Short Amino Acid Sequences Research Articles

SAP(E) – A cell-penetrating polyproline helix at lipid interfaces

Cell-penetrating peptides (CPPs) are short membrane-permeating amino acid sequences that can be used to deliver cargoes, e.g. drugs, into cells. The mechanism for CPP internalization is still subject of ongoing research. An interesting family of CPPs is the sweet arrow peptides – SAP(E) – which are known to adopt a polyproline II helical secondary structure. SAP(E) peptides stand out among CPPs because they carry a net negative charge while most CPPs are positively charged, the latter being conducive to electrostatic interaction with generally negatively charged membranes. For SAP(E)s, an internalization mechanism has been proposed, based on polypeptide aggregation on the cell surface, followed by an endocytic uptake. However, this process has not yet been observed directly – since peptide–membrane interactions are inherently difficult to monitor on a molecular scale. Here, we use sum frequency generation (SFG) vibrational spectroscopy to investigate molecular interactions of SAP(E) with differently charged model membranes, in both mono- and bi-layer configurations. The data suggest that the initial binding mechanism is accompanied by structural changes of the peptide. Also, the peptide-model membrane interaction depends on the charge of the lipid headgroup with phosphocholine being a favorable binding site. Moreover, while direct penetration has also been observed for some CPPs, the spectroscopy reveals that for SAP(E), its interaction with model membranes remains limited to the headgroup region, and insertion into the hydrophobic core of the lipid layer does not occur.

Bioreducible branched poly(modified nona-arginine) cell-penetrating peptide as a novel gene delivery platform

Cell-penetrating peptides (CPPs) have been widely used to deliver nucleic acid molecules. Generally, CPPs consisting of short amino acid sequences have a linear structure, resulting in a weak complexation and low transfection efficacy. To overcome these drawbacks, a novel type of CPP is required to enhance the delivery efficacy while maintaining its safe use at the same time.Herein, we report that a bioreducible branched poly-CPP structure capable of responding to reducing conditions attained both outstanding delivery effectiveness and selective gene release in carcinoma cells. Branched structures provide unusually strong electrostatic attraction between DNA and siRNA molecules, thereby improving the transfection capability through a tightly condensed form. We designed a modified type of nona-arginine (mR9) and synthesized a branched-mR9 (B-mR9) using disulfide bonds. A novel B-mR9/pDNA polyplex exhibited redox-cleavability and high transfection efficacy compared to conventional CPPs, with higher cell viability as well. B-mR9/VEGF siRNA polyplex exhibited significant serum stability and high gene-silencing effects in vitro. Furthermore, the B-mR9 polyplex showed outstanding tumor accumulation and inhibition ability in vivo. The results suggest that the bioreducible branched poly CPP has great potential as a gene delivery platform.

PARTICIPATION OF Y89 AND Y97 IN THE CONJUGATING ACTIVITY OF Drosophila melanogaster GLUTATHIONE S-TRANSFERASE D3 (DmGSTD3).

Drosophila melanogaster glutathione S-transferase D3 (DmGSTD3) has a shorter amino acid sequence as compared to other GSTs known in the fruit flies. This is due to the 15 amino acid N-terminal truncation in which normally active amino acid residue is located. The work has made use of homology modeling to visualize the arrangement of amino acid side chains in the glutathione (GSH) substrate cavity. The identified amino acids were then replaced with amino acids without functional groups in the side chains and the mutants were analyzed kinetically. Homology modeling revealed that the side chains of Y89 and Y97 were shown facing toward the substrate cavity proposing their possible role in catalyzing the conjugation. Y97A and Y89A GSH gave large changes in Km (twofold increase), Vmax (fivefold reduction), and Kcat /Km values for GSH suggesting their significant role in the conjugation reaction. The replacement at either positions has not affected the affinity of the enzyme toward 1-chloro-2,4-dinitrobenzene as no significant change in values of Kmax was observed. The replacement, however, had significantly reduced the catalytic efficiency of both mutants with (Kcat /Km )(GSH) and (Kcat /Km )(CDNB) of eight- and twofold reduction. The recombinant DmGSTD3 has shown no activity toward 1,2-dichloro-4-nitrobenzene, 2,4-hexadienal, 2,4-heptadienal, p-nitrobenzyl chloride, ethacrynic acid, and sulfobromophthalein. Therefore, it was evident that DmGSTD3 has made use of distal amino acids Y97 and Y89 for GSH conjugation.

Nouveaux aspects de la compatibilité HLA en transplantation

In organ transplantation, HLA compatibility between a donor and a recipient is currently assessed through the comparison of their HLA antigens and the sole count of incompatible HLA antigens. Similarly, antibodies were originally described as antigenic-specific. With solid phase assays detection of anti-HLA antibodies and crystallographic studies, it is now recognized that anti-HLA antibodies are not specific for antigens, but for epitopes, i.e. short polymorphic sequences of amino acids that are more often in positions accessible to allo-antibodies. These epitopes, due to the distribution of HLA molecules polymorphism, may be shared by different HLA antigens. This explains why an immunization towards a given HLA antigen can lead to synthesis of antibodies reactive towards other antigens sharing one or more epitopes. Similarly, structural comparison of the HLA molecules of a recipient and his donor defines the epitope load, i.e. the number of incompatible epitopes. This epitope load is correlated with the risk of developing antibodies specific for the donor after transplantation. New tools, such as the HLAMatchmaker software, allow to determine the epitopic load and to analyze the epitopic specificity of alloantibodies. These tools will possibly lead to rethink the method of graft allocation, or at least to take differently into account HLA compatibility in allocation algorythms.

Structural and transcriptional characterization of a novel member of the soybean urease gene family

In plants, ureases have been related to urea degradation, to defense against pathogenic fungi and phytophagous insects, and to the soybean−Bradyrhizobium japonicum symbiosis. Two urease isoforms have been described for soybean: the embryo-specific, encoded by Eu1 gene, and the ubiquitous urease, encoded by Eu4. A third urease-encoding locus exists in the completed soybean genome. The gene was designated Eu5 and the putative product of its ORF as SBU-III. Phylogenetic analysis shows that 41 plant, moss and algal ureases have diverged from a common ancestor protein, but ureases from monocots, eudicots and ancient species have evolved independently. Genomes of ancient organisms present a single urease-encoding gene and urease-encoding gene duplication has occurred independently along the evolution of some eudicot species. SBU-III has a shorter amino acid sequence, since many gaps are found when compared to other sequences. A mutation in a highly conserved amino acid residue suggests absence of ureolytic activity, but the overall protein architecture remains very similar to the other ureases. The expression profile of urease-encoding genes in different organs and developmental stages was determined by RT-qPCR. Eu5 transcripts were detected in seeds one day after dormancy break, roots of young plants and embryos of developing seeds. Eu1 and Eu4 transcripts were found in all analyzed organs, but Eu4 expression was more prominent in seeds one day after dormancy break whereas Eu1 predominated in developing seeds. The evidence suggests that SBU-III may not be involved in nitrogen availability to plants, but it could be involved in other biological role(s).

Aggregation and Fibrillation of α-Synuclein by Polymorph Patchy Particles

The intricate aggregation dynamics of alpha-synuclein, the amyloidogenic protein implicated in Parkinson's disease, is simulated with two novel highly coarse-grained models. First, the flexible protein is modelled as a single particle that smoothly transforms between a spherical state, for the disordered protein in solution, to a sphero-cylindrical state, for the mis-folded protein in an amyloid fibril [1]. The internal interactions favor the disordered state, while the transition to the misfolded state enables stabilization by strong inter-particle interactions. Brownian Dynamics is used to simulate the coupled translational and rotational motions of these anisotropic particles [2,3], as well as the internal dynamics of the particles. The simulations show the formation of oligomers and fibrils from solution, inter-conversions between these aggregates, and fibril-stimulated transformation of oligomers into fibrils. Second, the protein is modelled as a flexible chain of polymorph particles, with the variability of the particles representing the dynamical secondary structure of short amino acid sequences. This model has been used to explore the free-energy profile of a protein as it folds and attaches to the end of a fibril [4].[1] I.M.Ilie, W.K. den Otter and W.J. Briels, submitted.[2] I.M.Ilie, W.J.Briels and W.K. den Otter, J. Chem. Phys. 142, 114103 (2015).[3] I.M.Ilie, W.K. den Otter and W.J. Briels, J. Chem. Phys. 141, 065101 (2014).[4] I.M.Ilie, W.K. den Otter and W.J. Briels, in preparation.

Fine mapping of a salmonid E2 alphavirus neutralizing epitope.

In this study, we aimed to characterize the epitope recognized by the neutralizing 17H23 mAb directed against the E2 glycoprotein of most of salmonid alphavirus (SAV) subtypes and widely used in several laboratories to routinely diagnose SAV. We hypothesized that the 17H23 epitope was located in the major domain B, previously identified in the E2 of mammalian alphaviruses as the domain recognized by most of the E2 neutralizing mAbs. Indeed, the SAV E2 domain B counterpart is contained in the protein domain previously characterized as being recognized by mAb 17H23. Thus, to precisely characterize the 17H23 epitope, we developed an alanine scanning mutagenesis approach coupled with the generation of the respective recombinant SAV (rSAV) by using the available infectious cDNA. Ten mutant rSAVs termed A-J from E2 aa 223-236 were produced and characterized in vitro using indirect immunofluorescence assays on virus-infected cells with mAbs 17H23, 51B8 (another non-neutralizing anti-E2 mAb) and 19F3 directed against the non-structural protein nsp1. Two of the mutant rSAVs (G and H) escaped neutralization by mAb 17H23. In addition, we showed that when juvenile trout were infected by bath immersion with the rSAV mutants, some of them were either totally (D, E and G) or partially (H) attenuated. Together, the data from the in vitro and in vivo experiments indicated that the putative 17H23 amino acid sequence epitope comprised the short amino acid sequence (227)FTSDS(231).

Recent trends in the analysis of bioactive peptides in milk and dairy products.

Food-derived constituents represent important sources of several classes of bioactive compounds. Among them peptides have gained great attention in the last two decades thanks to the scientific evidence of their beneficial effects on health in addition to their established nutritional value. Several functionalities for bioactive peptides have been described, including antioxidative, antihypertensive, anti-inflammatory, immunomodulatory, and antimicrobial activity. They are now considered as novel and potential dietary ingredients to promote human health, though in some cases they may also have detrimental effects on health. Bioactive peptides can be naturally occurring, produced in vitro by enzymatic hydrolysis, and formed in vivo during gastrointestinal digestion of proteins. Thus, the need to gain a better understanding of the positive health effects of food peptides has prompted the development of analytical strategies for their isolation, separation, and identification in complex food matrices. Dairy products and milk are potential sources of bioactive peptides: several of them possess extra-nutritional physiological functions that qualify them to be classified under the functional food label. In this trends article we briefly describe the state-of-the-art of peptidomics methods for the identification and discovery of bioactive peptides, also considering recent progress in their analysis and highlighting the difficulty in the analysis of short amino acid sequences and endogenous peptides.

Protein transduction therapy into cochleae via the round window niche in guinea pigs

Cell-penetrating peptides (CPPs) are short sequences of amino acids that facilitate the penetration of conjugated cargoes across mammalian cell membranes, and as such, they may provide a safe and effective method for drug delivery to the inner ear. Simple polyarginine peptides have been shown to induce significantly higher cell penetration rates among CPPs. Herein, we show that a peptide consisting of nine arginines (“9R”) effectively delivered enhanced green fluorescent protein (EGFP) into guinea pig cochleae via the round window niche without causing any deterioration in auditory function. A second application, 24 hours after the first, prolonged the presence of EGFP. To assess the feasibility of protein transduction using 9R-CPPs via the round window, we used “X-linked inhibitor of apoptosis protein” (XIAP) bonded to a 9R peptide (XIAP-9R). XIAP-9R treatment prior to acoustic trauma significantly reduced putative hearing loss and the number of apoptotic hair cells loss in the cochleae. Thus, the topical application of molecules fused to 9R-CPPs may be a simple and promising strategy for treating inner ear diseases.

Does Elevated Alpha-fetoprotein During Pregnancy Protect Against Breast Cancer Later in Life? A Commentary

Elevated maternal serum alpha-fetoprotein (sAFP) has been associated with a future breast cancer risk reduction in previously pregnant women. Past reports have indicated that the presence of high sAFP concentrations in the second and third pregnancy trimester could convey a protective effect against breast cancer later in life. During pregnancy, elevations of sAFP can be attributed to: a) twins and multiple pregnancies; b) fetal defects such as spina bifida and anencephaly; c) gestational hypertension; d) pre-eclampsia; and e) ethnicity. AFP is also elevated in adults with DNA repair diseases such as ataxia telangiectasia and Fanconi anemia. Normally, AFP is a growth enhancing agent; however, a conformationally-altered form of AFP (known as transformed AFP) as well as AFPderived peptides have been shown to be growth-inhibitory agents. It has further been demonstrated that various third domain AFP short amino acid sequence stretches can bind and interact with mutated and/or misfolded proteins of the cell cycle DNA repair process that erroneously allow replication of cells with damaged DNA. Thus, there appears to be at least three possible mechanisms working singly or in concert that might explain the observed breast cancer risk reduction in later the life of previously pregnant women.

Interplay Between the Immune System and Colorectal Carcinoma - Towards Tumor-Specific Peptide-Based Vaccination for Any HLA-Type

Introduction: Peptide-basedcancer vaccines targeting tumor-associated antigens comprised of synthetic peptides have shown promising clinical results and have progressed to Phase III clinical testing in several tumor entities, including colorectal carcinoma (CRC). As of yet, these vaccines have typically been limited to HLA-A*02 positive patients, which means that more than half of the patient pool will not be eligible for those treatment strategies. The development of novel strategies for vaccine design, which are applicable to a larger fraction of potential patients, is therefore required. CRC seems to be particularly suitable for antigen-specific immunotherapy as it is among those cancers with highest rates of genetic mutations leading to alterations in protein metabolism. In this study, we aimed to identify HLA class I and II tumor-associated antigens from CRC covering the most frequent HLA-allotypes, in order to develop a peptide warehouse containing HLA-specific peptide panels that can be compiled in a patient-individualized manner, according to the respective patient's HLA-typing.Methods: The HLA presented immunopeptidome of 30 primary CRC samples and matched autologous non-malignant colon tissue was analyzed after HLA-immunoprecipitation by uHPLC tandem mass spectrometry. Identified source proteins and peptides were cross-evaluated with an in-house database of ligandome data derived from different benign tissues to preclude off-tumor presentation of HLA-ligands in order to to prevent any possible induction of autoimmunity by antigen-specific T cells against these ligands. Furthermore, we evaluated pre-existing antigen-specific T cell responses against these novel CRC-associated peptides in a cohort of 25 CRC patients.Results: About 17,000 MHC class I presented peptides could be identified on CRC and were cross-evaluated with our in-house database containing 39,000 MHC class I peptides presented on non-malignant tissue. The ligands identified were derived from 7,500 unique tumor-associated proteins and from 11,500 tumor-exclusive source proteins, respectively. Moreover, about 2,000 different MHC class II presented peptides from more than 1,600 source proteins were identified on CRC tissue. Interestingly, MHC class II peptides could contain shorter amino-acid sequences which - after further proteasomal degradation - might also bind to MHC class I molecules. For clinical applicability, we prioritized the identified ligands according to their frequencies of presentation and detectability on CRC. For representation in the HLA-specific peptide sets, a cutoff expression on at least 25% of allotype-matched tumors was required. This guided the assembly of a peptide vaccine warehouse consisting of 40 highly specific CRC-associated HLA ligands. Therefore, this selected set of HLA-specific peptides allows vaccination against CRC for about 95% of Caucasian patients. So far, we detected specific T cell reactivity against two peptides (detection of 10-fold and 17-fold increase of IFNγ-producing T cells representing spots as compared to background in enzyme linked immunospot assays - ELISPOT) in CRC patients.Conclusions: We here provide for the first time a comprehensive evaluation of HLA-ligandomes in CRC across the most frequent Caucasian HLA-alleles. We developed a HLA-allotype specific warehouse of newly identified CRC associated HLA-ligands, which can be individually assembled according to patient specific HLA-alleles. This approach expands the applicability of peptide-based cancer vaccines to literally every CRC patient. DisclosuresNo relevant conflicts of interest to declare.

Involvement of loop 5 lysine residues and the N-terminal β-hairpin of the ribotoxin hirsutellin A on its insecticidal activity.

Ribotoxins are cytotoxic members of the family of fungal extracellular ribonucleases best represented by RNase T1. They share a high degree of sequence identity and a common structural fold, including the geometric arrangement of their active sites. However, ribotoxins are larger, with a well-defined N-terminal β-hairpin, and display longer and positively charged unstructured loops. These structural differences account for their cytotoxic properties. Unexpectedly, the discovery of hirsutellin A (HtA), a ribotoxin produced by the invertebrate pathogen Hirsutella thompsonii, showed how it was possible to accommodate these features into a shorter amino acid sequence. Examination of HtA N-terminal β-hairpin reveals differences in terms of length, charge, and spatial distribution. Consequently, four different HtA mutants were prepared and characterized. One of them was the result of deleting this hairpin [Δ(8-15)] while the other three affected single Lys residues in its close spatial proximity (K115E, K118E, and K123E). The results obtained support the general conclusion that HtA active site would show a high degree of plasticity, being able to accommodate electrostatic and structural changes not suitable for the other previously known larger ribotoxins, as the variants described here only presented small differences in terms of ribonucleolytic activity and cytotoxicity against cultured insect cells.

Adhesion and spreading of osteoblast-like cells on surfaces coated with laminin-derived bioactive core peptides.

Functional peptides are attractive as novel therapeutic reagents because their amino acid sequences are flexible in adopting and mimicking the local functional features of proteins. These peptides are of low molecular weight, synthetically versatile and inexpensive to produce, suggesting that they can be used as drug targeting, potent, stable and bioavailable agents. A short bioactive peptide is expected to be more beneficial in regenerative medicine than an entire protein because of the lower antigenicity of short amino acid sequences. We detected core peptides from human laminin that are involved in adhesion and spreading, which are the first steps of various cells including osteogenic cells, in becoming functional. In this experiment, we detected adhesion and spreading of osteoblast-like cells seeded on the core peptide-coated surface. These in vitro data are related to the research article, entitled “Identification of a bioactive core sequence from human laminin and its applicability to tissue engineering” (Yeo et al., 2015) [1].

Meat and fermented meat products as a source of bioactive peptides.

Bioactive peptides are short amino acid sequences, that upon release from the parent protein may play different physiological roles, including antioxidant, antihypertensive, antimicrobial, and other bioactivities. They have been identified from a range of foods, including those of animal origin, e.g., milk and muscle sources (with pork, beef, or chicken and various species of fish and marine organism). Bioactive peptides are encrypted within the sequence of the parent protein molecule and latent until released and activated by enzymatic proteolysis, e.g. during gastrointestinal digestion or food processing. Bioactive peptides derived from food sources have the potential for incorporation into functional foods and nutraceuticals. The aim of this paper is to present an overview of the muscle-derived bioactive peptides, especially those of fermented meats and the potential benefits of these bioactive compounds to human health.

The VQ Motif-Containing Protein Family of Plant-Specific Transcriptional Regulators.

The VQ motif-containing proteins (designated as VQ proteins) are a class of plant-specific proteins with a conserved and single short FxxhVQxhTG amino acid sequence motif. VQ proteins regulate diverse developmental processes, including responses to biotic and abiotic stresses, seed development, and photomorphogenesis. In this Update, we summarize and discuss recent advances in our understanding of the regulation and function of VQ proteins and the role of the VQ motif in mediating transcriptional regulation and protein-protein interactions in signaling pathways. Based on the accumulated evidence, we propose a general mechanism of action for the VQ protein family, which likely defines a novel class of transcriptional regulators specific to plants.

Amphiphilic Residues 29-44 of DREAM N-Termini Mediate Calmodulin:DREAM Complex Formation.

DREAM (downstream regulatory element antagonist modulator) is a neuronal calcium sensor that has been shown to modulate gene expression as well as to be involved in numerous neuronal processes. In this report, we show that association of calcium-bound calmodulin (CaM) with DREAM is mediated by a short amphipathic amino acid sequence located between residues 29 and 44 on DREAM. The association of CaM with a peptide analogous to DREAM(29-44) or to full-length DREAM protein is calcium-dependent with a dissociation constant of 136 nM or 3.4 μM, respectively. Thermodynamic and kinetic studies show that the observed decrease in affinity for the native protein is due to electrostatic interactions between the basic N-terminus and an electronegative surface on DREAM. These results are further supported by circular dichroism, binding studies, and molecular dynamics simulations. Additionally, fluorescence anisotropy decay measurements show a rotational correlation time of 10.8 ns for a complex of CaM with a DREAM(29-44) peptide, supporting a wraparound semispherical model with 1:1 stoichiometry. Furthermore, the interaction between an IEDANS-labeled CaM construct with DREAM is best modeled as a heterotetramer that adopts an elongated conformation with a correlation time of 45 ns in the presence of Ca(2+). We also demonstrate that association of CaM with DREAM eliminates the nonspecific interaction of DREAM with the DRE double-stranded DNA sequence of the human prodynorphin gene. This work provides molecular insight into the CaM:DREAM complex and its potential role in modulation of gene expression.

Spider wrapping silk fibre architecture arising from its modular soluble protein precursor.

Spiders store spidroins in their silk glands as high concentration aqueous solutions, spinning these dopes into fibres with outstanding mechanical properties. Aciniform (or wrapping) silk is the toughest spider silk and is devoid of the short amino acid sequence motifs characteristic of the other spidroins. Using solution-state NMR spectroscopy, we demonstrate that the 200 amino acid Argiope trifasciata AcSp1 repeat unit contrasts with previously characterized spidroins, adopting a globular 5-helix bundle flanked by intrinsically disordered N- and C-terminal tails. Split-intein-mediated segmental NMR-active isotope-enrichment allowed unambiguous demonstration of modular and malleable “beads-on-a-string” concatemeric behaviour. Concatemers form fibres upon manual drawing with silk-like morphology and mechanical properties, alongside secondary structuring and orientation consistent with native AcSp1 fibres. AcSp1 structural stability varies locally, with the fifth helix denaturing most readily. The structural transition of aciniform spidroin from a mostly α-helical dope to a mixed α-helix/β-sheet-containing fibre can be directly related to spidroin architecture and stability.

Synthesis and in vitro and in vivo evaluation of an (18)F-labeled neuropeptide Y analogue for imaging of breast cancer by PET.

Imaging of Y1R expression in breast cancer is still a challenging task. Herein, we report a suitable (18)F-labeled high-molecular-weight glycopeptide for imaging of peripheral neuropeptide Y (NPY) Y1 receptor (Y1R)-positive tumors by preclinical small-animal positron emission tomography (PET). The Y1R-preferring NPY [F(7),P(34)]NPY analogue was functionalized with an alkyne-bearing propargylglycine (Pra) in position 4. The corresponding fluoroglycosylated (FGlc) peptide analogue [Pra(4)(FGlc),F(7),P(34)]NPY and its (18)F-labeled analogue were synthesized by click chemistry-based fluoroglycosylation. The radiosynthesis was performed by (18)F-fluoroglycosylation starting from the 2-triflate of the β-mannosylazide and the alkyne peptide [Pra(4),F(7),P(34)]NPY. The radiosynthesis of the(18)F-labeled analogue was optimized using a minimum amount of peptide precursor (40 nmol), proceeding with an overall radiochemical yield of 20-25% (nondecay corrected) in a total synthesis time of 75 min with specific activities of 40-70 GBq/μmol. In comparison to NPY and [F(7),P(34)]NPY, in vitro Y1R and Y2R activation studies with the cold [Pra(4)(FGlc),F(7),P(34)]NPY on stably transfected COS-7 cells displayed a high potency for the induction of Y1R-specific inositol accumulation (pEC50 = 8.5 ± 0.1), whereas the potency at Y2R was significantly decreased. Internalization studies on stably transfected HEK293 cells confirmed a strong glycopeptide-mediated Y1R internalization and a substantial Y1R subtype selectivity over Y2R. In vitro autoradiography with Y1R-positive MCF-7 tumor tissue slices indicated high specific binding of the (18)F-labeled glycopeptide, when binding was reduced by 95% ([Pra(4),F(7),P(34)]NPY) and by 86% (BIBP3226 Y1R antagonist) in competition studies. Biodistribution and small-animal PET studies on MCF-7 breast tumor-bearing nude mice revealed radiotracer uptake in the MCF-7 tumor of 1.8%ID/g at 20 min p.i. and 0.7%ID/g at 120 min p.i. (n = 3-4), increasing tumor-to-blood ratios from 1.2 to 2.4, and a tumor retention of 76 ± 4% (n = 4; 45-90 min p.i.). PET imaging studies with MCF-7 tumor-bearing nude mice demonstrated uptake of the (18)F-labeled glycopeptide in the tumor region at 60 min p.i., whereas only negligible tumor uptake was observed in animals injected with a nonbinding (18)F-labeled glycopeptide pendant as a measure of nonspecific binding. In conclusion, PET imaging experiments with the (18)F-labeled NPY glycopeptide revealed Y1R-specific binding uptake in MCF-7 tumors in vivo together with decreased kidney uptake compared to DOTA-derivatives of this peptide. We consider this glycopeptide to be a potent lead peptide for the design of improved (18)F-glycopeptides with shorter amino acid sequences that would further facilitate PET imaging studies of Y1R-positive breast tumors.

Binding of Halictine Antimicrobial Peptides to Model Membranes Composed of POPC:POPG Phospholipids

Halictine antimicrobial peptides were isolated from the venom of the bee Halictus sexcinctus. As described in previous studies, halictinemolecules exhibit antimicrobial action against Gram-positive and -negative bacterial cells. However, they have also a considerable hemolytic activity (LC50 ∼80 µM). Halictine 1 (HAL-1) has 12 amino acid residues (GMWSKILGHLIR-NH2), and +3 charges. Other similar sequence, halictine 2 (HAL-2), has also 12 amino acid residues (GKWMSLLKHILK-NH2), and +4 charges. The interaction of these peptides with model membranes composed of POPC:POPG (3:1, and 1:1 molar ratio) phospholipids were monitored using ITC and optical microscopy. For both bilayer composition, halictines were able to bind to the vesicles, resulting mainly in an exothermic process detected by ITC (ΔHpep = −6.8 kcal/mol, and ΔHpep = −4.5 kcal/mol for HAL-1; ΔHpep = −3.4 kcal/mol, and ΔHpep = −5.1 kcal/mol for HAL-2, for titrations with 3:1, and 1:1 POPC:POPG LUVs respectively). HAL-1 displayed endothermic peaks larger than HAL-2, with an additional heat signal at the end of the binding process that remains unclear. Giant unilamellar vesicles (GUVs) were monitored by optical microscopy, which allowed us to observe a peptide-induced permeabilization of these GUVs, followed by vesicle bursts, which was observed for membranes composed of 3:1, and 1:1 POPC:POPG. It could be an indirect evidence of pore formation, culminating in the total collapse of the GUVs. ITC and microscopy experiments were done with low peptide concentrations (30 µM and 20 µM, respectively), below the lethal dose (LC50) of halictines, estimated as hemolytic. The results suggest that HAL-1 and HAL-2, besides the short amino acid sequences, are efficient to bind to model membranes and, thus, to promote pore formation even with low POPG ratio and also low peptide concentration.

Molecular Basis of Laminin-Integrin Interactions.

Laminins are composed of three polypeptide chains, designated as α, β, and γ. The C-terminal region of laminin heterotrimers, containing coiled-coil regions, short tails, and laminin globular (LG) domains, is necessary and sufficient for binding to integrins, which are the major laminin receptor class. Laminin recognition by integrins critically requires the α chain LG domains and a glutamic acid residue of the γ chain at the third position from the C-terminus. Furthermore, the C-terminal region of the β chain contains a short amino acid sequence that modulates laminin affinity for integrins. Thus, all three of the laminin chains act cooperatively to facilitate integrin binding. Mammals possess 5 α (α1-5), 3 β (β1-3), and 3 γ (γ1-3) chains, combinations of which give rise to 16 distinct laminin isoforms. Each isoform is expressed in a tissue-specific and developmental stage-specific manner, exerting its functions through binding of integrins. In this review, we detail the current knowledge surrounding the molecular basis and physiological relevance of specific interactions between laminins and integrins, and describe the mechanisms underlying laminin action through integrins.