuclear proteins are targeted by SUMO, and the functional consequences of this modification are diverse. For most proteins, however, the functional and structural consequences of modification by specific SUMO isomers are unclear. Conjugation of SUMO to thymine-DNA glycosylase (TDG) induces the dissociation of TDG from its product DNA. Structure determination of the TDG central region conjugated to SUMO-1 previously suggested a mechanism in which the SUMOylation-induced conformational change in the C-terminal region of TDG releases TDG from tight binding to its product DNA. Here, we have determined the crystal structure of the central region of TDG conjugated to SUMO-3. The overall structure of SUMO-3-conjugated TDG is similar to the previously reported structure of TDG conjugated to SUMO-1, despite the relatively low level of amino acid sequence similarity between SUMO-3 and SUMO-1. The two structures revealed that the sequence of TDG that resembles the SUMO-binding motif (SBM) can form an intermolecular beta-sheet with either SUMO-1 or SUMO-3. Structural comparison with the canonical SBM shows that this SBM-like sequence of TDG retains all of the characteristic interactions of the SBM, indicating sequence diversity in the SBM.     

Protein arginine methylation of non-histone proteins and its role in diseases

Protein arginine methyltransferases (PRMTs) are a family of enzymes that can methylate arginine residues on histones and other proteins. PRMTs play a crucial role in influencing various cellular functions, including cellular development and tumorigenesis. Arginine methylation by PRMTs is found on both nuclear and cytoplasmic proteins. Recently, there is increasing evidence regarding post-translational modifications of non-histone proteins by PRMTs, illustrating the previously unknown importance of PRMTs in the regulation of various cellular functions by post-translational modifications. In this review, we present the recent developments in the regulation of non-histone proteins by PRMTs.     

Target protein for eucaryotic arginine-specific ADP-ribosyltransferase

Among ADP-ribosyltransferases reported in eucaryotes, arginine-specific transferases from turkey erythrocytes, chicken heterophils and rabbit skeletal muscle have been purified and extensively studied. They were reported to modify a number of proteinsin vitro. ADP-ribosylation of Ha-ras-p21 and transducin by the turkey erythrocyte transferase inhibits their GTPase and GTP-binding activities. Chicken heterophil enzyme modifies several substrate proteins for protein kinases and decreases the phosphate-acceptor activity. Rabbit skeletal muscle Ca²⁺-ATPase is inhibited by ADP-ribosylation catalyzed by the muscle transferase. Three transferases all ADP-ribosylate small molecular weight guanidino compounds such as arginine, arginine methylester and agmatine and poly-L-arginine and nuclear histones. However, the observation that muscle transferase did not ADP-ribosylate casein or actin, both of which can be modified by the heterophil transferase under the same conditions indicates that substrate specificity of these two enzymes are different. Substrate-dependent effects were observed with polyions of nucleotides such that polyanions stimulate the ADP-ribosylation of possible target protein, p33 by chicken heterophil transferase but has no effect on the modification of casein by the same enzyme.