Bioinformatic Analysis of Immobilized Enzymes: Towards a Better Understanding of The Guiding Forces

Protein bioinformatics has been applied to a myriad of opportunities in biocatalysis from enzyme engineering to enzyme discovery, but its application in enzyme immobilization is still very limited. Enzyme immobilization brings clear advantages in terms of sustainability and cost-efficiency but is still limited in its implementation. This, because it is a technique that remains tied to a quasi-blind protocol of trial and error, and therefore, is regarded as a time-intensive and costly approach. Here, we present the use of a set of bioinformatic tools to rationalize the results of protein immobilization that have been previously described. The study of proteins with these new tools allows the discovery of key driving forces in the process of immobilization that explain the obtained results, moving us a step closer to the final goal: predictive enzyme immobilization protocols.     

From Protein Structure to Function via Computational Tools and Approaches

The three-dimensional structures of proteins are often considered fundamental for understanding their function. Yet, because of the complexity of protein structure, extracting specific functional information from structures can be a considerable challenge. Here, we present selected approaches and tools that we have developed to study and connect protein sequence, structure, and function spaces. First, we consider a global perspective of structure space and view the protein data bank (PDB) as a database. We highlight challenges in searching protein structure space and in using the PDB as the starting point for computational structural studies. Then we describe a function-oriented view and show examples of how multiple protein structures can be used to extract insights about the function and specificity of proteins at the family level.     

Human Prostatic Acid Phosphatase: Structure,Function and Regulation

Human prostatic acid phosphatase (PAcP) is a 100 kDa glycoprotein composed of two subunits. Recent advances demonstrate that cellular PAcP (cPAcP) functions as a protein tyrosine phosphatase by dephosphorylating ErbB-2/Neu/HER-2 at the phosphotyrosine residues in prostate cancer (PCa) cells, which results in reduced tumorigenicity. Further, the interaction of cPAcP and ErbB-2 regulates androgen sensitivity of PCa cells. Knockdown of cPAcP expression allows androgen-sensitive PCa cells to develop the castration-resistant phenotype, where cells proliferate under an androgen-reduced condition. Thus, cPAcP has a significant influence on PCa cell growth. Interestingly, promoter analysis suggests that PAcP expression can be regulated by NF-κB, via a novel binding sequence in an androgen-independent manner. Further understanding of PAcP function and regulation of expression will have a significant impact on understanding PCa progression and therapy.     

Function Prediction and Analysis of Mycobacterium tuberculosis Hypothetical Proteins

High-throughput biology technologies have yielded complete genome sequences and functional genomics data for several organisms, including crucial microbial pathogens of humans, animals and plants. However, up to 50% of genes within a genome are often labeled "unknown", "uncharacterized" or "hypothetical", limiting our understanding of virulence and pathogenicity of these organisms. Even though biological functions of proteins encoded by these genes are not known, many of them have been predicted to be involved in key processes in these organisms. In particular, for Mycobacterium tuberculosis, some of these "hypothetical" proteins, for example those belonging to the Pro-Glu or Pro-Pro-Glu (PE/PPE) family, have been suspected to play a crucial role in the intracellular lifestyle of this pathogen, and may contribute to its survival in different environments. We have generated a functional interaction network for Mycobacterium tuberculosis proteins and used this to predict functions for many of its hypothetical proteins. Here we performed functional enrichment analysis of these proteins based on their predicted biological functions to identify annotations that are statistically relevant, and analysed and compared network properties of hypothetical proteins to the known proteins. From the statistically significant annotations and network information, we have tried to derive biologically meaningful annotations related to infection and disease. This quantitative analysis provides an overview of the functional contributions of Mycobacterium tuberculosis "hypothetical" proteins to many basic cellular functions, including its adaptability in the host system and its ability to evade the host immune response.     

Bioinformatic Tools for the Analysis and Prediction of ncRNA Interactions

Noncoding RNAs (ncRNAs) play prominent roles in the regulation of gene expression via their interactions with other biological molecules such as proteins and nucleic acids. Although much of our knowledge about how these ncRNAs operate in different biological processes has been obtained from experimental findings, computational biology can also clearly substantially boost this knowledge by suggesting possible novel interactions of these ncRNAs with other molecules. Computational predictions are thus used as an alternative source of new insights through a process of mutual enrichment because the information obtained through experiments continuously feeds through into computational methods. The results of these predictions in turn shed light on possible interactions that are subsequently validated experimentally. This review describes the latest advances in databases, bioinformatic tools, and new in silico strategies that allow the establishment or prediction of biological interactions of ncRNAs, particularly miRNAs and lncRNAs. The ncRNA species described in this work have a special emphasis on those found in humans, but information on ncRNA of other species is also included.     

Proteins of Unknown Function in the Protein Data Bank (PDB): An Inventory of True Uncharacterized Proteins and Computational Tools for Their Analysis

Proteins of uncharacterized functions form a large part of many of the currently available biological databases and this situation exists even in the Protein Data Bank (PDB). Our analysis of recent PDB data revealed that only 42.53% of PDB entries (1084 coordinate files) that were categorized under “unknown function” are true examples of proteins of unknown function at this point in time. The remainder 1465 entries also annotated as such appear to be able to have their annotations re-assessed, based on the availability of direct functional characterization experiments for the protein itself, or for homologous sequences or structures thus enabling computational function inference.     

Identification of CDPK Gene Family in Solanum habrochaites and Its Function Analysis under Stress

Tomato is an important vegetable crop. In the process of tomato production, it will encounter abiotic stress, such as low temperature, drought, and high salt, and biotic stress, such as pathogen infection, which will seriously affect the yield of tomato. Calcium-dependent protein kinase (CDPK) is a class of major calcium signal receptor which has an important regulatory effect on the perception and decoding of calcium signals. CDPK plays a key role in many aspects of plant growth, such as the elongation of pollen tubes, plant growth, and response to biotic and abiotic stress. While some studies have concentrated on Arabidopsis and pepper, Solanum habrochaites is a wild species relative of cultivated tomato and there is no report on CDPK in Solanum habrochaites to date. Using tomato genomic data, this study identified 33 members of the CDPK gene family. Evolutionary analysis divides family members into four Asian groups, of which the CDPK family members have 11 gene replication pairs. Subcellular location analysis showed that most proteins were predicted to be located in the cytoplasm, and less protein existed on the cell membrane. Not all CDPK family members have a transmembrane domain. Cis regulatory elements relating to light, hormones, and drought stress are overrepresented in the promoter region of the CDPK genes in Solanum habrochaites. The expression levels of each gene under biotic stress and abiotic stress were quantified by qRT-PCR. The results showed that members of the CDPK family in Solanum habrochaites respond to different biotic and abiotic stresses. Among them, the expression of ShCDPK6 and ShCDPK26 genes change significantly. ShCDPK6 and ShCDPK26 genes were silenced using VIGS (virus-induced gene silencing), and the silenced plants illustrated reduced stress resistance to Botrytis cinerea, cold, and drought stress. The results of this study will provide a basis for the in-depth study of the CDPK gene family in Solanum habrochaites, laying the foundation for further analysis of the function of the gene family.     

Exploring the Ability of Cyclic Peptides to Target SAM Domains: A Computational and Experimental Study

Sterile alpha motif (SAM) domains are protein interaction modules with a helical fold. SAM–SAM interactions often adopt the mid-loop (ML)/end-helix (EH) model, in which the C-terminal helix and adjacent loops of one SAM unit (EH site) bind the central regions of another SAM domain (ML site). Herein, an original strategy to attack SAM–SAM associations is reported. It relies on the design of cyclic peptides that target a region of the SAM domain positioned at the bottom side of the EH interface, which is thought to be important for the formation of a SAM–SAM complex. This strategy has been preliminarily tested by using a model system of heterotypic SAM–SAM interactions involving the erythropoietin-producing hepatoma kinase A2 (EphA2) receptor and implementing a multidisciplinary plan made up of computational docking studies, experimental interaction assays (by NMR spectroscopy and surface plasmon resonance techniques) and conformational analysis (by NMR spectroscopy and circular dichroism). This work further highlights how only a specific balance between flexibility and rigidity may be needed to generate modulators of SAM–SAM interactions.     

Role of Two Exceptional trans Adenylation Domains and MbtH-like Proteins in the Biosynthesis of the Nonribosomal Peptide WS9324A from Streptomyces calvus ATCC 13382

Nonribosomal peptide synthetases (NRPS) are organized in a modular arrangement. Usually, the modular order corresponds to the assembly of the amino acids in the respective peptide, following the collinearity rule. The WS9326A biosynthetic gene cluster from Streptomyces calvus shows deviations from this rule. Most interesting is the presence of two trans adenylation domains that are located downstream of the modular NRPS arrangement. Adenylation domains are responsible for the activation of their respective amino acids. In this study, we confirmed the involvement of the trans adenylation domains in WS9326A biosynthesis by performing gene knockout experiments and by observing the selective adenylation of their predicted amino acid substrates in vitro. We conclude that the trans adenylation domains are essential for WS9326A biosynthesis. Moreover, both adenylation domains are observed to have MbtH-like protein dependency. Overall, we conclude that the trans adenylation domains are essential for WS9326A biosynthesis.     

Atomic Resolution Analysis of the Defect Chemistry and Microdomain Structure of Brownmillerite-Type Strontium Cobaltite

To investigate the fundamental aspects of vacancy ordering in oxygen-transporting ceramic membranes, we have performed atomic resolution analysis of individual domains in brownmillerite-type SrCoO_3−δ. Electron energy loss spectroscopy indicates that the Co valence state in adjacent planes can be 2+ and 4+. This charge localization is accompanied by oxygen deficiency and the formation of ordered octahedral and tetrahedral coordinated Co sites. At microdomain boundaries, Z -contrast images reveal a structural relaxation of the octahedral site with the reduction of the Co valence state from 4+ to 3+ and the incorporation of extra oxygen vacancies.     

Identification and Functional Analysis of Tomato TPR Gene Family

Tomato (Solanum lycopersicum) as an important vegetable grown around the world is threatened by many diseases, which seriously affects its yield. Therefore, studying the interaction between tomato and pathogenic bacteria is biologically and economically important. The TPR (Tetratricopeptide repeat) gene family is a class of genes containing TPR conserved motifs, which are widely involved in cell cycle regulation, gene expression, protein degradation and other biological processes. The functions of TPR gene in Arabidopsis and wheat plants have been well studied, but the research on TPR genes in tomato is not well studied. In this study, 26 TPR gene families were identified using bioinformatics based on tomato genome data, and they were analyzed for subcellular localization, phylogenetic evolution, conserved motifs, tissue expression, and GO (Gene Ontology) analysis. The qRT-PCR was used to detect the expression levels of each member of the tomato TPR gene family (SlTPRs) under biological stress (Botrytis cinerea) and abiotic stress such as drought and abscisic acid (ABA). The results showed that members of the tomato TPR family responded to various abiotic stresses and Botrytis cinerea stress, and the SlTPR2 and SlTPR4 genes changed significantly under different stresses. Using VIGS (Virus-induced gene silencing) technology to silence these two genes, the silenced plants showed reduced disease resistance. It was also shown that TPR4 can interact with atpA which encodes a chloroplast ATP synthase CF1 α subunit. The above results provide a theoretical basis for further exploring the molecular mechanism of TPR-mediated resistance in disease defense, and also provide a foundation for tomato disease resistance breeding.     

Identification and Analysis of Stress-Associated Proteins (SAPs) Protein Family and Drought Tolerance of ZmSAP8 in Transgenic Arabidopsis

Stress-associated proteins (SAPs), a class of A20/AN1 zinc finger proteins, play vital roles in plant stress response. However, investigation of SAPs in maize has been very limited. Herein, to better trace the evolutionary history of SAPs in maize and plants, 415 SAPs were identified in 33 plant species and four species of other kingdoms. Moreover, gene duplication mode exploration showed whole genome duplication contributed largely to SAP gene expansion in angiosperms. Phylogeny reconstruction was performed with all identified SAPs by the maximum likelihood (ML) method and the SAPs were divided into five clades. SAPs within the same clades showed conserved domain composition. Focusing on maize, nine ZmSAPs were identified. Further promoter cis-elements and stress-induced expression pattern analysis of ZmSAPs indicated that ZmSAP8 was a promising candidate in response to drought stress, which was the only AN1-AN1-C2H2-C2H2 type SAP in maize and belonged to clade I. Additionally, ZmSAP8 was located in the nucleus and had no transactivation activity in yeast. Overexpressing ZmSAP8 enhanced the tolerance to drought stress in Arabidopsis thaliana, with higher seed germination and longer root length. Our results should benefit the further functional characterization of ZmSAPs.     

The Potential Role of REG Family Proteins in Inflammatory and Inflammation-Associated Diseases of the Gastrointestinal Tract

Regenerating gene (REG) family proteins serve as multifunctional secretory molecules with trophic, antiapoptotic, anti-inflammatory, antimicrobial and probably immuno-regulatory effects. Since their discovery, accumulating evidence has clarified the potential roles of the REG family in the occurrence, progression and development of a wide range of inflammatory and inflammation-associated diseases of the gastrointestinal (GI) tract. However, significant gaps still exist due to the undefined nature of certain receptors, regulatory signaling pathways and possible interactions among distinct Reg members. In this narrative review, we first describe the structural features, distribution pattern and purported regulatory mechanisms of REG family proteins. Furthermore, we summarize the established and proposed roles of REG proteins in the pathogenesis of various inflammation-associated pathologies of the GI tract and the body as a whole, focusing particularly on carcinogenesis in the ulcerative colitis—colitic cancer sequence and gastric cancer. Finally, the clinical relevance of REG products in the context of diagnosis, treatment and prognostication are also discussed in detail. The current evidence suggests a need to better understanding the versatile roles of Reg family proteins in the pathogenesis of inflammatory-associated diseases, and their broadened future usage as therapeutic targets and prognostic biomarkers is anticipated.     

热变性对蛋白质结构及泡沫行为的影响

以牛血清白蛋白(BSA)和鸡蛋白蛋白(albumin)为模型蛋白,研究了热变性对蛋白质起泡性能和分子结构的影响. 结果表明,热变性后的BSA起泡能力下降,泡沫稳定性有增强的趋势. 热变性使BSA分子表面巯基含量下降,分子之间发生缔合,表面疏水性下降. 而albumin在热变性后的起泡能力及泡沫稳定性都大大提高,热变性使albumin分子展开,表面巯基含量增加. 实验证明,蛋白质分子的表面疏水性是决定其起泡能力的重要因素之一,蛋白质分子之间的相互作用对泡沫的稳定性有很大的影响.     

关于教育部高等学校战略性新兴产业化学化工类相关本科新专业的分析

文章结合教高[2010]7号文和教高司函[2010]176号文,从新专业属性和新专业布点情况方面分析了2010年教育部同意设置的高等学校战略性新兴产业相关本科新专业,同时对广东省内各高校战略性新兴产业相关专业申报和批复情况进行了分析,旨在为今后各高校进行专业设置和专业申报提供参考,同时为企业今后引进相关人才提供指引.分...     

Analysis of Phosphoinositide-Binding Properties and Subcellular Localization of GFP-Fusion Proteins

Specific protein‐phosphoinositide (PI) interactions are known to play a key role in the targeting of proteins to specific cellular membranes. Investigation of these interactions would be greatly facilitated if GFP‐fusion proteins expressed in mammalian cells and used for their subcellular localization could also be employed for in vitro lipid binding. In this study, we found that lysates of cells overexpressing GFP‐fusion proteins could be used for in vitro protein‐PI binding assays. We applied this approach to examine the PI‐binding properties of Aplysia Sec7 protein (ApSec7) and its isoform ApSec7(VPKIS), in which a VPKIS sequence is inserted into the PH domain of ApSec7. EGFP‐ApSec7 but not EGFP‐ApSec7(VPKIS) did specifically bind to PI(3,4,5)P₃ in an in vitro lipid‐coated bead assay. Overexpression of EGFP‐ApSec7 but not EGFP‐ApSec7(VPKIS) did induce neurite outgrowth in Aplysia sensory neurons. Structure modeling analysis revealed that the inserted VPKIS caused misfolding around the PI(3,4,5)P₃‐binding pocket of ApSec7 and disturbed the binding of PI(3,4,5)P₃ to the pleckstrin homology (PH) domain. Our data indicate that plasma membrane localization of EGFP‐ApSec7 via the interaction between its PH domain and PI(3,4,5)P₃ might play a key role in neurite outgrowth in Aplysia.     

Structure and Function of the Zinc Binding Protein ZrgA from Vibrio cholerae

ATP binding cassette (ABC) transporters are the primary means by which bacteria acquire trace elements from the environment. They rely on solute binding proteins (SBPs) to bind the relevant substrate and deliver it to the integral membrane permease for ATP-powered import into the cytoplasm. SBPs of cluster A-I are known to facilitate the transport of essential metals zinc, manganese, and iron, and many have been characterized to date. A group of ABC transporter operons dubbed zinc-regulated genes (zrg) have recently been shown to transport zinc with putative SBPs (zrgA) bearing no homology to the classical cluster A-I family, and a recent crystal structure of a representative protein from Pseudomonas aeruginosa shows no structural similarity to classical SBPs. Thus, the ZrgA proteins appear to represent a newly discovered family of zinc SBPs widespread among Gram-negative bacteria, including human pathogens. Here, we have determined the crystal structure of ZrgA from Vibrio cholerae and characterized its zinc binding in vitro and function in vivo. We also assessed the role of a histidine-rich sequence that appears to be a hallmark of ZrgA proteins that is particularly long in V. cholerae ZrgA. The results show that the zrgA gene is critical to the function of the operon, consistent with a function as an SBP in this system. Further, the His-rich region is not essential to the function of ZrgA, but it does provide additional zinc binding sites in vitro. The structure and zinc binding data for ZrgA reveal interesting differences between it and its homologue from P. aeruginosa, illustrating diversity within this little-studied protein family.