The primary fibrin polymerization pocket: Three-dimensional structure of a 30-kDa C-terminal γ chain fragment complexed with the peptide Gly-Pro-Arg-Pro

After vascular injury, a cascade of serine protease activations leads to the conversion of the soluble fibrinogen molecule into fibrin. The fibrin monomers then polymerize spontaneously and noncovalently to form a fibrin gel. The primary interaction of this polymerization reaction is between the newly exposed N-terminal Gly-Pro-Arg sequence of the α chain of one fibrin molecule and the C-terminal region of a γ chain of an adjacent fibrin(ogen) molecule. In this report, the polymerization pocket has been identified by determining the crystal structure of a 30-kDa C-terminal fragment of the fibrin(ogen) γ chain complexed with the peptide Gly-Pro-Arg-Pro. This peptide mimics the N terminus of the α chain of fibrin. The conformational change in the protein upon binding the peptide is subtle, with electrostatic interactions primarily mediating the association. This is consistent with biophysical experiments carried out over the last 50 years on this fundamental polymerization reaction.

High-affinity binding of the Drosophila Numb phosphotyrosine-binding domain to peptides containing a Gly-Pro-(p)Tyr motif

The phosphotyrosine-binding (PTB) domain is a recently identified protein module that has been characterized as binding to phosphopeptides containing an NPXpY motif (X = any amino acid). We describe here a novel peptide sequence recognized by the PTB domain from Drosophila Numb (dNumb), a protein involved in cell fate determination and asymmetric cell division during the development of the Drosophila nervous system. Using a Tyr-oriented peptide library to screen for ligands, the dNumb PTB domain was found to bind selectively to peptides containing a YIGPYφ motif (φ represents a hydrophobic residue). A synthetic peptide containing this sequence bound specifically to the isolated dNumb PTB domain in solution with a dissociation constant (Kd) of 5.78 ± 0.74 μM. Interestingly, the affinity of this peptide for the dNumb PTB domain was increased (Kd = 1.41 ± 0.10 μM) when the second tyrosine in the sequence was phosphorylated. Amino acid substitution studies of the phosphopeptide demonstrated that a core motif of sequence GP(p)Y is required for high-affinity binding to the dNumb PTB domain. Nuclear magnetic resonance experiments performed on isotopically labeled protein complexed with either Tyr- or pTyr-containing peptides suggest that the same set of amino acids in the dNumb PTB domain is involved in binding both phosphorylated and nonphosphorylated forms of the peptide. The in vitro selectivity of the dNumb PTB domain is therefore markedly different from those of the Shc and IRS-1 PTB domains, in that it interacts preferentially with a GP(p)Y motif, rather than NPXpY, and does not absolutely require ligand phosphorylation for binding. Our results suggest that the PTB domain is a versatile protein module, capable of exhibiting varied binding specificities.

The mechanism of maturation of insulin-like growth factor-1

This study, to elucidate the role of des(1-3)IGF-I in the maturation of IGF-I,used two strategies. The first was to detect the presence of enzymes in tissues, which would act on IGF-I to produce des(1-3)IGF-I, and the second was to detect the potential products of such enzymic activity, namely Gly-Pro-Glu(GPE), Gly-Pro(GP) and des(l- 3)IGF-I. No neutral tripeptidyl peptidase (TPP II), which would release the tripeptide GPE from IGF-I, was detected in brain, urine nor in red or white blood cells. The TPPlike activity which was detected, was attributed to a combined action of a dipeptidyl peptidase (DPP N) and an aminopeptidase (AP A). A true TPP II was, however, detected in platelets. Two purified TPP II enzymes were investigated but they did not release GPE from IGF-I under a variety of conditions. Consequently, TPP II seemed unlikely to participate in the formation of des(1-3)IGF-I. In contrast, an acidic tripeptidyl peptidase activity (TPP I) was detected in brain and colostrum, the former with a pH optimum of 4.5 and the latter 3.8. It seems likely that such an enzyme would participate in the formation of des( 1-3 )IGF-I in these tissues in vitro, ie. that des(1-3)IGF-I may have been produced as an artifact in the isolation of IGF-I from brain and colostrum in acidic conditions. This contrasts with suggestions of an in vivo role for des(1-3)IGF-I, as reported by others. The activity of a dipeptidyl peptidase N (DPP N) from urine, which should release the dipeptide GP from IGF-I, was assessed under a variety of conditions and with a variety of additives and potential enzyme stimulants, but there was no release of GP. The DPP N also exhibited a transferase activity with synthetic substrates in the presence of dipeptides, at lower concentrations than previously reported for other acceptors or other proteolytic enzymes. In addition, a low concentration of a product,possibly the tetrapeptide Gly-Pro-Gly-Leu, was detected with the action of the enzyme on IGF-I in the presence of the dipeptide Gly-Leu. As part of attempts to detect tissue production of des(1-3)IGF-I, a monoclonal antibody (MAb ), directed towards the GPE- end ofiGF-I was produced by immunisation with a 10-mer covalently attached to a carrier protein. By the use of indirect ELISA and inhibitor studies, the MAb was shown to selectively recognise peptides with anNterminal GPE- sequence, and applied to the indirect detection of des(1-3)IGF-I. The concentration of GPE in brain, measured by mass spectrometry ( MS), was low, and the concentration of total IGF-I (measured by ELISA with a commercial polyclonal antibody [P Ab]) was 40 times higher at 50 nmol/kg. This also, was not consistent with the action of a tripeptidyl peptidase in brain that converted all IGF-I to des(1-3)IGF-I plus GPE. Contrasting ELISA results, using the MAb prepared in this study, suggest an even higher concentration of intact IGF-I of 150 nmollkg. This would argue against the presence of any des( 1-3 )IGF-I in brain, but in turn, this indicates either the presence of other substances containing a GPE amino-terminus or other cross reacting epitope. Although the results of the specificity studies reported in Chapter 5 would make this latter possibility seem unlikely, it cannot be completely excluded. No GP was detected in brain by MS. No GPE was detected in colostrum by capillary electrophoresis (CE) but the interference from extraneous substances reduced the detectability of GPE by CE and this approach would require further, prior, purification and concentration steps. A molecule, with a migration time equal to that of the peptide GP, was detected in colostrum by CE, but the concentration (~ 10 11mo/L) was much higher than the IGF-I concentration measured by radio-immunoassay using a PAb (80 nmol/L) or using a Mab (300-400 nmolL). A DPP IV enzyme was detected in colostrum and this could account for the GP, derived from substrates other than IGF-1. Based on the differential results of the two antibody assays, there was no indication of the presence of des(1-3)IGF-I in brain or colostrum. In the absence of any enzyme activity directed towards the amino terminus of IGF-I and the absence any potential products, IGF-I, therefore, does not appear to "mature" via des(1-3)IGF-I in the brain, nor in the neutral colostrum. In spite of these results which indicate the absence of an enzymic attack on IGF-I and the absence of the expected products in tissues, the possibility that the conversion of IGF-I may occur in neutral conditions in limited amounts, cannot be ruled out. It remains possible that in the extracellular environment of the membrane, a complex interaction of IGF-I, binding protein, aminopeptidase(s) and receptor, produces des(1- 3)IGF-I as a transient product which is bound to the receptor and internalised.

THE CORRECTED PRIMARY STRUCTURE OF CHICKEN (AVIAN) PANCREATIC-POLYPEPTIDE

Chicken (avian) pancreatic polypeptide was the first member of the pancreatic polypeptide (PP)/neuropeptide Y (NPY) superfamily to be discovered and structurally-characterised. In this 36 amino acid residue, C-terminally amidated peptide, residues 22 and 23 were identified as Asp and Asn, respectively. However, sequencing of chicken PP using modem automated gas-phase sequencing technology has revealed that the original primary structure is incorrect in that residue 22 is Asn and that residue 23 is Asp. After digestion of chicken PP with endoproteinase Asp-N, fragments of chicken PP corresponding in molecular mass to residues 16-22 and 23-36, were unequivocally identified. The corrected primary structure of chicken PP is therefore: Gly-Pro-Ser-Gln-Pro-Thr-Tyr-Pro-Gly-Asp-Asp-Ala-Pro-Val-Glu-Asp-Leu-Ile-Arg-Phe-Tyr-Asn-Asp-Leu-Gln-Gln-Tyr-Leu-Asn-Val-Val-Thr-Arg-His-Arg-Tyr-NH2.

Identification and Functional Analysis of a Novel Tryptophyllin Peptide from the Skin of the Red-eye Leaf Frog, Agalychnis callidryas

Amphibian skin has proved repeatedly to be a largely untapped source of bioactive peptides and this is especially true of members of the Phyllomedusinae subfamily of frogs native to South and Central America. Tryptophyllins are a group of peptides mainly found in the skin of members of this genus. In this study, a novel tryptophyllin (TPH) type 3 peptide, named AcT-3, has been isolated and structurally-characterised from the skin secretion and lyophilised skin extract of the red-eye leaf frog, Agalychnis callidryas. The peptide was identified in and purified from the skin secretion by reverse-phase HPLC. MALDI-TOF mass spectrometry and MS/MS fragmentation sequencing established its primary structure as: pGlu-Gly-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Phe-Leu-Pro-Glu, with a non-protonated molecular mass of 1538.19Da. The mature peptide possessed the canonical N-terminal pGlu residue that arises from post-translational modification of a Gln residue. The deduced open-reading frame consisted of 63 amino acid residues encoding a highly-conserved signal peptide of approximately 22 amino acid residues, an intervening acidic spacer peptide domain, a single AcT-3 encoding domain and a C terminal processing site. A synthetic replicate of AcT-3 was found to antagonise the effect of BK on rat tail artery smooth muscle and to contract the intestinal smooth muscle preparations. It was also found that AcT-3 could dose-dependently inhibit the proliferation of human prostate cancer cell lines after 72h incubation.

Mapping and molecular modeling of S-adenosyl-L-methionine binding sites in N-methyltransferase domains of the multifunctional polypeptide cyclosporin synthetase

We employed a highly specific photoaffinity labeling procedure, using 14C-labeled S-adenosyl-L-methionine (AdoMet) to define the chemical structure of the AdoMet binding centers on cyclosporin synthetase (CySyn). Tryptic digestion of CySyn photolabeled with either [methyl-14C]AdoMet or [carboxyl-14C]AdoMet yielded the sequence H2N-Asn-Asp-Gly-Leu-Glu-Ser-Tyr-Val-Gly-Ile-Glu-Pro-Ser-Arg-COOH (residues 10644-10657), situated within the N-methyltransferase domain of module 8 of CySyn. Radiosequencing detected Glu10654 and Pro10655 as the major sites of derivatization. [carboxyl-14C]AdoMet in addition labeled Tyr10650. Chymotryptic digestion generated the radiolabeled peptide H2N-Ile-Gly-Leu-Glu-Pro-Ser-Gln-Ser-Ala-Val-Gln-Phe-COOH, corresponding to amino acids 2125-2136 of the N-methyltransferase domain of module 2. The radiolabeled amino acids were identified as Glu2128 and Pro2129, which are equivalent in position and function to the modified residues identified with tryptic digestions in module 8. Homology modeling of the N-methyltransferase domains indicates that these regions conserve the consensus topology of the AdoMet binding fold and consensus cofactor interactions seen in structurally characterized AdoMet-dependent methyltransferases. The modified sequence regions correspond to the motif II consensus sequence element, which is involved in directly complexing the adenine and ribose components of AdoMet. We conclude that the AdoMet binding to nonribosomal peptide synthetase N-methyltransferase domains obeys the consensus cofactor interactions seen among most structurally characterized low molecular weight AdoMet-dependent methyltransferases.

Design, synthesis and characterization of a hexapeptide bio-inspired by acetylcholinesterase and its interaction with pesticide dichlorvos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Experimental adaptation of wild-type canine distemper virus (CDV) to the human entry receptor CD150.

Canine distemper virus (CDV), a close relative of measles virus (MV), is widespread and well known for its broad host range. When the goal of measles eradication may be achieved, and when measles vaccination will be stopped, CDV might eventually cross the species barrier to humans and emerge as a new human pathogen. In order to get an impression how fast such alterations may occur, we characterized required adaptive mutations to the human entry receptors CD150 (SLAM) and nectin-4 as first step to infect human target cells. Recombinant wild-type CDV-A75/17(red) adapted quickly to growth in human H358 epithelial cells expressing human nectin-4. Sequencing of the viral attachment proteins (hemagglutinin, H, and fusion protein, F) genes revealed that no adaptive alteration was required to utilize human nectin-4. In contrast, the virus replicated only to low titres (10(2) pfu/ml) in Vero cells expressing human CD150 (Vero-hSLAM). After three passages using these cells virus was adapted to human CD150 and replicated to high titres (10(5) pfu/ml). Sequence analyses revealed that only one amino acid exchange in the H-protein at position 540 Asp→Gly (D540G) was required for functional adaptation to human CD150. Structural modelling suggests that the adaptive mutation D540G in H reflects the sequence alteration from canine to human CD150 at position 70 and 71 from Pro to Leu (P70L) and Gly to Glu (G71E), and compensates for the gain of a negative charge in the human CD150 molecule. Using this model system our data indicate that only a minimal alteration, in this case one adaptive mutation, is required for adaptation of CDV to the human entry receptors, and help to understand the molecular basis why this adaptive mutation occurs.

Quantitative assessment of enzyme specificity in vivo: P2 recognition by Kex2 protease defined in a genetic system

The specificity of the yeast proprotein-processing Kex2 protease was examined in vivo by using a sensitive, quantitative assay. A truncated prepro-α-factor gene encoding an α-factor precursor with a single α-factor repeat was constructed with restriction sites for cassette mutagenesis flanking the single Kex2 cleavage site (-SLDKR↓EAEA-). All of the 19 substitutions for the Lys (P2) residue in the cleavage site were made. The wild-type and mutant precursors were expressed in a yeast strain lacking the chromosomal genes encoding Kex2 and prepro-α-factor. Cleavage of the 20 sites by Kex2, expressed at the wild-type level, was assessed by using a quantitative-mating assay with an effective range greater than six orders of magnitude. All substitutions for Lys at P2 decreased mating, from 2-fold for Arg to >106-fold for Trp. Eviction of the Kex2-encoding plasmid indicated that cleavage of mutant sites by other cellular proteases was not a complicating factor. Mating efficiencies of strains expressing the mutant precursors correlated well with the specificity (kcat/KM) of purified Kex2 for comparable model peptide substrates, validating the in vivo approach as a quantitative method. The results support the conclusion that KM, which is heavily influenced by the nature of the P2 residue, is a major determinant of cleavage efficiency in vivo. P2 preference followed the rank order: Lys > Arg > Thr > Pro > Glu > Ile > Ser > Ala > Asn > Val > Cys > AsP > Gln > Gly > His > Met > Leu > Tyr > Phe > Trp.

The crude skin secretion of the pepper frog leptodactylus labyrinthicus is rich in metallo and serine peptidases

Peptidases are ubiquitous enzymes involved in diverse biological processes. Fragments from bioactive peptides have been found in skin secretions from frogs, and their presence suggests processing by peptidases. Thus, the aim of this work was to characterize the peptidase activity present in the skin secretion of Leptodactylus labyrinthicus. Zymography revealed the presence of three bands of gelatinase activity of approximately 60 kDa, 66 kDa, and 80 kDa, which the first two were calcium-dependent. These three bands were inhibited either by ethylenediaminetetraacetic acid (EDTA) and phenathroline; thus, they were characterized as metallopeptidases. Furthermore, the proteolytic enzymes identified were active only at pH 6.0–10.0, and their activity increased in the presence of CHAPS or NaCl. Experiments with fluorogenic substrates incubated with skin secretions identified aminopeptidase activity, with cleavage after leucine, proline, and alanine residues. This activity was directly proportional to the protein concentration, and it was inhibited in the presence of metallo and serine peptidase inhibitors. Besides, the optimal pH for substrate cleavage was determined to be 7.0–8.0. The results of the in gel activity assay showed that all substrates were hydrolyzed by a 45 kDa peptidase. Gly-Pro-AMC was also cleaved by a peptidase greater than 97 kDa. The data suggest the presence of dipeptidyl peptidases (DPPs) and metallopeptidases; however, further research is necessary. In conclusion, our work will help to elucidate the implication of these enzymatic activities in the processing of the bioactive peptides present in frog venom, expanding the knowledge of amphibian biology.

Stereochemical restrictions on the occurrence of amino acid residues in the collagen structure

The primary structure of collagen is characterized by the repeating tripeptide sequence (Gly-R2-R3)n. The results of theoretical studies, carried out using contact criteria to compute the stereochemically allowed orientations for various side chains at locations 2 and 3, are reported here. It is found that side chains with only γ-atoms, as in valine, serine and threonine, or with only one δ-methyl group, as in isoleucine, can occur equally well at locations 2 and 3, as is actually the case in collagen. Side chains with two Cδ-atoms, as in leucine and phenyl-alanine, can also be accommodated at both positions. However, if they occur as R3 their freedom of orientation is severely restricted in the presence of a proline residue as R2 in a neighbouring chain. If water molecules bound to the chains of the triple helix are assumed to be present, then location 3 is virtually impossible for leucine and phenylalanine residues. Location 2 is, however, unaffected, and their presence as R2 can help to shield the water molecules from disturbance by the solvent medium. This may be the reason for the preferential occurrence of Leu and Phe residues in location 2 in the collagen triplets, although the polypeptides (Gly-Pro-Leu)n and (Gly-Pro-Phe)n form collagen-like structures.

Porlyl oligopeptidase in development

Prolyl oligopeptidase (POP, prolyl endopeptidase, EC 3.4.21.26) is a serine-type peptidase (family S9 of clan SC) hydrolyzing peptides shorter than 30 amino acids. POP has been found in various mammalian and bacterial sources and it is widely distributed throughout different organisms. In human and rat, POP enzyme activity has been detected in most tissues, with the highest activity found mostly in the brain. POP has gained scientific interest as being involved in the hydrolyzis of many bioactive peptides connected with learning and memory functions, and also with neurodegenerative disorders. In drug or lesion induced amnesia models and in aged rodents, POP inhibitors have been able to revert memory loss. POP may have a fuction in IP3 signaling and it may be a possible target of mood stabilizing substances. POP may also have a role in protein trafficking, sorting and secretion. The role of POP during ontogeny has not yet been resolved. POP enzyme activity and expression have shown fluctuation during development. Specially high enzyme activities have been measured in the brain during early development. Reduced neuronal proliferation and differentation in presence of POP inhibitor have been reported. Nuclear POP has been observed in proliferating peripheral tissues and in cell cultures at the early stage of development. Also, POP coding mRNA is abundantly expressed during brain ontogeny and the highest levels of expression are associated with proliferative germinal matrices. This observation indicates a special role for POP in the regulation of neurogenesis during development. For the experimental part, the study was undertaken to investigate the expression and distribution of POP protein and enzymatic activity of POP in developing rat brain (from embryonic day 14 to post natal day 7) using immunohistochemistry, POP enzyme activity measurements and western blot-analysis. The aim was also to find in vivo confirmation of the nuclear colocalization of POP during early brain ontogeny. For immunohistochemistry, cryosections from the brains of the fetuses/rats were made and stained using specific antibody for POP and fluorescent markers for POP and nuclei. The enzyme activity assay was based on the fluorescence of 7- amino-4-methylcoumarin (AMC) generated from the fluorogenic substrate succinyl-glycyl-prolyl-7-amino-4-methylcoumarin (Suc-Gly-Pro-AMC) by POP. The amounts of POP protein and the specifity of POP antibody in rat embryos was confirmed by western blot analysis. We observed that enzymatic activity of POP is highest at embryonic day 18 while the protein amounts reach their peak at birth. POP was widely present throughout the developmental stages from embryonic day 14 to parturition day, although the POP-immunoreactivity varied abundantly. At embryonic days 14 and 18 notably amounts of POP was distributed at proliferative germinal zones. Furthermore, POP was located in the nucleus early in the development but is transferred to cytosol before birth. At P0 and P7 the POP-immunoreactivity was also widely observed, but the amount of POP was notably reduced at P7. POP was present in cytosol and in intercellular space, but no nuclear POP was observed. These findings support the idea of POP being involved in specific brain functions, such as neuronal proliferation and differentation. Our results in vivo confirm the previous cell culture results supporting the role of POP in neurogenesis. Moreover, an inconsistency of POP protein amounts and enzymatic activity late in the development suggests a strong regulation of POP activity and a possible non-hydrolytic role at that stage.

The Design and Construction of Synthetic Protein Mimics

A strategy for the modular construction of synthetic protein mimics based on the ability non-protein amino acids to act as stereochemical directors of polypeptide chain folding, is described. The use of alpha-aminoisobutyric acid (Aib) to construct stereochemically rigid helices has been exemplified by crystallographic and spectroscopic studies of several apolar peptides, ranging in length from seven to sixteen residues. The problem of linker design in elaborating alpha,alpha motifs has been considered. Analysis of protein crystal structure data provides a guide to choosing linking sequences. Attempts at constructing linked helical motifs using linking Gly-Pro segments have been described. The use of flexible linkers, like epsilon-aminocaproic acid has been examined and the crystallographic and solution state analysis of a linked helix motif has been presented. The use of bulky sidechain modifications on a helical scaffold, as a means of generating putative binding sites has been exemplified by a crystal structure of a peptide packed in a parallel zipper arrangement.

酸性抗菌肽及其基因和在制药中的应用

本发明涉及一种酸性抗菌肽及其基因和在制药中的应用，属于生物医学领域。酸性抗菌肽，是中国两栖类动物大蹼铃蟾基因编码的一种单链多肽，分子量2021.4，等电点4.8，多肽氨基酸全序列一级结构为：Ile Leu Gly Pro Val LeuGly Leu Val Ser Asp Thr Leu Asp Asp Val Leu Gly Ile Leu-AMIDATION。编码酸性抗菌肽的基因由632个核苷酸组成，其中编码成熟酸性抗菌肽为第418-477位核苷酸。人工化学合成的酸性抗菌肽具有显著的抑制细菌生长的作用，可以作为制备抗微生物感染疾病药物的应用。