Extraction of consensus protein patterns in regions containing non-proline cis peptide bonds and their functional assessment

<p>Abstract</p> <p>Background</p> <p>In peptides and proteins, only a small percentile of peptide bonds adopts the <it>cis </it>configuration. Especially in the case of amide peptide bonds, the amount of <it>cis </it>conformations is quite limited thus hampering systematic studies, until recently. However, lately the emerging population of databases with more 3D structures of proteins has produced a considerable number of sequences containing non-proline <it>cis </it>formations (<it>cis</it>-nonPro).</p> <p>Results</p> <p>In our work, we extract regular expression-type patterns that are descriptive of regions surrounding the <it>cis</it>-nonPro formations. For this purpose, three types of pattern discovery are performed: i) exact pattern discovery, ii) pattern discovery using a chemical equivalency set, and iii) pattern discovery using a structural equivalency set. Afterwards, using each pattern as predicate, we search the Eukaryotic Linear Motif (ELM) resource to identify potential functional implications of regions with <it>cis</it>-nonPro peptide bonds. The patterns extracted from each type of pattern discovery are further employed, in order to formulate a pattern-based classifier, which is used to discriminate between <it>cis</it>-nonPro and <it>trans</it>-nonPro formations.</p> <p>Conclusions</p> <p>In terms of functional implications, we observe a significant association of <it>cis</it>-nonPro peptide bonds towards ligand/binding functionalities. As for the pattern-based classification scheme, the highest results were obtained using the structural equivalency set, which yielded 70% accuracy, 77% sensitivity and 63% specificity.</p

Identification of endogenous small peptides involved in rice immunity through transcriptomics- and proteomics-based screening

Small signalling peptides, generated from larger protein precursors, are important components to orchestrate various plant processes such as development and immune responses. However, small signalling peptides involved in plant immunity remain largely unknown. Here, we developed a pipeline using transcriptomics- and proteomics-based screening to identify putative precursors of small signalling peptides: small secreted proteins (SSPs) in rice, induced by rice blast fungus Magnaporthe oryzae and its elicitor, chitin. We identified 236 SSPs including members of two known small signalling peptide families, namely rapid alkalinization factors and phytosulfokines, as well as many other protein families that are known to be involved in immunity, such as proteinase inhibitors and pathogenesis-related protein families. We also isolated 52 unannotated SSPs and among them, we found one gene which we named immune response peptide (IRP) that appeared to encode the precursor of a small signalling peptide regulating rice immunity. In rice suspension cells, the expression of IRP was induced by bacterial peptidoglycan and fungal chitin. Overexpression of IRP enhanced the expression of a defence gene, PAL1 and induced the activation of the MAPKs in rice suspension cells. Moreover, the IRP protein level increased in suspension cell medium after chitin treatment. Collectively, we established a simple and efficient pipeline to discover SSP candidates that probably play important roles in rice immunity and identified 52 unannotated SSPs that may be useful for further elucidation of rice immunity. Our method can be applied to identify SSPs that are involved not only in immunity but also in other plant functions

Part I: Crystallization of A Type IV Pilin from Pseudomonas Aeruginosa. Part II: Characterization of a Peptidyl-Prolyl-Cis,Trans-Isomerase Through X-Ray Crystallography

Within a host, pathogenic bacteria employ several mechanisms that enhance their survival and motility. These mechanisms include their ability to adhere, replicate (in order to avoid eradication), and secretion of virulent proteins. The study of proteins involved in bacterial pathogenesis provides us with a more thorough understanding of their mechanism and function, which can lead to the development of more effective therapeutics. In this study proteins secreted by gram-negative bacteria are explored, specifically those from Pseudomonas aeruginosa and Helicobacter pylori. P. aeruginosa is a common opportunistic pathogen associated with 10% of hospital infections, mainly owed to their ability to bind to biotic and abiotic surfaces. Type IV pili secreted by P. aeruginosa are associated with adhesion, motility and DNA transfer. Part I details the cloning to crystallization of KB7, a pilin secreted by P. aeruginosa. The second bacteria studied in Part II is H. pylori, associated with gastric ulcers and gastric inflammation compromising 50% of the global population, where severity of infection is highly dependent on the strain of H. pylori, and the individual infected. The secreted protein HP0175 from H. pylori binds to Toll-Like Receptor 4 and activates a cascade of mechanisms leading to apoptosis, as well as triggering the innate immune response. HP0175 is also classified as a peptidyl-prolyl cis,trans-isomerase involved in the isomerization of proline peptide bonds preceding the N-terminal. Here, apo-HP0175 was crystallized to 2.09 in space group P3221 with one monomer in the asymmetric unit; the dimer is generated through symmetry mates. A comparison to indole-2-carboxylic acid bound HP0175 shows N- and C- terminal helix extensions upon interaction of the catalytic residues in the binding pocket. Helix extension supports other parvulin findings that N- and C- terminal helices stabilize proteins undergoing catalysis by protein-protein interaction

Biophysical studies of TIMP-1

This study had two aspects. The first was the production and purification of TIMP-l. The second was a series of biophysical studies of TIMP-l and a TIMP-l derived peptide. A monoclonal antibody affinity column was developed and used to purifY large quantities of human TIMP-l for further experiments. Two E.coli expression systems were studied to determine whether they would be suitable for large scale production of recombinant protein. In the first system TIMP-I was to be secreted as a fusion protein which could be cleaved, leaving a free N-terminus. It was discovered that it was not possible to cleave off the fusion protein. In the second system, the protein was secreted, without additions to the periplasm. Although active protein, with the correct N-tenninus, was obtained, the yields were too low to be of use for large scale expression. Secondary structure analysis by CD and FTIR showed TIMP-l to be a mostly f3- sheet protein (approaching 50%) with around 20% a-helix. A temperature study using these techniques found that little change occurs until temperatures of over 60°C where the protein aggregates. The small changes appear to be a general loosening of the structure. In analyses of the surface of TIMP-l, additional carbohydrate was identified (other than the two N-linked chains) using Con-A probing of Western blots. TIMP-l purified from WI-38 foetal lung fibroblast cells can be separated into two pools by Concanavalin A-Sepharose chromatography. These two pools were found to have a different set of pIs and a different monosaccharide composition. The use of NMR paramagnetic probes identified a hydrophobic region exposed on the surface of TIMP-I. This region probably includes a tyrosine residue and either a tryptophan or phenylalanine. The presence of an exposed hydrophobic region was also shown in binding studies using the fluorescent probe ANS. These studies identified a single, low affinity binding site. An additional study with the N-terminal fragment of type-I collagenase found no binding sites on the enzyme, but a change in fluorescence occurred when TIMP-I was present. A peptide was designed based on the N-terminal sequence of TIMP-I. High homology, susceptibility to mutation and an interesting resemblance to the Bowman-Birk family of inhibitors suggested that this peptide might be inhibitory. It was found to have only a weak inhibitory activity against gelatinase. NMR studies of this peptide in water showed a large number of conformers as a result of stabilisation of the cis isomer of its proline residues. This preference for the cis form was retained for one proline in the solvent, TFE. Preliminary NMR studies were also carried out which concluded that TIMP-I should be suitable for further structural studies using isotopic labelling

A study of seed storage protein accumulation by ectopic expression in Arabidopsis

Understanding the mechanisms plants utilize for seed storage protein (SSP) synthesis, transport and deposition have the potential rewards of enabling high yields of modified or foreign proteins. Hayashi et al. (1999) indicated that the machinery devoted to the synthesis of protein storage vacuoles in cotyledon cells can be induced in vegetative tissue by the constitutive expression of a pumpkin 2S albumin phosphinothricin-acetyl-transferase gene fusion (pumpkin 2S-PAT) resulting in the biogenesis of precursor-accumulating (PAC) vesicles in Arabidopsis leaves. This discovery was the impetus behind the work described which sought to examine this phenomenon further by ectopically evoking SSP trafficking and vesicle biogenesis machinery in leaves. With the aim of elucidating the mechanisms necessary to evoke PAC vesicle biogenesis, a suite of constructs including the pumpkin 2S-PAT and analogous napin-PAT and napin-GFP variants were synthesized. Analysis of these transgenes in Arabidopsis revealed that the pumpkin 2S albumin has a capacity unique from napin peptides to result in fusion protein accumulation. Further, the truncated pumpkin 2S albumin peptide and the pumpkin 2S albumin C-terminus were found to direct deposition to vesicles; however, the C-terminus alone was not enough to direct deposition to vesicles unless combined with a significantly shortened napin peptide. An increased ER protein throughput was correlated to trafficking of the fusion protein by Golgi-independent mechanisms resulting in stable accumulation of the unprocessed protein whereas less ER throughput indicated passage through the Golgi-dependent pathway resulting in accumulation of a processed variant. At the level of gene expression, as examined by a microarray study, both inducible and constitutive ectopic expression of pumpkin 2S-PAT resulted in substantial perturbations of the endomembrane system affecting protein folding, flowering time and ER-associated biosynthetic functions which indicated that modulation of flowering time and photoperiodism are highly dependent on protein trafficking and vacuolar biogenesis mechanisms and that high ER protein throughput occurs at the expense of biosynthesis and cessation of ER functioning

Structural and functional studies of the terminal protein complex of the human complement system

The membrane attack complex (MAC: C5blC61C71C81C9n) is the terminal complex of the mammalian complement cascade. Its principal function is to form a self-assembling, potentially cytolytic pore spanning the membrane of a foreign cell. However, inappropriate complement activation and subsequent aberrant MAC action is responsible for tissue damage in several human pathologies.1 The C-terminal domains or modules that are exclusive to the C6 and C7 components of the MAC, form a putative molecular arm. These modules consist of two complement control protein modules (CCPs) followed by a pair of Factor I-like Modules (FIMs). The C-terminal molecular arms of C6 and C7 are important for linking the complement activation cascade with MAC assembly and thereby ensuring MAC assembly occurs only when needed. They interact with the C345C domain of C5 and C5b. Currently there are no high-resolution structural data for any of the MAC components but efforts are underway to solve the structures of the individual domains. For instance, the structure of the central MAC/perforin domain of C8 has been determined, so has the structure of C5 and the FIM domains of C7. However , the lack of structural information for the MAC is being balanced by an increased understanding of mechanism, and eventually towards the rational design of therapies designed to supress MAC formation.This report describes the preparation from bacterial and yeast cells of ¹³C,¹⁵N-labelled samples of the C7 CCP-pair and of a triple module consisting of the second CCP followed by the FIM-pair, as well as other constructs from the C6 and C7 C-terminal arms. The NMR samples were used to solve 3D solutions of structures, thus allowing for reconstruction of the four-module C-terminus of C7. Efforts were aimed at obtaining additional structural information for C7. This included SAXS analysis as well as the development of a novel approach using chemical cross-linking followed by tryptic digestion and mass spectrometry-based identification of cross-linked peptides