Improving binding affinity and stability of peptide ligands by substituting glycines with D-amino acids.

Improving the binding affinity and/or stability of peptide ligands often requires testing of large numbers of variants to identify beneficial mutations. Herein we propose a type of mutation that promises a high success rate. In a bicyclic peptide inhibitor of the cancer-related protease urokinase-type plasminogen activator (uPA), we observed a glycine residue that has a positive ϕ dihedral angle when bound to the target. We hypothesized that replacing it with a D-amino acid, which favors positive ϕ angles, could enhance the binding affinity and/or proteolytic resistance. Mutation of this specific glycine to D-serine in the bicyclic peptide indeed improved inhibitory activity (1.75-fold) and stability (fourfold). X-ray-structure analysis of the inhibitors in complex with uPA showed that the peptide backbone conformation was conserved. Analysis of known cyclic peptide ligands showed that glycine is one of the most frequent amino acids, and that glycines with positive ϕ angles are found in many protein-bound peptides. These results suggest that the glycine-to-D-amino acid mutagenesis strategy could be broadly applied.

Inhibition of mouse mammary tumor virus-induced T cell responses in vivo by antibodies to an open reading frame protein.

Minor lymphocyte stimulating (Mls) antigens specifically stimulate T cell responses that are restricted to particular T cell receptor (TCR) beta chain variable domains. The Mls phenotype is genetically controlled by an open reading frame (orf) located in the 3' long terminal repeat of mouse mammary tumor virus (MMTV); however, the mechanism of action of the orf gene product is unknown. Whereas predicted orf amino acid sequences show strong overall homology, the 20-30 COOH-terminal residues are strikingly polymorphic. This polymorphic region correlates with TCR V beta specificity. We have generated monoclonal antibodies to a synthetic peptide encompassing the 19 COOH-terminal amino acid residues of Mtv-7 orf, which encodes the Mls-1a determinant. We show here that these antibodies block Mls responses in vitro and can interfere specifically with thymic clonal deletion of Mls-1a reactive V beta 6+ T cells in neonatal mice. Furthermore, the antibodies can inhibit V beta 6+ T cell responses in vivo to an infectious MMTV that shares orf sequence homology and TCR specificity with Mtv-7. These results confirm the predicted extracellular localization of the orf COOH terminus and imply that the orf proteins of both endogenous and exogenous MMTV interact directly with TCR V beta.

Evaluation of two long synthetic merozoite surface protein 2 peptides as malaria vaccine candidates.

Merozoite surface protein 2 (MSP2) is a promising vaccine candidate against Plasmodium falciparum blood stages. A recombinant 3D7 form of MSP2 was a subunit of Combination B, a blood stage vaccine tested in the field in Papua New Guinea. A selective effect in favour of the allelic family not represented by the vaccine argued for a MSP2 vaccine consisting of both dimorphic variants. An alternative approach to recombinant manufacture of vaccines is the production of long synthetic peptides (LSP). LSP exceeding a length of well over 100 amino acids can now be routinely synthesized. Synthetic production of vaccine antigens cuts the often time-consuming steps of protein expression and purification short. This considerably reduces the time for a candidate to reach the phase of clinical trials. Here we present the evaluation of two long synthetic peptides representing both allelic families of MSP2 as potential vaccine candidates. The constructs were well recognized by human immune sera from different locations and different age groups. Furthermore, peptide-specific antibodies in human immune sera were associated with protection from clinical malaria. The synthetic fragments share major antigenic properties with native MSP2. Immunization of mice with these antigens yielded high titre antibody responses and monoclonal antibodies recognized parasite-derived MSP2. Our results justify taking these candidate poly-peptides into further vaccine development.

Positional scanning-synthetic peptide library-based analysis of self- and pathogen-derived peptide cross-reactivity with tumor-reactive Melan-A-specific CTL.

Synthetic combinatorial peptide libraries in positional scanning format (PS-SCL) have recently emerged as a useful tool for the analysis of T cell recognition. This includes identification of potentially cross-reactive sequences of self or pathogen origin that could be relevant for the understanding of TCR repertoire selection and maintenance, as well as of the cross-reactive potential of Ag-specific immune responses. In this study, we have analyzed the recognition of sequences retrieved by using a biometric analysis of the data generated by screening a PS-SCL with a tumor-reactive CTL clone specific for an immunodominant peptide from the melanocyte differentiation and tumor-associated Ag Melan-A. We found that 39% of the retrieved peptides were recognized by the CTL clone used for PS-SCL screening. The proportion of peptides recognized was higher among those with both high predicted affinity for the HLA-A2 molecule and high predicted stimulatory score. Interestingly, up to 94% of the retrieved peptides were cross-recognized by other Melan-A-specific CTL. Cross-recognition was at least partially focused, as some peptides were cross-recognized by the majority of CTL. Importantly, stimulation of PBMC from melanoma patients with the most frequently recognized peptides elicited the expansion of heterogeneous CD8(+) T cell populations, one fraction of which cross-recognized Melan-A. Together, these results underline the high predictive value of PS-SCL for the identification of sequences cross-recognized by Ag-specific T cells.

Alpha 3 domain mutants of peptide/MHC class I multimers allow the selective isolation of high avidity tumor-reactive CD8 T cells.

The goal of adoptive T cell therapy in cancer is to provide effective antitumor immunity by transfer of selected populations of tumor Ag-specific T cells. Transfer of T cells with high TCR avidity is critical for in vivo efficacy. In this study, we demonstrate that fluorescent peptide/MHC class I multimeric complexes incorporating mutations in the alpha3 domain (D227K/T228A) that abrogate binding to the CD8 coreceptor can be used to selectively isolate tumor Ag-specific T cells of high functional avidity from both in vitro expanded and ex vivo T cell populations. Sorting, cloning, and expansion of alpha3 domain mutant multimer-positive CD8 T cells enabled rapid selection of high avidity tumor-reactive T cell clones. Our results are relevant for ex vivo identification and isolation of T cells with potent antitumor activity for adoptive T cell therapy.

The Fanconi anemia protein FANCM can promote branch migration of Holliday junctions and replication forks.

Fanconi anemia (FA) is a genetically heterogeneous cancer-prone disorder associated with chromosomal instability and cellular hypersensitivity to DNA crosslinking agents. The FA pathway is suspected to play a crucial role in the cellular response to DNA replication stress. At a molecular level, however, the function of most of the FA proteins is unknown. FANCM displays DNA-dependent ATPase activity and promotes the dissociation of DNA triplexes, but the physiological significance of this activity remains elusive. Here we show that purified FANCM binds to Holliday junctions and replication forks with high specificity and promotes migration of their junction point in an ATPase-dependent manner. Furthermore, we provide evidence that FANCM can dissociate large recombination intermediates, via branch migration of Holliday junctions through 2.6 kb of DNA. Our data suggest a direct role for FANCM in DNA processing, consistent with the current view that FA proteins coordinate DNA repair at stalled replication forks.

The mitochondrial targeting function of randomly generated peptide sequences correlates with predicted helical amphiphilicity.

A pool of oligonucleotides encoding a start methionine and nine random amino acids was inserted at the 5'-end of the gene for the yeast cytochrome oxidase subunit IV lacking its own mitochondrial targeting sequence. Approximately one-quarter of the randomly generated sequences targeted subunit IV to its correct intramitochondrial location in vivo. Sequence analysis of 89 randomly generated sequences showed that their efficiencies as mitochondrial targeting signals correlated with the potential to fold into an amphiphilic alpha-helix. Functional targeting sequences were enriched in arginine and isoleucine residues but contained few aspartate, glutamate, and proline residues. Nonfunctional sequences predicted to have significant helical amphiphilicity often had at least one acidic or multiple helix-breaking residues that would be expected to interfere with targeting functioning. These results support the hypothesis that the signal for targeting a protein into the mitochondrial matrix is usually a positively charged amphiphilic helix.

Production of renewable polymers from crop plants

Plants produce a range of biopolymers for purposes such as maintenance of structural integrity, carbon storage, and defense against pathogens and desiccation. Several of these natural polymers are used by humans as food and materials, and increasingly as an energy carrier. In this review, we focus on plant biopolymers that are used as materials in bulk applications, such as plastics and elastomers, in the context of depleting resources and climate change, and consider technical and scientific bottlenecks in the production of novel or improved materials in transgenic or alternative crop plants. The biopolymers discussed are natural rubber and several polymers that are not naturally produced in plants, such as polyhydroxyalkanoates, fibrous proteins and poly-amino acids. In addition, monomers or precursors for the chemical synthesis of biopolymers, such as 4-hydroxybenzoate, itaconic acid, fructose and sorbitol, are discussed briefly

Malaria vaccines - The long synthetic peptide approach: Technical and conceptual advancements.

This review describes the advances in malaria antigen discovery and vaccine development using the long synthetic peptide platforms that have been made available during the past 5 years. The most recent technical developments regarding peptide synthesis with the optimized production of large synthetic fragments are discussed. Clinical trials of long synthetic peptides are also reviewed. These trials demonstrated that long synthetic peptides are safe and immunogenic when formulated with various adjuvants. In addition, long synthetic peptides can elicit an antibody response in humans and have demonstrated inhibitory activity against parasite growth in vitro. Finally, new approaches to exploit the abundance of genomic data and the flexibility and speed of peptide synthesis are proposed.

Oxidative folding of synthetic polypeptides S-protected as tert-butylthio derivatives.

A new method for oxidative folding of synthetic polypeptides assembled by stepwise solid phase synthesis is introduced. Folding is obtained in excellent yields by reacting S-tert-butylthiolated polypeptides with a 100-fold molar excess of cysteine at 37 degrees C in a slightly alkaline buffer containing chaotropic salts, and in the presence of air-oxygen. This novel protocol has been applied to the folding of S-tert-butylthiolated human thymus and activation-regulated chemokine (hu-TARC) derivatives as well as to larger segments of Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins. Folded P. falciparum polypeptides have been used as substrates of endoproteinase Glu-C (Glu-C) and endoproteinase Asp-N (Asp-N) in an attempt to identify their disulfide connectivities. Particular practical advantages of the present method are (i) easy purification and storage of the S-protected peptide derivatives, (ii) elimination of the risk of cysteine alkylation during the acidolytic cleavage deprotection and resin cleavage steps, (iii) possibility to precisely evaluate the extent of folding and disulfide bond formation by mass spectrometry, and (iv) facile recovery of the final folded product.

Synthetic RGD-containing alpha-helical coiled coil peptides promote integrin-dependent cell adhesion.

Integrin receptors are the main mediators of cell adhesion to the extracellular matrix. They bind to their ligands by interacting with short amino acid sequences, such as the RGD sequence. Soluble, small RGD-based peptides have been used to block integrin-binding to ligands, thereby interfering with cell adhesion, migration and survival, while substrate-immobilized RGD sequences have been used to enhance cell binding to artificial surfaces. This approach has several important medical applications, e.g. in suppression of tumor angiogenesis or stimulation of bone formation around implants. However, the relatively weak affinity of short RGD-containing peptides often results in incomplete integrin inhibition or ineffective ligation. In this work, we designed and synthesized several new multivalent RGD-containing molecules and tested their ability to inhibit or to promote integrin-dependent cell adhesion when used in solution or immobilized on substrates, respectively. These molecules consist of an oligomeric structure formed by alpha-helical coiled coil peptides fused at their amino-terminal ends with an RGD-containing fragment. When immobilized on a substrate, these peptides specifically promoted integrin alphaVbeta3-dependent cell adhesion, but when used in solution, they blocked alphaVbeta3-dependent cell adhesion to the natural substrates fibronectin and vitronectin. One of the peptides was nearly 10-fold more efficient than fibronectin or vitronectin in promoting cell adhesion, and almost 100-fold more efficient than a linear RGD tripeptide in blocking adhesion. These results indicate that alpha-helical coiled coil peptides carrying an amino-terminal RGD motif can be used as soluble antagonists or surface-immobilized agonists to efficiently inhibit or promote integrin alphaVbeta3-mediated cell adhesion, respectively.

Guayule and Russian dandelion as alternative sources of natural rubber.

Natural rubber, obtained almost exclusively from the Para rubber tree (Hevea brasiliensis), is a unique biopolymer of strategic importance that, in many of its most significant applications, cannot be replaced by synthetic rubber alternatives. Several pressing motives lead to the search for alternative sources of natural rubber. These include increased evidence of allergenic reactions to Hevea rubber, the danger that the fungal pathogen Microcyclus ulei, causative agent of South American Leaf Blight (SALB), might spread to Southeast Asia, which would severely disrupt rubber production, potential shortages of supply due to increasing demand and changes in land use, and a general trend towards the replacement of petroleum-derived chemicals with renewables. Two plant species have received considerable attention as potential alternative sources of natural rubber: the Mexican shrub Guayule (Parthenium argentatum Gray) and the Russian dandelion (Taraxacum koksaghyz). This review will summarize the current production methods and applications of natural rubber (dry rubber and latex), the threats to the production of natural rubber from the rubber tree, and describe the current knowledge of the production of natural rubber from guayule and Russian dandelion.

Vaccine potentials of an intrinsically unstructured fragment derived from the blood stage-associated Plasmodium falciparum protein PFF0165c.

We have identified new malaria vaccine candidates through the combination of bioinformatics prediction of stable protein domains in the Plasmodium falciparum genome, chemical synthesis of polypeptides, in vitro biological functional assays, and association of an antigen-specific antibody response with protection against clinical malaria. Within the predicted open reading frame of P. falciparum hypothetical protein PFF0165c, several segments with low hydrophobic amino acid content, which are likely to be intrinsically unstructured, were identified. The synthetic peptide corresponding to one such segment (P27A) was well recognized by sera and peripheral blood mononuclear cells of adults living in different regions where malaria is endemic. High antibody titers were induced in different strains of mice and in rabbits immunized with the polypeptide formulated with different adjuvants. These antibodies recognized native epitopes in P. falciparum-infected erythrocytes, formed distinct bands in Western blots, and were inhibitory in an in vitro antibody-dependent cellular inhibition parasite-growth assay. The immunological properties of P27A, together with its low polymorphism and association with clinical protection from malaria in humans, warrant its further development as a malaria vaccine candidate.

Inhibition of lymphocyte mediated cytotoxicity by perforin antisense oligonucleotides.

The granule/perforin exocytosis model of CTL mediated cytolysis proposes that CTL, upon recognition of the specific targets, release the cytolytic, pore-forming protein perforin into the intercellular space which then mediates the cytotoxic effect. However, direct evidence for the involvement of perforin is still lacking, and indeed, recent results even seem incompatible with the model. To determine directly the role of perforin in CTL cytotoxicity, perforin antisense oligonucleotides were exogenously added during the stimulation of mouse spleen derived T cells and human peripheral blood lymphocytes (PBL), respectively. Perforin protein expression in lymphocytes was reduced by up to 65%, and cytotoxicity of stimulated T cells by as much as 69% (5.7-fold). These results provide the first experimental evidence for a crucial role of perforin in lymphocyte mediated cytotoxicity.

Pseudomonas fluorescens CHA0 produces enantio-pyochelin, the optical antipode of the Pseudomonas aeruginosa siderophore pyochelin.

The siderophore pyochelin is made by a thiotemplate mechanism from salicylate and two molecules of cysteine. In Pseudomonas aeruginosa, the first cysteine residue is converted to its D-isoform during thiazoline ring formation whereas the second cysteine remains in its L-configuration, thus determining the stereochemistry of the two interconvertible pyochelin diastereoisomers as 4'R, 2''R, 4''R (pyochelin I) and 4'R, 2''S, 4''R (pyochelin II). Pseudomonas fluorescens CHA0 was found to make a different stereoisomeric mixture, which promoted growth under iron limitation in strain CHA0 and induced the expression of its biosynthetic genes, but was not recognized as a siderophore and signaling molecule by P. aeruginosa. Reciprocally, pyochelin promoted growth and induced pyochelin gene expression in P. aeruginosa, but was not functional in P. fluorescens. The structure of the CHA0 siderophore was determined by mass spectrometry, thin-layer chromatography, NMR, polarimetry, and chiral HPLC as enantio-pyochelin, the optical antipode of the P. aeruginosa siderophore pyochelin. Enantio-pyochelin was chemically synthesized and confirmed to be active in CHA0. Its potential biosynthetic pathway in CHA0 is discussed.