Human tissue kallikrein S1 subsite recognition of non-natural basic amino acids

We explored the unique substrate specificity of the primary S(1) subsite of human urinary kallikrein (hK1), which accepts both Phe and Arg, using internally quenched fluorescent peptides Abz-F-X-S-R-Q-EDDnp and Abz-G-F-S-P-F-X-S-S-R-P-Q-EDDnp [Abz is o-aminobenzoic acid; EDDnp is N-(2,4-dinitrophenyl)ethylenediamine], which were based on the human kininogen sequence at the C-terminal region of bradykinin. Position X, which in natural sequence stands for Arg, received the following synthetic basic non-natural amino acids: 4-(aminomethyl)phenylalanine (Amf), 4-guanidine phenylalanine (Gnf), 4-(aminomethyl)-N-isopropylphenylalanine (Iaf), N(im)-(dimethyl)histidine [H(2Me)], 3-pyridylalanine (Pya), 4-piperidinylalanine (Ppa), 4-(aminomethyl)cyclohexylalanine (Ama), and 4-(aminocyclohexyl)alanine (Aca). Only Abz-F-Amf-S-R-Q-EDDnp and Abz-F-H(2Me)]-S-R-Q-EDDnp were efficiently hydrolyzed, and all others were resistant to hydrolysis. However, Abz-F-Ama-S-R-Q-EDDnp inhibited hK1 with a K(i) of 50 nM with high specificity compared to human plasma kallikrein, thrombin, plasmin, and trypsin. The Abz-G-F-S-P-F-X-S-S-R-P-Q-EDDnp series were more susceptible to hK1, although the peptides with Gnf, Pya, and Ama were resistant to it. Unexpectedly, the peptides in which X is His, Lys, H(2Me), Amf, Iaf, Ppa, and Aca were cleaved at amino or at carboxyl sites of these amino acids, indicating that the S(1)' subsite has significant preference for basic residues. Human plasma kallikrein did not hydrolyze any peptide of this series except the natural sequence where X is Arg. In conclusion, the S(1) subsite of hK1 accepts amino acids with combined basic and aromatic side chain, although for the S(1)-P(1) interaction the preference is for aliphatic and basic side chains.     

The specific binding of peptide ligands to cardiomyocytes derived from mouse embryonic stem cells

Purification of pluripotent stem cell (PSC)‐derived cardiomyocytes is critical for the application of cardiomyocytes both in clinical and basic research. Finding a specific cell marker is a promising method for purifying induced cells. The present study employed phage display technology to search for particular cell markers that could bind specifically to PSC‐derived cardiomyocytes. After three rounds of biopanning, several peptides were obtained. The ELISA results show the no. 3 sequence peptide (QPFTTSLTPPAR), and other four sequences having a consensus motif [SS(Q)PPQ(S)], no. 9, 11, 14, and 10, have relatively high affinity and specificity to cardiomyocytes. Immunofluorescence confirmed that the selected peptides could bind specifically to the PSC‐derived cardiomyocytes. Competition tests with chemically synthesized peptides revealed the binding ability was caused by the peptide itself. Western blot analysis proved the phages were both bound to two 17 kDa cardiomyocyte membrane proteins and the no. 9 sequence showed a 55 kDa protein that was not observed in the no. 3 sequence. These results suggest that the selected peptides specifically target receptors on PSC‐derived cardiomyocyte membranes. The results will pave the way for further studies of cell surface markers and their applications, such as labeling, purification, and as vehicles for drug delivery. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Screening and characterization of anti‐SEB peptides using a bacterial display library and microfluidic magnetic sorting

Bacterial peptide display libraries enable the rapid and efficient selection of peptides that have high affinity and selectivity toward their targets. Using a 15‐mer random library on the outer surface of Escherichia coli (E.coli), high‐affinity peptides were selected against a staphylococcal enterotoxin B (SEB) protein after four rounds of biopanning. On‐cell screening analysis of affinity and specificity were measured by flow cytometry and directly compared to the synthetic peptide, off‐cell, using peptide‐ELISA. DNA sequencing of the positive clones after four rounds of microfluidic magnetic sorting (MMS) revealed a common consensus sequence of (S/T)CH(Y/F)W for the SEB‐binding peptides R338, R418, and R445. The consensus sequence in these bacterial display peptides has similar amino acid characteristics with SEB peptide sequences isolated from phage display. The K_d measured by peptide‐ELISA off‐cell was 2.4 nM for R418 and 3.0 nM for R445. The bacterial peptide display methodology using the semiautomated MMS resulted in the discovery of selective peptides with affinity for a food safety and defense threat. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Journal of Molecular Recognition published by John Wiley & Sons, Ltd.     

Kinetic and functional characterisation of the heparin‐binding peptides from human transglutaminase 2

Transglutaminase 2 (TG2) is an autoantigen in celiac disease (CD) and it has multiple biologic functions including involvement in cell adhesion through interactions with integrins, fibronectin (FN), and heparan sulfate proteoglycans. We aimed to delineate the heparin‐binding regions of human TG2 by studying binding kinetics of the predicted heparin‐binding peptides using surface plasmon resonance method. In addition, we characterized immunogenicity of the TG2 peptides and their effect on cell adhesion. The high‐affinity binding of human TG2 to the immobilized heparin was observed, and two TG2 peptides, P1 (amino acids 202–215) and P2 (261–274), were found to bind heparin. The amino acid sequences corresponding to the heparin‐binding peptides were located close to each other on the surface of the TG2 molecule as part of the α‐helical structures. The heparin‐binding peptides displayed increased immunoreactivity against serum IgA of CD patients compared with other TG2 peptides. The cell adhesion reducing effect of the peptide P2 was revealed in Caco‐2 intestinal epithelial cell attachment to the FN and FN‐TG2 coated surfaces. We propose that TG2 amino acid sequences 202–215 and 261–274 could be involved in binding of TG2 to cell surface heparan sulfates. High immunoreactivity of the corresponding heparin‐binding peptides of TG2 with CD patient's IgA supports the previously described role of anti‐TG2 autoantibodies interfering with this interaction. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Determination of protease subsite preference on SPOT peptide array by fluorescence quenching‐based assay

A peptide SPOT array was synthesized on a glass chip and used to determine protease subsite preference. To synthesize a peptide array for positional scanning, the ratio of the isokinetic concentration was determined for every Fmoc‐amino acid except Cys. Based on this ratio, a peptide array consisting of Dabcyl‐X‐X‐P₂‐Arg‐X‐X‐X‐Lys(FITC) (X: equimolar mixture of 19 amino acids, P₂: one of 19 amino acids) was synthesized on a chitosan‐grafted glass chip. Subsequently, the peptide substrates on the array were hydrolyzed by thrombin to screen for subsite specificity using a fluorescence quenching‐based assay. The P₂ subsite specificity of thrombin was screened by the fluorescence images obtained after hydrolysis. Pro at the P₂ subsite showed the highest specificity for thrombin based on both the fluorescence quenching‐based assay and the solution phase assay. From these results, we confirmed that our mixture‐based peptide SPOT array format on the chitosan‐grafted glass chips could be used to determine protease subsite preference. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Chronotherapeutic Dose Schedule of Phenytoin and Carbamazepine in Epileptic Patients

The objective of this study was to compare the efficacy and safety of a chronotherapeutic dosing schedule of phenytoin and carbamazepine versus a conventional dosing schedule for the treatment of tonic‐clonic epileptic patients. Of 148 epileptic subjects found to have subtherapeutic trough drug levels (subtherapeutic group, STG), 103 subjects who completed the study were randomized to either STG I (n=51) for treatment by the conventional dosing schedule (tablet phenytoin 100–400 mg/day OD or BD, tablet carbamazepine 200–800 mg BD, or both, equally divided doses with no fixed time of drug intake), with a dose increment but no change in usual time of drug administration allowed; or to STG II (n=52), with no dose increment permitted but a shift in all or most (two‐thirds or three‐fourths) of the daily dose of one or both medications to 20:00 h. The 62 patients who experienced drug toxicity reactions (toxicity group, TG) and who had serum drug levels in the toxic range were assigned to TG I for dose reduction or TG II for dose reduction and drug administration at 20:00 h. Those 16 subjects in STG I and 47 subjects in STG II who initially evidenced subtherapeutic trough drug concentrations exhibited therapeutic drug levels by the end of four weeks of treatment (p<0.01). A significantly greater number of TG II, as compared to TG I, subjects who experienced toxic reactions showed improved drug tolerance. There were no poor responders and more good responders (control of epilepsy for one year) in STG II compared to STG I subjects. The findings of this study indicate that a chronotherapeutic dosing schedule of phenytoin and carbamazepine involving the administration of most or all the daily dose of medication(s) at 20:00 h can improve the response of diurnally active epileptic patients not responding to standard doses, achieve therapeutic drug levels, and reduce toxic manifestations in subjects having drug concentrations beyond the therapeutic range.     

Peptide array‐based characterization and design of ZnO‐high affinity peptides

Peptides with both an affinity for ZnO and the ability to generate ZnO nanoparticles have attracted attention for the self‐assembly and templating of nanoscale building blocks under ambient conditions with compositional uniformity. In this study, we have analyzed the specific binding sites of the ZnO‐binding peptide, EAHVMHKVAPRP, which was identified using a phage display peptide library. The peptide binding assay against ZnO nanoparticles was performed using peptides synthesized on a cellulose membrane using the spot method. Using randomized rotation of amino acids in the ZnO‐binding peptide, 125 spot‐synthesized peptides were assayed. The peptide binding activity against ZnO nanoparticles varied greatly. This indicates that ZnO binding does not depend on total hydrophobicity or other physical parameters of these peptides, but rather that ZnO recognizes the specific amino acid alignment of these peptides. In addition, several peptides were found to show higher binding ability compared with that of the original peptides. Identification of important binding sites in the EAHVMHKVAPRP peptide was investigated by shortened, stepwise sequence from both termini. Interestingly, two ZnO‐binding sites were found as 6‐mer peptides: HVMHKV and HKVAPR. The peptides identified by amino acid substitution of HKVAPR were found to show high affinity and specificity for ZnO nanoparticles. Biotechnol. Bioeng. 2010;106: 845–851. © 2010 Wiley Periodicals, Inc.     

Synthesis of pathological and nonpathological human exon 1 huntingtin

Huntington's disease (HD) is a neurodegenerative disorder that affects approximately 1 in 10 000 individuals. The underlying gene mutation was identified as a CAG‐triplet repeat expansion in the gene huntingtin. The CAG sequence codes for glutamine, and in HD, an expansion of the polyglutamine (poly‐Q) stretch above 35 glutamine residues results in pathogenicity. It has been demonstrated in various animal models that only the expression of exon 1 huntingtin, a 67‐amino acid‐long polypeptide plus a variable poly‐Q stretch, is sufficient to cause full HD‐like pathology. Therefore, a deeper understanding of exon 1 huntingtin, its structure, aggregation mechanism and interaction with other proteins is crucial for a better understanding of the disease. Here, we describe the synthesis of a 109‐amino acid‐long exon 1 huntingtin peptide including a poly‐Q stretch of 42 glutamines. This microwave‐assisted solid phase peptide synthesis resulted in milligram amounts of peptide with high purity. We also synthesized a nonpathogenic version of exon 1 huntingtin (90‐amino acid long including a poly‐Q stretch of 23 glutamine residues) using the same strategy. In circular dichroism spectroscopy, both polypeptides showed weak alpha‐helical properties with the longer peptide showing a higher helical degree. These model peptides have great potential for further biomedical analyses, e.g. for large‐scale pre‐screenings for aggregation inhibitors, further structural analyses as well as protein–protein interaction studies. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Evolution of Class-Specific Peptides Targeting a Hot Spot of the Gαs Subunit

The four classes of heterotrimeric G-protein α subunits act as molecular routers inside cells, gating signals based on a bound guanosine nucleotide (guanosine 5′-triphosphate versus guanosine 5′-diphosphate). Ligands that specifically target individual subunits provide new tools for monitoring and modulating these networks, but are challenging to design due to the high sequence homology and structural plasticity of the Gα-binding surface. Here we have created an mRNA display library of peptides based on the short Gα-modulating peptide R6A-1 and selected variants that target a convergent protein-binding surface of Gαs·guanosine 5′-diphosphate. After selection/evolution, the most Gαs-specific peptide, Gαs(s)-binding peptide (GSP), was used to design a second-generation library, resulting in several new affinity- and selectivity-matured peptides denoted as mGSPs. The two-step evolutionary walk from R6A-1 to mGSP-1 resulted in an 8000-fold inversion in binding specificity, altered seven out of nine residues in the starting peptide core, and incorporated both positive and negative design steps. The resulting mGSP-1 peptide shows remarkable selectivity and affinity, exhibiting little or no binding to nine homologous Gα subunits or human H-Ras, and even discriminates the Gαs splice variant Gαs(l). Selected peptides make specific contacts with the effector-binding region of Gα, which may explain an interesting bifunctional activity observed in GSP. Overall, our work demonstrates a design of simple, linear, highly specific peptides that target a protein-binding surface of Gαs and argues that mRNA display-based selection/evolution is a powerful route for targeting protein families with high class specificity and state specificity.     

Phage display selection of efficient glutamine-donor substrate peptides for transglutaminase 2

Understanding substrate specificity and identification of natural targets of transglutaminase 2 (TG2), the ubiquitous multifunctional cross-linking enzyme, which forms isopeptide bonds between protein-linked glutamine and lysine residues, is crucial in the elucidation of its physiological role. As a novel means of specificity analysis, we adapted the phage display technique to select glutamine-donor substrates from a random heptapeptide library via binding to recombinant TG2 and elution with a synthetic amine-donor substrate. Twenty-six Gln-containing sequences from the second and third biopanning rounds were susceptible for TG2-mediated incorporation of 5-(biotinamido)penthylamine, and the peptides GQQQTPY, GLQQASV, and WQTPMNS were modified most efficiently. A consensus around glutamines was established as pQX(P,T,S)l, which is consistent with identified substrates listed in the TRANSDAB database. Database searches showed that several proteins contain peptides similar to the phage-selected sequences, and the N-terminal glutamine-rich domain of SWI1/SNF1-related chromatin remodeling proteins was chosen for detailed analysis. MALDI/TOF and tandem mass spectrometry-based studies of a representative part of the domain, SGYGQQGQTPYYNQQSPHPQQQQPPYS (SnQ1), revealed that Q(6), Q(8), and Q(22) are modified by TG2. Kinetic parameters of SnQ1 transamidation (K(M)(app) = 250 microM, k(cat) = 18.3 sec(-1), and k(cat)/K(M)(app) = 73,200) classify it as an efficient TG2 substrate. Circular dichroism spectra indicated that SnQ1 has a random coil conformation, supporting its accessibility in the full-length parental protein. Added together, here we report a novel use of the phage display technology with great potential in transglutaminase research.     

Advantages of mRNA display selections over other selection techniques for investigation of protein–protein interactions

mRNA display is a genotype–phenotype conjugation method that allows for amplification-based, iterative rounds of in vitro selection to be applied to peptides and proteins. mRNA display can be used to display both long natural protein and short synthetic peptide libraries with unusually high diversities for the investigation of protein–protein interactions. Here, we summarize the advantages of mRNA display by comparing it with other widely used peptide or protein-selection techniques, and discuss various applications of this technique in studying protein–protein interactions.     

S(1)' and S(2)' subsite specificities of human plasma kallikrein and tissue kallikrein 1 for the hydrolysis of peptides derived from the bradykinin domain of human kininogen

The S(1)' and S(2)' subsite specificities of human tissue kallikrein 1 (KLK1) and human plasma kallikrein (HPK) were examined with the peptide series Abz-GFSPFRXSRIQ-EDDnp and Abz-GFSPFRSXRIQ-EDDnp [X=natural amino acids or S(PO(3)H(2))]. KLK1 efficiently hydrolyzed most of the peptides except those containing negatively charged amino acids at P(1)' and P(2)' positions. Abz-GFSPFRSSRIQ-EDDnp, as in human kininogen, is the best substrate for KLK1 and exclusively cleaved the R-S bond. All other peptides were cleaved also at the F-R bond. The synthetic human kininogen segment Abz-MISLMKRPPGFSPFRS(390)S(391)RI-NH(2) was hydrolyzed by KLK1 first at R-S and then at M-K bonds, releasing Lys-bradykinin. In the S(390) and S(391) phosphorylated analogs, this order of hydrolysis was inverted due to the higher resistance of the R-S bond. Abz-MISLMKRPPG-FSPFRSS(PO(3)H(2))(391)RI-NH(2) was hydrolyzed by KLK1 at M-K and mainly at the F-R bond, releasing des-(Arg(9))-Lys-Bk which is a B1 receptor agonist. HPK cleaved all the peptides at R and showed restricted specificity for S in the S(1)' subsite, with lower specificity for the S(2)' subsite. Abz-MISLMKRPPGFSPFRSSRI-NH(2) was efficiently hydrolyzed by HPK under bradykinin release, while the analogs containing S(PO(3)H(2)) were poorly hydrolyzed. In conclusion, S(1)' and S(2)' subsite specificities of KLK1 and HPK showed peculiarities that were observed with substrates containing the amino acid sequence of human kininogen.     

Identification of the major TG4 cross‐linking sites in the androgen‐dependent SVS I exclusively expressed in mouse seminal vesicle

SVS I was exclusively expressed in seminal vesicle in which the protein was immunolocalized primarily to the luminal epithelium of mucosal folds. The developmental profile of its mRNA expression was shown to be androgen‐dependent, manifesting a positive correlation with the animal's maturation. There are 43 glutamine and 43 lysine residues in one molecule of SVS I, which is one of the seven major monomer proteins tentatively assigned on reducing SDS–PAGE during the resolution of mouse seminal vesicle secretion. Based on the fact that SVS I‐deduced protein sequence consists of 796 amino acid residues, we produced 7 recombinant polypeptide fragments including residues 1–78/F1, residues 79–259/F2, residues 260–405/F3, residues 406–500/F4, residues 501–650/F5, residues 651–715/F6, and residues 716–796/F7, and measured the covalent incorporation of 5‐(biotinamido)pentylamine (BPNH₂) or biotin‐TVQQEL (A25 peptide) to each of F1‐to‐F7 by type 4 transglutaminase (TG₄) from the coagulating gland secretion. F2 was active to a greater extent than the other fragments during the BPNH₂‐glutamine incorporation, and a relatively low extent of A25‐lysine cross link was observed with all of the seven fragments. The MS analysis of BPNH₂‐F2 conjugate identified Q²³² and Q²⁵⁴ as the two major TG₄ cross‐linking sites. This was substantiated by the result that much less BPNH₂ was cross‐linked to any one of the three F2 mutants, including Q232G and Q254G obtained from single‐site mutation, and Q232G/Q254G from double‐site mutation. J. Cell. Biochem. 107: 899–907, 2009. © 2009 Wiley‐Liss, Inc.     

A new direction for anticoagulants: Inhibiting fibrin assembly with PEGylated fibrin knob mimics

Current anticoagulants target coagulation factors upstream from fibrin assembly and polymerization (i.e., formation of fibrin clot). While effective, this approach requires constant patient monitoring since pharmacokinetics and pharmacodynamics vary from patient to patient. To address these limitations, we developed an alternative anticoagulant that effectively inhibits fibrin polymerization. Specifically, we investigated PEGylated fibrin knob “A” peptides, evaluating the effect of both polyethylene glycol (PEG) chain length (0, 2, 5, and 10–30 kDa) and knob peptide sequence (GPRPAAC, GPRPFPAC, and GPRPPERC) on inhibiting fibrin polymerization (i.e., clot formation). Thrombin‐initiated clotting assays with purified fibrinogen were performed to compare clot formation with each peptide–PEG conjugate. Results indicated a biphasic effect of PEG chain length, whereby, active‐PEG conjugates demonstrated increasingly enhanced inhibition of fibrin polymerization from 0 to 5 kDa PEG. However, the anticoagulant activity diminished to control levels for PEG chains above 5 kDa. Ultimately, we observed a 10‐fold enhancement of anticoagulant activity with active peptides PEGylated with 5 kDa PEG compared to non‐PEGylated knob peptides. The sequence of the active peptide significantly influenced the anticoagulant properties only at the highest 1:100 molar ratio where GPRPFPAC‐5 kDa PEG and GPRPPERC‐5 kDa PEG demonstrated significantly lower percent clottable protein than GPRPAAC‐5 kDa PEG. Moreover, human plasma treated with the active 5 kDa PEG conjugate exhibited delayed prothrombin time to within the therapeutic range specified for oral anticoagulants. Collectively, this study demonstrated the utility of PEGylated fibrin knob peptides as potential anticoagulant therapeutics. Biotechnol. Bioeng. 2011;108: 2424–2433. © 2011 Wiley Periodicals, Inc.     

运用 mRNA 体外展示技术筛选胸苷酸合成酶 RNA 亲和肽

以体外选择方法筛选不同功能的核酸、肽和蛋白质是近年的研究热点, mRNA 体外展示是一种新兴的高效多肽选择技术,其基本原理是通过含嘌呤霉素寡核苷酸的 Linker 使 mRNA 与它编码的肽或蛋白质共价结合,形成 mRNA- 蛋白质融合体,这一方法已用于多种功能肽的鉴定 . 以 mRNA 体外展示技术进行了由大容量多肽库中 (>1013) 筛选胸苷酸合成酶 (thymidylate synthase , TS) RNA 亲和肽的研究,通过精密的实验设计,建立了一套完整有效的筛选方法,并对实验条件进行了优化 . 已进行了 8 轮筛选,结果表明,以 mRNA 体外展示技术获得的多肽分子,可以与 TS mRNA 亲和 . 将测序结果与初始肽库进行比较,发现亲和肽中碱性氨基酸及芳香族氨基酸含量明显增加,说明其在与 RNA 结合中具有重要作用 . mRNA 展示技术作为一种大容量文库的体外筛选方法,将广泛应用于与固定化靶物质具高度亲和性及特异性的多肽和蛋白质的筛选 .     

A β1‐6/β1‐3 galactosidase from Bifidobacterium animalis subsp. lactis Bl‐04 gives insight into sub‐specificities of β‐galactoside catabolism within Bifidobacterium

The Bifidobacterium genus harbours several health promoting members of the gut microbiota. Bifidobacteria display metabolic specialization by preferentially utilizing dietary or host‐derived β‐galactosides. This study investigates the biochemistry and structure of a glycoside hydrolase family 42 (GH42) β‐galactosidase from the probiotic Bifidobacterium animalis subsp. lactis Bl‐04 (BlGal42A). BlGal42A displays a preference for undecorated β1‐6 and β1‐3 linked galactosides and populates a phylogenetic cluster with close bifidobacterial homologues implicated in the utilization of N‐acetyl substituted β1‐3 galactosides from human milk and mucin. A long loop containing an invariant tryptophan in GH42, proposed to bind substrate at subsite + 1, is identified here as specificity signature within this clade of bifidobacterial enzymes. Galactose binding at the subsite ? 1 of the active site induced conformational changes resulting in an extra polar interaction and the ordering of a flexible loop that narrows the active site. The amino acid sequence of this loop provides an additional specificity signature within this GH42 clade. The phylogenetic relatedness of enzymes targeting β1‐6 and β1‐3 galactosides likely reflects structural differences between these substrates and β1‐4 galactosides, containing an axial galactosidic bond. These data advance our molecular understanding of the evolution of sub‐specificities that support metabolic specialization in the gut niche.     

Mass spectral comparison of the neuropeptide complement of the stomatogastric ganglion and brain in the adult and embryonic lobster, Homarus americanus

Neuropeptides in the stomatogastric ganglion (STG) and the brain of adult and late embryonic Homarus americanus were compared using a multi-faceted mass spectral strategy. Overall, 29 neuropeptides from 10 families were identified in the brain and/or the STG of the lobster. Many of these neuropeptides are reported for the first time in the embryonic lobster. Neuropeptide extraction followed by liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry enabled confident identification of 24 previously characterized peptides in the adult brain and 13 peptides in the embryonic brain. Two novel peptides (QDLDHVFLRFa and GPPSLRLRFa) were de novo sequenced. In addition, a comparison of adult to embryonic brains revealed the presence of an incompletely processed form of Cancer borealis tachykinin-related peptide 1a (CabTRP 1a, APSGFLGMRG) only in the embryonic brain. A comparison of adult to embryonic STGs revealed that QDLDHVFLRFa was present in the embryonic STG but absent in the adult STG, and CabTRP 1a exhibited the opposite trend. Relative quantification of neuropeptides in the STG revealed that three orcokinin family peptides (NFDEIDRSGFGF, NFDEIDRSGFGFV, and NFDEIDRSGFGFN), a B-type allatostatin (STNWSSLRSAWa), and an orcomyotropin-related peptide (FDAFTTGFGHS) exhibited higher signal intensities in the adult relative to the embryonic STG. RFamide (Arg-Phe-amide) family peptide (DTSTPALRLRFa), [Val¹]SIFamide (VYRKPPFNGSIFa), and orcokinin-related peptide (VYGPRDIANLY) were more intense in the embryonic STG spectra than in the adult STG spectra. Collectively, this study expands our current knowledge of the H. americanus neuropeptidome and highlights some intriguing expression differences that occur during development.     

The acylaminoacyl peptidase from Aeropyrum pernix K1 thought to be an exopeptidase displays endopeptidase activity

Mammalian acylaminoacyl peptidase, a member of the prolyl oligopeptidase family of serine peptidases, is an exopeptidase, which removes acylated amino acid residues from the N terminus of oligopeptides. We have investigated the kinetics and inhibitor binding of the orthologous acylaminoacyl peptidase from the thermophile Aeropyrum pernix K1 (ApAAP). Complex pH-rate profiles were found with charged substrates, indicating a strong electrostatic effect in the surroundings of the active site. Unexpectedly, we have found that oligopeptides can be hydrolysed beyond the N-terminal peptide bond, demonstrating that ApAAP exhibits endopeptidase activity. It was thought that the enzyme is specific for hydrophobic amino acids, in particular phenylalanine, in accord with the non-polar S1 subsite of ApAAP. However, cleavage after an Ala residue contradicted this notion and demonstrated that P1 residues of different nature may bind to the S1 subsite depending on the remaining peptide residues. The crystal structures of the complexes formed between the enzyme and product-like inhibitors identified the oxyanion-binding site unambiguously and demonstrated that the phenylalanine ring of the P1 peptide residue assumes a position different from that established in a previous study, using 4-nitrophenylphosphate. We have found that the substrate-binding site extends beyond the S2 subsite, being capable of binding peptides with a longer N terminus. The S2 subsite displays a non-polar character, which is unique among the enzymes of this family. The S3 site was identified as a hydrophobic region that does not form hydrogen bonds with the inhibitor P3 residue. The enzyme-inhibitor complexes revealed that, upon ligand-binding, the S1 subsite undergoes significant conformational changes, demonstrating the plasticity of the specificity site.     

Interaction structure of the complex between neuroprotective factor humanin and Alzheimer's β‐amyloid peptide revealed by affinity mass spectrometry and molecular modeling

Humanin (HN) is a linear 24‐aa peptide recently detected in human Alzheimer's disease (AD) brain. HN specifically inhibits neuronal cell death in vitro induced by ß‐amyloid (Aß) peptides and by amyloid precursor protein and its gene mutations in familial AD, thereby representing a potential therapeutic lead structure for AD; however, its molecular mechanism of action is not well understood. We report here the identification of the binding epitopes between HN and Aß(1–40) and characterization of the interaction structure through a molecular modeling study. Wild‐type HN and HN‐sequence mutations were synthesized by SPPS and the HPLC‐purified peptides characterized by MALDI‐MS. The interaction epitopes between HN and Aß(1–40) were identified by affinity‐MS using proteolytic epitope excision and extraction, followed by elution and mass spectrometric characterization of the affinity‐bound peptides. The affinity‐MS analyses revealed HN(5–15) as the epitope sequence of HN, whereas Aß(17–28) was identified as the Aß interaction epitope. The epitopes and binding sites were ascertained by ELISA of the complex of HN peptides with immobilized Aß(1–40) and by ELISA with Aß(1–40) and Aß‐partial sequences as ligands to immobilized HN. The specificity and affinity of the HN‐Aß interaction were characterized by direct ESI‐MS of the HN‐Aß(1–40) complex and by bioaffinity analysis using a surface acoustic wave biosensor, providing a K_D of the complex of 610 n m . A molecular dynamics simulation of the HN‐Aß(1–40) complex was consistent with the binding specificity and shielding effects of the HN and Aß interaction epitopes. These results indicate a specific strong association of HN and Aß(1–40) polypeptide and provide a molecular basis for understanding the neuroprotective function of HN. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

In vitro selection of state-specific peptide modulators of G protein signaling using mRNA display

The G protein regulatory (GPR) motif is a approximately 20-residue conserved domain that acts as a guanine dissociation inhibitor (GDI) for G(i/o)(alpha) subunits. Here, we describe the isolation of peptides derived from a GPR consensus sequence using mRNA display selection libraries. Biotinylated G(i)(alpha)(1), modified at either the N or C terminus, serves as a high-affinity binding target for mRNA-displayed GPR peptides. In vitro selection using mRNA display libraries based on the C terminus of the GPR motif revealed novel peptide sequences with conserved residues. Surprisingly, selected peptides contain mutations to a highly conserved Arg in the GPR motif, previously shown to be crucial for binding and inhibition activities. The dominant peptide from the selection, R6A, and a minimal 9-mer peptide, R6A-1, do not contain Arg residues yet retain high affinity (K(D) = 60 and 200 nM, respectively) and specificity for the GDP-bound state of G(i)(alpha)(1), as measured by surface plasmon resonance. The selected peptides also maintain GDI activity for G(i)(alpha)(1), inhibiting both the exchange of GDP in GTPgammaS binding assays and the AlF(4)(-)-stimulated enhancement of intrinsic tryptophan fluorescence. The kinetics of GDI activity, however, are different for the selected peptides and demonstrate biphasic kinetics, suggesting a complex mechanism for inhibition. Like the GPR motif, the R6A and R6A-1 peptides compete with G(betagamma) subunits for binding to G(i)(alpha)(1), suggesting their use as activators of G(betagamma) signaling.