Peptides Of Different Lengths Research Articles

Exploring the Unexpected Gelation of Tripeptides in a Binary Mixture of Water and Ethanol

Hydrogels formed by peptides of different length are a special class of materials that have been heavily researched in the past due to their inherent biodegradability. In this context, some low molecular weight di- or tripeptides with aromatic residues and terminal groups have been shown to form gels after self-assembling into large supramolecular structures above critical concentrations in the centimolar range. Contrary to expectations, our group recently discovered that cationic glycylanalylglycine (GAG), a tripeptide of rather limited hydrophobicity, forms a gel in 55 mol% ethanol/45 mol% water at room temperature if the concentration exceeds 200 mM.

Immunofocusing using conformationally constrained V3 peptide immunogens improves HIV-1 neutralization

V3-directed antibodies are present in practically all HIV-1 infected patients and in individuals vaccinated with gp120. The levels of maternal V3-directed antibodies were recently shown to correlate with reduced mother to child transmission, and V3 IgGs were found to be a negative correlate of risk in the RV-144 human trial. mAb directed to the tip of the V3 are capable of broad neutralization of Tier-1 and some Tier-2 viruses. Here we report an immunofocusing approach using conformationally constrained V3 peptides of different lengths. Immunofocusing with short constrained V3 peptides following immunizations with long constrained V3 peptides resulted in sera with improved neutralization of Tier-1B viruses in comparison with immunizations with the long constrained peptide alone. Immunizations only with the short constrained peptide were ineffective. Our results demonstrate that immunofocusing with constrained V3 peptides of different lengths could improve the induction of HIV-1 neutralizing antibodies.

The Impact of the ‘Austrian’ Mutation of the Amyloid Precursor Protein Transmembrane Helix is Communicated to the Hinge Region

AbstractThe transmembrane helix of the amyloid precursor protein is subject to proteolytic cleavages by γ‐secretase at different sites resulting in Aβ peptides of different length and toxicity. A number of point mutations within this transmembrane helix alter the cleavage pattern thus enhancing production of toxic Aβ peptide species that are at the root of familial Alzheimer's disease. Here, we investigated how one of the most devastating mutations, the ‘Austrian’ mutation T43I, affects this transmembrane helix. Site‐resolved deuterium/hydrogen amide exchange experiments reveal that the mutation destabilizes amide hydrogen bonds in the hinge which connects dimerization and cleavage regions. Weaker intrahelical hydrogen bonds at the hinge may enhance helix bending and thereby affect recognition of the transmembrane substrate by the enzyme and/or presentation of its cleavage sites to the catalytic cleft.

Amyloidogenic amyloid-β-peptide variants induce microbial agglutination and exert antimicrobial activity.

Amyloid-β (Aβ) peptides are the main components of the plaques found in the brains of patients with Alzheimer’s disease. However, Aβ peptides are also detectable in secretory compartments and peripheral blood contains a complex mixture of more than 40 different modified and/or N- and C-terminally truncated Aβ peptides. Recently, anti-infective properties of Aβ peptides have been reported. Here, we investigated the interaction of Aβ peptides of different lengths with various bacterial strains and the yeast Candida albicans. The amyloidogenic peptides Aβ1-42, Aβ2-42, and Aβ3p-42 but not the non-amyloidogenic peptides Aβ1-40 and Aβ2-40 bound to microbial surfaces. As observed by immunocytochemistry, scanning electron microscopy and Gram staining, treatment of several bacterial strains and Candida albicans with Aβ peptide variants ending at position 42 (Aβx-42) caused the formation of large agglutinates. These aggregates were not detected after incubation with Aβx-40. Furthermore, Aβx-42 exerted an antimicrobial activity on all tested pathogens, killing up to 80% of microorganisms within 6 h. Aβ1-40 only had a moderate antimicrobial activity against C. albicans. Agglutination of Aβ1-42 was accelerated in the presence of microorganisms. These data demonstrate that the amyloidogenic Aβx-42 variants have antimicrobial activity and may therefore act as antimicrobial peptides in the immune system.

SNebula, a network-based algorithm to predict binding between human leukocyte antigens and peptides

Understanding the binding between human leukocyte antigens (HLAs) and peptides is important to understand the functioning of the immune system. Since it is time-consuming and costly to measure the binding between large numbers of HLAs and peptides, computational methods including machine learning models and network approaches have been developed to predict HLA-peptide binding. However, there are several limitations for the existing methods. We developed a network-based algorithm called sNebula to address these limitations. We curated qualitative Class I HLA-peptide binding data and demonstrated the prediction performance of sNebula on this dataset using leave-one-out cross-validation and five-fold cross-validations. This algorithm can predict not only peptides of different lengths and different types of HLAs, but also the peptides or HLAs that have no existing binding data. We believe sNebula is an effective method to predict HLA-peptide binding and thus improve our understanding of the immune system.

Peripheral Immune Signatures in Alzheimer Disease.

According to the current paradigm, the main cause of AD is the accumulation of neurotoxic amyloid beta (Aβ) peptide aggregates resulting from the cleavage of the amyloid precursor protein into peptides of different length, with the 42 amino acid long Aβ42 being the most toxic form. Aβ can aggregate and form plaques in the brain. It further promotes the hyperphosphorylation of the tau protein which forms characteristic neurofibrillary tangles and thereby loses its important role in axonal transport and contributes to neurodegeneration. Therefore, treatments have targeted Aβ, but clinical trials of immunotherapies caused severe side effects and showed that Aβ clearance alone did not result in any cognitive improvement. This leads to the question: what else promotes AD pathology? Here, we review data on systemic inflammation and the possible roles that the immune system might play in AD. Microglia and astrocytes are activated and secrete inflammatory cytokines and chemokines. Via a disturbed blood-brain barrier, peripheral immune cells are activated and recruited towards inflamed brain lesions and amyloid plaques, but due to the chronic nature of the amyloid burden and their reduced function, these cells are not able to control inflammation and the associated detrimental immune responses. In addition, age-related inflammation and chronic infection with herpes viruses might contribute to the systemic inflammation and exacerbate attempts to restore the balance of inflammation.

CPAD, Curated Protein Aggregation Database: A Repository of Manually Curated Experimental Data on Protein and Peptide Aggregation.

Accurate distinction between peptide sequences that can form amyloid-fibrils or amorphous β-aggregates, identification of potential aggregation prone regions in proteins, and prediction of change in aggregation rate of a protein upon mutation(s) are critical to research on protein misfolding diseases, such as Alzheimer’s and Parkinson’s, as well as biotechnological production of protein based therapeutics. We have developed a Curated Protein Aggregation Database (CPAD), which has collected results from experimental studies performed by scientific community aimed at understanding protein/peptide aggregation. CPAD contains more than 2300 experimentally observed aggregation rates upon mutations in known amyloidogenic proteins. Each entry includes numerical values for the following parameters: change in rate of aggregation as measured by fluorescence intensity or turbidity, name and source of the protein, Uniprot and Protein Data Bank codes, single point as well as multiple mutations, and literature citation. The data in CPAD has been supplemented with five different types of additional information: (i) Amyloid fibril forming hexa-peptides, (ii) Amorphous β-aggregating hexa-peptides, (iii) Amyloid fibril forming peptides of different lengths, (iv) Amyloid fibril forming hexa-peptides whose crystal structures are available in the Protein Data Bank (PDB) and (v) Experimentally validated aggregation prone regions found in amyloidogenic proteins. Furthermore, CPAD is linked to other related databases and resources, such as Uniprot, Protein Data Bank, PUBMED, GAP, TANGO, WALTZ etc. We have set up a web interface with different search and display options so that users have the ability to get the data in multiple ways. CPAD is freely available at http://www.iitm.ac.in/bioinfo/CPAD/. The potential applications of CPAD have also been discussed.

Fluorescence and phosphorescence of tryptophan in peptides of different length and sequence

To interpret accurately protein fluorescence and phosphorescence, it is essential to achieve a better understanding of the luminescence properties of tryptophan (Trp, or W) in peptides. In published literature data on luminescence of peptides of varied length are scarce. This article describes studies of fluorescence and phosphorescence properties of the eight Trp-containing synthetic peptides: WAK, AWK, SWA, KYLWE, AVSWK, WVSWAK, WAKLAWE, and AVSWAKLARE. The aim was to investigate which factors influence the fluorescence yield and phosphorescence-spectra and lifetimes. Absorption spectra, room temperature fluorescence emission and corresponding excitation spectra and time-resolved phosphorescence spectra (77K) have been recorded; the dependence of the fluorescence quantum yield on the specific peptide and its variation with the wavelength of excitation has been studied. The changes in fluorescence yield and shape of phosphorescence spectra are explained in terms of internal electron and proton transfer. The structured phosphorescence spectrum originates from proton transfer occurring upon excitation of Trp, while electron transfer gives rise to a non-structured luminescence spectrum. There is also electron transfer from higher vibronic S1 states. In the peptides there is higher probability of electron transfer than in Trp alone. The obtained data are interpreted in light of the peptides' sequence, length and conformation.

Bending Moduli of Ternary Mixture Models of the Cell Plasma Membrane

Cells must be able to maintain and change membrane shape. This membrane curvature can be influenced by any lateral phase heterogeneity. Ternary mixtures of high-melting lipid, low-melting lipid, and cholesterol exhibit a region of liquid-ordered (Lo)+ liquid-disordered (Ld) phase coexistence analogous to raft + non-raft behavior in cells. Although curvature can induce separation and sorting in vitro, a number of highly-curved membranes in vivo have raft-like composition, which is puzzling since the raft-like compositions have greater rigidity in lipid model mixtures. We find that including transmembrane helical WALP peptides as a component of the model mixtures greatly changes their bending behavior. Measurements were made by shape analysis of giant unilamellar vesicles with both fluorescence and phase contrast microscopy. Vesicles of a single phase were made by the gentle hydration method to obtain a more narrow distribution of vesicle tensions than is obtained by electroformation. We find that a WALP peptide component rigidifies the liquid-disordered phase whereas the liquid-ordered phase is unaffected. This rigidifying effect might be related to the monolayers sliding relative to each other. Further, we have investigated the effects of peptides of different lengths and results suggest a role for hydrophobic mismatch of the peptide component relative to the bilayer thickness.

Linker engineering for fusion protein construction: Improvement and characterization of a GLP-1 fusion protein

Protein engineering has been successfully applied in protein drug discovery. Using this technology, we previously have constructed a fusion protein by linking the globular domain of adiponectin to the C-terminus of a glucagon-like peptide-1 (GLP-1) analog. Herein, to further improve its bioactivity, we reconstructed this fusion protein by introducing linker peptides of different length and flexibility. The reconstructed fusion proteins were overexpressed in Escherichia coli and purified using nickel affinity chromatography. Their agonist activity towards receptors of GLP-1 and adiponectin were assessed in vitro by using luciferase assay and AMP-activated protein kinase (AMPK) immunoblotting, respectively. The effects of the selected fusion protein on glucose and lipid metabolism were evaluated in mice. The fusion protein reconstructed using a linker peptide of AMGPSSGAPGGGGS showed high potency in activating GLP-1 receptor and triggering AMPK phosphorylation via activating the adiponectin receptor. Remarkably, the optimized fusion protein was highly effective in lowering blood glucose and lipids in mice. Collectively, these findings demonstrate that the bioactivity of this GLP-1 fusion protein can be significantly promoted by linker engineering, and indicate that the optimized GLP-1 fusion protein is a promising lead structure for anti-diabetic drug discovery.

Investigation of the structure of anti-human seminal plasma protein single-chain antibody and its association with linker peptide length.

To enhance the activity of seminoprotein single-chain variable fragment (γ-Sm-ScFv) antibodies, modulation of the length of the linker peptide, which connects the variable region of the heavy chain (VH) and the light chain (VL) of single-chain antibodies, was performed in the present study. Homologous modeling of single VH and VL were performed, respectively. Subsequently, modeling of the whole ScFv sequence, which was previously modified with added linkers of different lengths was also performed, and the (Gly4Ser)n peptide chain structure was used as the linker. The similarities between VH and VL prior to and following the addition of the linker were compared by applying the algorithm of protein similarity, based on spherical coordinates layering. In addition, changes in the fore and aft distance, and diffusion radius were calculated using a MATLAB tool, based on which changes in structural stability were analyzed. Finally, the single-chain antibody was assessed in a nude mouse model. When n=3 or n=6, the similarity between the original distance and VH and VL were the highest, and the fore and aft distance and diffusion radius were relatively close. In addition, the nude mouse model indicated that, when n=3 or n=6, the inhibitory rate of the single-chain antibody against tumor cells was significantly higher, compared with the other linker peptides of different lengths. The effect of structural changes of the linker peptides in the single-chain antibodies on the whole antibody molecule was examined at different levels using a combination of mathematical modeling, bioinformatics methods and biological experiments. The findings of the present study may provide a foundation for further investigation into the preparation of single-chain antibodies.

Identification and relative quantification of phosphopeptides by a combination of multi‐protease digestion and isobaric labeling

The identification and the determination of the extent of protein phosphorylation are major prerequisites for the comparative analysis of this important posttranslational modification of proteins in different biological situations. High sequence coverages and the availability of straightforward quantification methods are necessary to achieve these goals. Phosphoproteins and non-phosphorylated analogues separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were digested using four different proteases (trypsin, chymotrypsin, elastase and GluC) and the digests were isobarically labeled using eight-plex iTRAQ. The combined labeled digests were subsequently enriched using titanium dioxide and both the phosphorylated and non-phosphorylated fractions were analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). The influence of different data analysis algorithms (Percolator or False Discovery Rate) on the outcome of analysis was investigated. Almost complete sequence coverage could be achieved upon application of a multi-protease approach. The formation of peptides of different lengths and physicochemical properties allowed the identification and the mapping of all phosphorylation sites in the investigated model proteins. The introduction of isobaric labels allowed quantification of different peptides of the same phosphorylation site with more than one peptide, leading to significantly improved statistical confidence. A workflow for the straightforward comparative analysis of protein phosphorylation in samples of low complexity, e.g. isolated proteins, was developed. The workflow is transferable to other posttranslational modifications.

Identification of Prolyl Endopeptidase (PEP) Inhibitory Peptides from Lactobacillus helveticus Digestion of Four Recombinant Bovine Caseins

Eurygaster integriceps Puton (Sunn pest) is the most damaging insect pest of wheat in areas of North Africa, Eastern Europe and Western and Central Asia. Sunn pest injects an enzyme characterized as a prolyl endylprotease (spPEP) into wheat grains that digests the gluten protein (Darkoh et al., 2010). Previous studies have shown that casein peptides generated by Lactobacillus species have inhibitory effects on mammalian and bacterial prolyl endoproteases (PEP) which makes casein a good natural candidate against spPEP. This study used four purified recombinant bovine casein genes, α1, α2, β and κ to identify which Lactobacillus generated peptides will inhibit the spPEP. Firstly, these four recombinant casein genes were expressed and purified. Then the peptides were generated by incubating each purified casein with Lactobacillus helveticus. These peptides were separated using High Performance Liquid Chromatography (HPLC). The inhibitory peptides were identified by incubating them with recombinant PEP enzyme in the presence the GPpNA substrate. Mass and sequence of the inhibitory peptides were determined using MALDI Mass Spectrophotometry.. Future studies will include the cloning and expression of the identified peptides of different lengths to determine specificity unique to spPEP. Funding: Office of Research and Sponsored Projects, SFASU and Ed and Gwen Cole, Nacogdoches, TX.

T cells recognizing a 11mer influenza peptide complexed to H-2D(b) show promiscuity for peptide length.

T-cell repertoire is selected according to self peptide-MHC (major histocompatibility complex) complexes in the thymus. Although most peripheral T cells recognize specific pathogen-derived peptides complexed to self-MHC exclusively, some possess cross-reactivity to other self or foreign peptides presented by self-MHC molecules; a phenomenon often termed T-cell receptor (TCR) promiscuity or degeneracy. TCR promiscuity has been attributed to various autoimmune conditions. On the other hand, it is considered a mechanism for a relatively limited TCR repertoire to deal with a potentially much larger antigenic peptide repertoire. Such property has also been utilized to bypass self-tolerance for cancer vaccine development. Although many studies explored such degeneracy for peptide of the same length, few studies reported such properties for peptides of different length. In this study, we finely characterized the CD8(+) T-cell response specific for a 11mer peptide derived from influenza A viral polymerase basic protein 2. The short-term T-cell line, despite possessing highly biased TCR, was able to react with multiple peptides of different length sharing the same core sequence. Out data clearly showed the importance of detailed and quantitative assessments for such T-cell specificity. Our data also emphasize the importance of biochemical demonstration of the naturally presented minimal peptide.

Single-Molecule Motions of MHC Class II Rely on Bound Peptides

The major histocompatibility complex (MHC) class II protein can bind peptides of different lengths in the region outside the peptide-binding groove. Peptide-flanking residues (PFRs) contribute to the binding affinity of the peptide for MHC and change the immunogenicity of the peptide/MHC complex with regard to T cell receptor (TCR). The mechanisms underlying these phenomena are currently unknown. The molecular flexibility of the peptide/MHC complex may be an important determinant of the structures recognized by certain T cells. We used single-molecule x-ray analysis (diffracted x-ray tracking (DXT)) and fluorescence anisotropy to investigate these mechanisms. DXT enabled us to monitor the real-time Brownian motion of the peptide/MHC complex and revealed that peptides without PFRs undergo larger rotational motions than peptides with PFRs. Fluorescence anisotropy further revealed that peptides without PFRs exhibit slightly larger motions on the nanosecond timescale. These results demonstrate that peptides without PFRs undergo dynamic motions in the groove of MHC and consequently are able to assume diverse structures that can be recognized by T cells.

Reconstructing the discontinuous and conformational β1/β3-loop binding site on hFSH/hCG by using highly constrained multicyclic peptides.

Making peptide-based molecules that mimic functional interaction sites on proteins remains a challenge in biomedical sciences. Here, we present a robust technology for the covalent assembly of highly constrained and discontinuous binding site mimics, the potential of which is exemplified for structurally complex binding sites on the "Cys-knot" proteins hFSH and hCG. Peptidic structures were assembled by Ar(CH2 Br)2-promoted peptide cyclizations, combined with oxime ligation and disulfide formation. The technology allows unprotected side chain groups and is applicable to peptides of different lengths and nature. A tetracyclic FSH mimic was constructed, showing >600-fold improved binding compared to linear or monocyclic controls. Binding of a tricyclic hCG mimic to anti-hCG mAb 8G5 was identical to hCG itself (IC50 =260 vs. 470 pM), whereas this mimic displayed an IC50 value of 149 nM for mAb 3468, an hCG-neutralizing antibody with undetectable binding to either linear or monocyclic controls.

Shredding the signal: targeting peptide degradation in mitochondria and chloroplasts.

The biogenesis and functionality of mitochondria and chloroplasts depend on the constant turnover of their proteins. The majority of mitochondrial and chloroplastic proteins are imported as precursors via their N-terminal targeting peptides. After import, the targeting peptides are cleaved off and degraded. Recent work has elucidated a pathway involved in the degradation of targeting peptides in mitochondria and chloroplasts, with two proteolytic components: the presequence protease (PreP) and the organellar oligopeptidase (OOP). PreP and OOP are specialized in degrading peptides of different lengths, with the substrate restriction being dictated by the structure of their proteolytic cavities. The importance of the intraorganellar peptide degradation is highlighted by the fact that elimination of both oligopeptidases affects growth and development of Arabidopsis thaliana.

Rational design of α-helix-stabilized exendin-4 analogues.

Exendin-4 (Ex4) is a potent glucagon-like peptide-1 receptor agonist, a drug regulating the plasma glucose level of patients suffering from type 2 diabetes. The molecule's poor solubility and its readiness to form aggregates increase the likelihood of unwanted side effects. Therefore, we designed Ex4 analogues with improved structural characteristics and better water solubility. Rational design was started from the parent 20-amino acid, well-folded Trp cage (TC) miniprotein and involved the step-by-step N-terminal elongation of the TC head, resulting in the 39-amino acid Ex4 analogue, E19. Helical propensity coupled to tertiary structure compactness was monitored and quantitatively analyzed by electronic circular dichroism and nuclear magnetic resonance (NMR) spectroscopy for the 14 peptides of different lengths. Both (15)N relaxation- and diffusion-ordered NMR measurements were established to investigate the inherent mobility and self-association propensity of Ex4 and E19. Our designed E19 molecule has the same tertiary structure as Ex4 but is more helical than Ex4 under all studied conditions; it is less prone to oligomerization and has preserved biological activity. These conditions make E19 a perfect lead compound for further drug discovery. We believe that this structural study improves our understanding of the relationship between local molecular features and global physicochemical properties such as water solubility and could help in the development of more potent Ex4 analogues with improved pharmacokinetic properties.

A novel T-cell receptor mimic defines dendritic cells that present an immunodominant West Nile virus epitope in mice.

We used a newly generated T-cell receptor mimic monoclonal antibody (TCRm MAb) that recognizes a known nonself immunodominant peptide epitope from West Nile virus (WNV) NS4B protein to investigate epitope presentation after virus infection in C57BL/6 mice. Previous studies suggested that peptides of different length, either SSVWNATTAI (10-mer) or SSVWNATTA (9-mer) in complex with class I MHC antigen H-2D(b) , were immunodominant after WNV infection. Our data establish that both peptides are presented on the cell surface after WNV infection and that CD8(+) T cells can detect 10- and 9-mer length variants similarly. This result varies from the idea that a given T-cell receptor (TCR) prefers a single peptide length bound to its cognate class I MHC. In separate WNV infection studies with the TCRm MAb, we show that in vivo the 10-mer was presented on the surface of uninfected and infected CD8α(+) CD11c(+) dendritic cells, which suggests the use of direct and cross-presentation pathways. In contrast, CD11b(+) CD11c(-) cells bound the TCRm MAb only when they were infected. Our study demonstrates that TCR recognition of peptides is not limited to certain peptide lengths and that TCRm MAbs can be used to dissect the cell-type specific mechanisms of antigen presentation in vivo.

Perturbations of the Straight Transmembrane α-Helical Structure of the Amyloid Precursor Protein Affect Its Processing by γ-Secretase

The amyloid precursor protein (APP) is a widely expressed type I transmembrane (TM) glycoprotein present at the neuronal synapse. The proteolytic cleavage by γ-secretase of its C-terminal fragment produces amyloid-β (Aβ) peptides of different lengths, the deposition of which is an early indicator of Alzheimer disease. At present, there is no consensus on the conformation of the APP-TM domain at the biological membrane. Although structures have been determined by NMR in detergent micelles, their conformation is markedly different. Here we show by using molecular simulations that the APP-TM region systematically prefers a straight α-helical conformation once embedded in a membrane bilayer. However, APP-TM is highly flexible, and its secondary structure is strongly influenced by the surrounding lipid environment, as when enclosed in detergent micelles. This behavior is confirmed when analyzing in silico the atomistic APP-TM population observed by residual dipolar couplings and double electron-electron resonance spectroscopy. These structural and dynamic features are critical in the proteolytic processing of APP by the γ-secretase enzyme, as suggested by a series of Gly(700) mutants. Affecting the hydration and flexibility of APP-TM, these mutants invariantly show an increase in the production of Aβ38 compared with Aβ40 peptides, which is reminiscent of the effect of γ-secretase modulators inhibitors.