Peptide phage display as a tool for drug discovery: targeting membrane receptors

Ligands selected from phage-displayed random peptide libraries tend to be directed to biologically relevant sites on the surface of the target protein. Consequently, peptides derived from library screenings often modulate the target protein's activity in vitro and in vivo and can be used as lead compounds in drug design and as alternatives to antibodies for target validation in both genomics and drug discovery. This review discusses the use of phage display to identify membrane receptor modulators with agonistic or antagonistic activities. Because isolating or producing recombinant membrane proteins for use as target molecules in library screening is often impossible, innovative selection strategies such as panning against whole cells or tissues, recombinant receptor ectodomains, or neutralizing antibodies to endogenous binding partners were devised. Prominent examples from a two-decade history of peptide phage display will be presented, focusing on the design of affinity selection experiments, methods for improving the initial hits, and applications of the identified peptides.     

Synchrotron radiation circular dichroism spectroscopy of proteins and applications in structural and functional genomics

The technique of Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy and its advantages over conventional circular dichroism spectroscopy are described in this tutorial review, as well as recent applications of the technique in structural and functional genomics.Circular dichroism (CD) spectroscopy is a well-established method in biological chemistry and structural biology, but its utility can be limited by the low flux of the light source in the far ultraviolet and vacuum ultraviolet wavelength regions in conventional CD instruments. The development of synchrotron radiation circular dichroism (SRCD), using the intense light of a synchrotron beam, has greatly expanded the utility of the method, especially as a tool for both structural and functional genomics. These applications take advantage of the enhanced features of SRCD relative to conventional CD: the ability to measure lower wavelength data containing more electronic transitions and hence more structural information, the higher signal-to-noise hence requiring smaller samples, the higher intensity enabling measurements in absorbing buffers and in the presence of lipids and detergents, and the ability to do faster measurements enabling high throughput and time-resolved spectroscopy.This article discusses recent developments in SRCD instrumentation, software, sample preparation and methods of analyses, with particular emphasis on their applications to the study of proteins. These advances have led to new applications in structural genomics (SG), including the potential for fold recognition as a means of target selection and the examination of membrane proteins, a class of proteins usually excluded from SG programmes. Other SG uses include detection of macromolecular interactions as a screen for complex formation, and examination of glycoproteins and sugar components. In functional genomics (FG) new applications include screening for ligand binding as a means of identifying function, and examination of structural differences in mutant proteins as a means of gaining insight into function.     

High-density synthetic peptide microarrays: emerging tools for functional genomics and proteomics

New approaches for manufacturing and application of peptide arrays on planar surfaces are emerging, thereby opening advanced opportunities to probe the expression and function of the proteome. In complementing DNA and protein array analyses, peptide fragment screening directly addresses functional protein interaction sites, leading to a detailed insight into the discovered molecular recognition events, placing them in the context of the whole genome, and even allowing rapid determination of the chemical nature of these interactions. This information can then be transferred into powerful small peptide tools that interfere with these interactions in vivo and help to link targets with phenotypes. With the spreading of new peptide array tools, peptide screening will extend its impact on modern genome-driven molecular biology. This will advance the systematic discovery and validation of new pharmaceutical targets as well as the development of potent molecular diagnostics for medical and ecological monitoring.     

Peptide nucleic acids (PNAs) with a functional backbone

The synthesis of 10 new T-PNA monomers derived from L-amino acids is presented. The monomers were incorporated into decameric PNA oligomers, and the hybridisation with RNA, DNA and PNA complements studied by thermal stability measurements.     

A new plasmid display technology for the in vitro selection of functional phenotype-genotype linked proteins

BACKGROUND: Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function. Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor kappa B (NF-kappa B) p50 to establish a phenotype-genotype link between the displayed protein and the encoding gene. RESULTS: A range of model fusion proteins to either the amino- or carboxy-terminus of NF-kappa B p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-kappa B p50. Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50-DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein-plasmid complexes with enrichment factors of up to 6000-fold per round. The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:10(8) and to enrich to near homogeneity a single functional protein from a phenotype-genotype linked Escherichia coli genomic library using in vitro functional selections. CONCLUSIONS: A new display technology is described which addresses the challenge of functional protein display. The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.     

Display cloning: functional identification of natural product receptors using cDNA-phage display.

BACKGROUND: The identification of cellular targets has traditionally been the starting point for natural product mode of action studies and has led to the understanding of many biological processes. Conventional experimental approaches have depended on cell-based screening and/or affinity chromatography. Although both of these techniques aid in the discovery of protein cellular targets, a method that couples protein identification with gene isolation would be extremely valuable. RESULTS: A procedure for the direct cloning of cellular proteins, based on their affinity for natural products, using cDNA phage display has been developed. The technique is referred to as display cloning because it involves the cloning of proteins displayed on the surface of a bacteriophage particle. The approach has been established by isolating a full-length gene clone of FKBP12 (FK506-binding protein) from a human brain cDNA library using a biotinylated FK506 probe molecule. During the affinity selection, the FKBP12 gene emerged as the dominant library member and was the only sequence identified after the second round of selection. CONCLUSIONS: The development of display cloning greatly facilitates the investigation of ligand-receptor interaction biology and natural product mode of action studies. This procedure utilizes heterologous protein display on infectious phage, which allows the amplification and repeated selection of putative sequences, leading to unambiguous target identification. In addition, the direct connection of a functional protein to its gene sequence eliminates the subsequent cloning step required with tissue homogenate or cell lysate affinity methods, allowing direct isolation of an expressible gene sequence.     

多肽片段连接方法及肽硫酯和肽醛的合成*

本文综述了多肽和蛋白质合成中的片段连接方法,这是近年来多肽和蛋白质合成领域中方法学上的重要进展.该方法使用非保护的多肽片段,无需酶或化学活化试剂,在缓冲溶液中能够高产率地获得多肽和蛋白质.还介绍了与多肽片段连接有关的肽硫酯和肽醛的合成方法.     

Node-based differential network analysis in genomics

Gene dependency networks often undergo changes in response to different conditions. Understanding how these networks change across two conditions is an important task in genomics research. Most previous differential network analysis approaches assume that the difference between two condition-specific networks is driven by individual edges. Thus, they may fail in detecting key players which might represent important genes whose mutations drive the change of network. In this work, we develop a node-based differential network analysis (N-DNA) model to directly estimate the differential network that is driven by certain hub nodes. We model each condition-specific gene network as a precision matrix and the differential network as the difference between two precision matrices. Then we formulate a convex optimization problem to infer the differential network by combing a D-trace loss function and a row-column overlap norm penalty function. Simulation studies demonstrate that N-DNA provides more accurate estimate of the differential network than previous competing approaches. We apply N-DNA to ovarian cancer and breast cancer gene expression data. The model rediscovers known cancer-related genes and contains interesting predictions.     

Peptide

Ohne Zusammenfassung     

Fungal glycoside hydrolases for saccharification of lignocellulose: outlook for new discoveries fueled by genomics and functional studies

Genome sequencing of a variety of fungi is a major initiative currently supported by the Department of Energy’s Joint Genome Institute. Encoded within the genomes of many fungi are upwards of 200+ enzymes called glycoside hydrolases (GHs). GHs are known for their ability to hydrolyze the polysaccharide components of lignocellulosic biomass. Production of ethanol and “next generation” biofuels from lignocellulosic biomass represents a sustainable route to biofuels production. However, this process has to become more economical before large scale operations are put into place. Identifying and characterizing GHs with improved properties for biomass degradation is a key factor for the development of cost effective processes to convert biomass to fuels and chemicals. With the recent explosion in the number of GH encoding genes discovered by fungal genome sequencing projects, it has become apparent that improvements in GH gene annotation processes have to be developed. This will enable more informed and efficient decision making with regard to selection and utilization of these important enzymes in bioprocess that produce fuels and chemicals from lignocellulosic feedstocks.     

Design, synthesis and functional analysis of dansylated polytheonamide mimic: an artificial Peptide ion channel

We report herein the design, total synthesis, and functional analysis of a novel artificial ion channel molecule, designated as dansylated polytheonamide mimic (3). The channel 3 was designed based on an exceptionally potent cytotoxin, polytheonamide B (1). Our strategy for the development of synthetic ion channels, which could be easily derivatized for various functions, involved two key features. First, the structure of 1 was simplified by replacing many of nonproteinogenic amino acid residues which required multistep synthesis by commercially available amino acids while retaining those residues necessary for folding. It significantly reduced the number of synthetic steps and facilitated a practical chemical construction of 3. Second, the introduction of propargyl glycine at residue 44 enabled facile installation of dansyl group as a reporter of the membrane localization of 3. Application of a newly designed protective group strategy provided efficient construction of the 37 amino acid sequence of residues 12-48 through one automatic solid-phase peptide synthesis. After peptide cleavage from the resin, 3 was synthesized via dansyl group introduction and one fragment-coupling reaction with residues 1-11, followed by the global deprotection. The simplified mimic 3 exhibited potent cytotoxicity toward p388 mouse leukemia cells (IC(50) = 12 nM), effectively induced ion transport across the lipid bilayers of liposomes, and displayed H(+) and Na(+) ion channel activities. Because of its simplified yet functional scaffold structure with a potential for diversification, our rationally designed ion channel molecule should be useful as a novel platform for developing various cytotoxic channel molecules with additional desired functions.     

From proteomics to genomics

Presently, science is moving from genomics to proteomics in order to get insight into the functional network of gene expression. Actually however, proteomics is much older than genomics and dates back to the introduction of the two-dimensional gel electrophoresis technique (2-DE) independently by Klose and O'Farrell. Based on this approach almost all cellular proteins can be separated. New developments in mass spectrometry allowed identification of single spots in the 2-DE protein pattern, including the underlying genes. Joachim Klose has focused his pioneering 2-DE studies on mouse models with special emphasis on quantitative protein variants. According to him, proteins are living molecules exhibiting a characteristic protein phenotype.     

化妆品中的肽化学*

随着科技进步和产业提升,人们不断从本源上追求美、从长效上保持美、从机制上激发美。众多肽类化合物,已被证实在人体内具有独特的生物学促进或抑制作用,也因此被广泛应用于美妆产品。以化妆品中生物活性肽的不同功效为主线,对其化学结构进行分析并阐述其中的生理作用机制。     

A novel class of small functional peptides that bind and inhibit human alpha-thrombin isolated by mRNA display

Here we report the in vitro selection of novel small peptide motifs that bind to human alpha-thrombin. We have applied mRNA display to select for thrombin binding peptides from an unbiased library of 1.2 x 10(11) different 35-mer peptides, each containing a random sequence of 15 amino acids. Two clones showed binding affinities ranging from 166 to 520 nM. A conserved motif of four amino acids, DPGR, was identified. Clot formation of human plasma is inhibited by the selected clones, and they downregulate the thrombin-mediated activation of protein C. The identified peptide motifs do not share primary sequence similarities to any of the known natural thrombin binding motifs. As new inhibitors for human thrombin open interesting possibilities in thrombosis research, our newly identified peptides may provide further insights into this field of investigation and may be possible candidates for the development of new anti-thrombotic agents.     

Peptide fibrillization

The fibrillization of peptides is relevant to many diseases based on the deposition of amyloids. The formation of fibrils is being intensively studied, especially in terms of nanotechnology applications, where fibrillar peptide hydrogels are used for cell scaffolds, as supports for functional and responsive biomaterials, biosensors, and nanowires. This Review is concerned with fundamental aspects of the self-assembly of peptides into fibrils, and discusses both natural amyloid-forming peptides and synthetic materials, including peptide fragments, copolymers, and amphiphiles.     

多肽纳米管

环状多肽是构成多肽纳米管的主要子结构,它的结构形式和骨架分子构象直接影响多肽纳米管的特性。作为有特殊电子和光学性质的多肽纳米管,它在化学、生物、材料和医学等方面有潜在的应用。本文就自组装多肽纳米管的结构和应用作了介绍。     

Peptide hydrolysis by the binuclear zinc enzyme aminopeptidase from Aeromonas proteolytica: a density functional theory study

Aminopeptidase from Aeromonas proteolytica (AAP) is a binuclear zinc enzyme that catalyzes the cleavage of the N-terminal amino acid residue of peptides and proteins. In this study, we used density functional methods to investigate the reaction mechanism of this enzyme. A model of the active site was constructed on the basis of the X-ray crystal structure of the native enzyme, and a model dipeptide was used as a substrate. It was concluded that the hydroxide is capable of performing a nucleophilic attack at the peptide carbonyl from its bridging position without the need to first become terminal. The two zinc ions are shown to have quite different roles. Zn2 binds the amino group of the substrate, thereby orienting it toward the nucleophile, while Zn1 stabilizes the alkoxide ion of the tetrahedral intermediate, thereby lowering the barrier for the nucleophilic attack. The rate-limiting step is suggested to be the protonation of the nitrogen of the former peptide bond, which eventually leads to the cleavage of the C-N bond.