Synthetic Peptide Combinatorial Library Research Articles

Non-prime- and prime-side profiling of Pro-Pro endopeptidase specificity using synthetic combinatorial peptide libraries and mass spectrometry.

A group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), possess the unique ability to hydrolyze proline-proline bonds in proteins. Since a protease's function is largely determined by its substrate specificity, methods that can extensively characterize substrate specificity are valuable tools for protease research. Previously, we achieved an in-depth characterization of PPEP prime-side specificity. However, PPEP specificity is also determined by the non-prime-side residues in the substrate. To gain a more complete insight into the determinants of PPEP specificity, we characterized the non-prime- and prime-side specificity of various PPEPs using a combination of synthetic combinatorial peptide libraries and mass spectrometry. With this approach, we deepened our understanding of the P3-P3' specificities of PPEP-1 and PPEP-2, while identifying the endogenous substrate of PPEP-2 as the most optimal substrate in our library data. Furthermore, by employing the library approach, we investigated the altered specificity of mutants of PPEP-1 and PPEP-2. Additionally, we characterized a novel PPEP from Anoxybacillus tepidamans, which we termed PPEP-4. Based on structural comparisons, we hypothesized that PPEP-4 displays a PPEP-1-like prime-side specificity, which was substantiated by the experimental data. Intriguingly, another putative PPEP from Clostridioides difficile, CD1597, did not display Pro-Pro endoproteolytic activity. Collectively, we characterized PPEP specificity in detail using our robust peptide library method and, together with additional structural information, provide more insight into the intricate mechanisms that govern protease specificity.

Early Selection of the Amino Acid Alphabet Was Adaptively Shaped by Biophysical Constraints of Foldability.

Whereas modern proteins rely on a quasi-universal repertoire of 20 canonical amino acids (AAs), numerous lines of evidence suggest that ancient proteins relied on a limited alphabet of 10 "early" AAs and that the 10 "late" AAs were products of biosynthetic pathways. However, many nonproteinogenic AAs were also prebiotically available, which begs two fundamental questions: Why do we have the current modern amino acid alphabet and would proteins be able to fold into globular structures as well if different amino acids comprised the genetic code? Here, we experimentally evaluate the solubility and secondary structure propensities of several prebiotically relevant amino acids in the context of synthetic combinatorial 25-mer peptide libraries. The most prebiotically abundant linear aliphatic and basic residues were incorporated along with or in place of other early amino acids to explore these alternative sequence spaces. The results show that foldability was likely a critical factor in the selection of the canonical alphabet. Unbranched aliphatic amino acids were purged from the proteinogenic alphabet despite their high prebiotic abundance because they generate polypeptides that are oversolubilized and have low packing efficiency. Surprisingly, we find that the inclusion of a short-chain basic amino acid also decreases polypeptides' secondary structure potential, for which we suggest a biophysical model. Our results support the view that, despite lacking basic residues, the early canonical alphabet was remarkably adaptive at supporting protein folding and explain why basic residues were only incorporated at a later stage of protein evolution.

A complete search of combinatorial peptide library greatly benefited from probabilistic incorporation of prior knowledge

The core of peptide detection in tandem mass spectrometry lies in associating fragment spectra with promising peptide candidates. We examined such detection in a synthetic combinatorial peptide library using four scoring metrics, against all theoretical peptides, and with a varying level of probabilistic prior knowledge—analyzing more than a trillion peptide-spectrum matches in total. Even after adjusting for peptide-length scoring bias, most MS/MS spectra had multiple at-least-as-good candidates as the correct peptide, showing that the highest spectral match was not a guarantee of correctness. As a remedy, we probabilistically integrated prior knowledge about expected cleavage behavior and expected peptide sequences into peptide scoring, reaching and even overcoming the performance of state-of-the-art de novo sequencing algorithms. Overall, we found that even partial and weak beliefs considerably improved peptide detection and are, in principle, generally applicable to any detection approach. Detection of peptides in a complete search thus often resulted in multiple admissible candidates near the maximal score, and the use of probabilistic prior knowledge substantially improved their discrimination.

Picomolar Affinity Antagonist and Sustained Signaling Agonist Peptide Ligands for the Adrenomedullin and Calcitonin Gene-Related Peptide Receptors.

The calcitonin receptor-like class B G protein-coupled receptor (CLR) mediates adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) functions including vasodilation, cardioprotection, and nociception. Receptor activity-modifying proteins (RAMP1-3) form heterodimers with CLR and determine its peptide ligand selectivity through an unresolved mechanism. The CGRP (RAMP1:CLR) and AM (RAMP2/3:CLR) receptors are proven or promising drug targets, but short AM and CGRP plasma half-lives limit their therapeutic utility. Here, we used synthetic peptide combinatorial library and rational design approaches to probe the ligand selectivity determinants and develop truncated AM and CGRP antagonist variants with receptor extracellular domain binding affinities that were enhanced ∼1000-fold into the low nanomolar range. Receptor binding studies and a high-resolution crystal structure of a novel library-identified AM variant bound to the RAMP2-CLR extracellular domain complex explained the increased affinities and defined roles for AM Lys46 and RAMP modulation of CLR conformation in the ligand selectivity mechanism. In longer AM and CGRP scaffolds that also bind the CLR transmembrane domain, the variants generated picomolar affinity antagonists, one with an estimated 12.5 h CGRP receptor residence time, and sustained signaling agonists "ss-AM" and "ss-CGRP" that exhibited persistent cAMP signaling after ligand washout. Sustained signaling was demonstrated in primary human umbilical vein endothelial cells and the SK-N-MC cell line, which endogenously express AM and CGRP receptors, respectively. This work clarifies the RAMP-modulated CLR ligand selectivity mechanism and provides AM and CGRP variants that are valuable pharmacological tools and may have potential as long-acting therapeutics.

Development of Picomolar Affinity Antagonists and Long‐acting Agonists for the Adrenomedullin and CGRP Receptors Using Combinatorial Peptide Library and Structure‐guided Design Approaches

Adrenomedullin (AM) and calcitonin gene‐related peptide (CGRP) have diverse functions in the cardiovascular, lymphatic, and nervous systems including regulation of vascular tone, cardioprotection, and pain transmission. These peptides signal through three heterodimeric receptors comprising the class B GPCR, the calcitonin receptor‐like receptor (CLR), and one of three receptor activity‐modifying proteins (RAMP1‐3) that determine ligand selectivity. CLR‐RAMP complexes are proven or promising drug targets for several diseases, however, the moderate binding affinities and short plasma half‐lives of AM and CGRP limit their utility as therapeutics. Here, we used synthetic peptide combinatorial library and rational structure‐guided design approaches to develop AM and CGRP variant peptides with dramatically enhanced receptor affinities. The binding of the variants to purified RAMP‐CLR extracellular domain (ECD) complexes was characterized and a high‐resolution crystal structure of an AM variant bound to RAMP2‐CLR ECD explained its enhanced affinity. Incorporation of the variants into different length AM and CGRP scaffolds yielded picomolar affinity antagonists or agonists that exhibited substantially prolonged cAMP signaling after ligand washout. These activities were demonstrated in a model cell line transiently expressing the receptors and in primary human cells or immortalized cell lines that naturally express the receptors. This work provides a suite of AM and CGRP variants that will be valuable pharmacological tools and may have promise as peptide therapeutics with long receptor residence times. In addition, the properties of the variants support a mechanism for RAMP function in which direct RAMP‐peptide contacts and allosteric modulation of CLR cooperate to determine ligand selectivity.Support or Funding InformationThis work was supported by grants NIH R01GM104251 and OCAST HR16‐005 (AAP) and AHA predoctoral fellowship 18PRE33990152 (JMB).

Identification of small peptides and glycinamide that inhibit melanin synthesis using a positional scanning synthetic peptide combinatorial library.

Antimelanogenic peptides are potentially useful to treat hyperpigmentation, but many peptides have limited application because of high cost and/or low activity. To identify small and potent peptide inhibitors of cellular melanin synthesis that are useful for cosmetic and medical applications. A positional scanning synthetic tetrapeptide combinatorial library was used for screening of potentially active peptides. Antimelanogenic activities of the peptide pools and individual peptides were evaluated in B16-F10 melanoma cells and human epidermal melanocytes treated with alpha-melanocyte-stimulating hormone (α-MSH). Predicted active tetrapeptide sequences were R-(F/L)-(C/W)-(G/R)-NH2 . Of the individual tetrapeptides tested, D3 (RFWG-NH2 ) and D5 (RLWG-NH2 ) exhibited high antimelanogenic activities. Tetrapeptide D9 (FRWG-NH2 ) with a sequence identical to that of a portion of α-MSH also showed antimelanogenic activity. Of the tripeptides tested, E5 (FWG-NH2 ), E6 (LWG-NH2 ) and E7 (RWG-NH2 ) were relatively more active. Dipeptide F1 (WG-NH2 ) and monopeptide G1 (G-NH2 , glycinamide) retained activity, but G2 (Ac-G-NH2 ) and G3 (glycine) did not. The antimelanogenic activities of peptides D3, E5, F1 and G1 were verified in α-MSH-stimulated human epidermal melanocytes. Commercially available G-NH2 ·HCl suppressed the phosphorylation levels of cAMP-responsive element binding protein, protein levels of microphthalmia-associated transcription factor and tyrosinase, l-tyrosine hydroxylase activity of tyrosinase, and the melanin levels in stimulated cells. Small peptides, including glycinamide and tryptophanyl glycinamide, are potent antimelanogenic agents with potential value for the treatment of skin hyperpigmentation.

Exploring the chemical space of peptides for drug discovery: a focus on linear and cyclic penta-peptides.

Peptide and peptide-like structures are regaining attention in drug discovery. Previous studies suggest that bioactive peptides have diverse structures and may have physicochemical properties attractive to become hit and lead compounds. However, chemoinformatic studies that characterize such diversity are limited. Herein, we report the physicochemical property profile and chemical space of four synthetic linear and cyclic combinatorial peptide libraries. As a case study, the analysis was focused on penta-peptides. The chemical space of the peptide and N-methylated peptides libraries was compared to compound data sets of pharmaceutical relevance. Results indicated that there is a major overlap in the chemical space of N-methylated cyclic peptides with inhibitors of protein-protein interactions and macrocyclic natural products available for screening. Also, there is an overlap between the chemical space of the synthetic peptides with peptides approved for clinical use (or in clinical trials), and to other approved drugs that are outside the traditional chemical space. Results further support that synthetic penta-peptides are suitable compounds to be used in drug discovery projects.

Abstract 207: Reliable identification of mutations in bottom-up proteomics

Abstract Knowledge of missense mutations is of significant biological importance as it provides valuable insights into alterations of phenotype. While identification of reference proteins is routinely performed in proteomics, identification of mutations remains still fairly uncommon. Well-established methods for reference peptides are generally insufficient for altered ones; e.g., utilizing mutant-augmented database search usually results in inadmissible proportion of false positives. This undesirable situation is, however, rather natural consequence of peptide fragmentation, its computational modelling and evaluation of their correspondence. Often, there are many peptides having the same or similar agreement with acquired spectrum, thereby preventing agreement-based decision. These situations happen frequently in identification of mutant peptides (e.g., homologous peptides with PTMs, semi-specific peptides), therefore their reliable identification requires additional treatment. To deeper understand properties of identification, we have formally studied generalized version of the identification problem and derived its optimal solution under particular assumptions. In proteomics, the selection of optimal solution can be always found in finite time; moreover, the strategy is straightforward to implement, enables parallelization and provides guarantees over claimed interpretations. The solution utilizes almost exclusively spectral data of product ions without additional LC/MS information. In practice, however, it is beneficial to employ precursor isotopic distribution analysis for correction of non-monoisotopic selection of precursor as that would otherwise systematically result in artefacts. The behaviour of proposed method was validated in a variety of scenarios that could be broadly categorized as direct (known spectral content) and indirect (partial knowledge of sample content). For direct validation, synthetic combinatorial peptide library of 400 peptides (all coded amino acids in 12. and 13. codon of KRAS peptide) was measured and interpreted; the sensitivity and specificity obtained was 0.81 and 0.99 respectively. Indirect validation was, at first, performed on in-house samples with RNA-Seq data available (HCT116), giving 97 missense RNA-Seq supported mutations. Similarly, the analysis was done on proteome data of NCI60 cell lines, resulting on average in 23 SNVs with maximum of 56 SNVs (HCC-2998). Identified alterations correspond, in general, to highly expressed genes. In summary, the approach was shown to reliably identify missense mutations in a range of cancer cell lines; its wide applicability enhances interpretation of standard bottom-up proteomics data. Citation Format: Miroslav Hruska, Lakshman Varanasi, Jiri Voller, Petr Dzubak, Marian Hajduch. Reliable identification of mutations in bottom-up proteomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 207. doi:10.1158/1538-7445.AM2017-207

Selection of Small Synthetic Antimicrobial Peptides Inhibiting Xanthomonas citri subsp. citri Causing Citrus Canker.

Citrus canker disease decreases the fruit quality and yield significantly, furthermore, emerging of streptomycin-resistant pathogens threatens the citrus industry seriously because of a lack of proper control agents. Small synthetic antimicrobial peptides (AMPs) could be a promising alternative. Fourteen hexapeptides were selected by using positional scanning of synthetic peptide combinatorial libraries. Each hexapeptide showed different antimicrobial spectrum against Bacillus, Pseudomonas, Xanthomonas, and Candida species. Intriguingly, BHC10 showed bactericidal activity exclusively on Xanthomonas citri subsp. citri (Xcc), while BHC7 was none-active exclusively against two Pseudomonas spp. at concentration of 100 μg/ml suggesting potential selectivity constrained in hexapeptide frame. Three hexapeptides, BHC02, 06 and 11, showed bactericidal activities against various Xcc strains at concentration of 10 μg/ml. When they were co-infiltrated with pathogens into citrus leaves the disease progress was suppressed significantly. Further study would be needed to confirm the actual disease control capacity of the selected hexapeptides.

159 - Identification of Antimelanogenic Peptides through Positional Scanning of a Synthetic Peptide Combinatorial Library

Antimelanogenic peptides are potentially useful as cosmeceutical ingredients in skin whitening agents. The purpose of this study was to identify peptide inhibitors of cellular melanogenesis through positional scanning of a synthetic peptide combinatorial library (PS-SPCL). A synthetic hexapeptide combinatorial library consisted of 6 positional sub-libraries, namely OXXXXX, XOXXXX, XXOXXX, XXXOXX, XXXXOX, and XXXXXO, where O positions are any one of the 19 amino acids excluding cysteine, and other X positions are equimolar mixtures of the 19 amino acids. The antimelanogenic effects of peptide mixtures were evaluated in murine melanoma B16-F10 cells stimulated with α-melanocyte-stimulating hormone (α-MSH) to identify the sequence of antimelanogenic hexapeptides. PS-SPCL identified the active hexapeptide sequences as (F/I)(N/S)(D/H/N)(H/N)L(G/F)-NH 2 . Of the hexapeptide mixtures and individual hexapeptides evaluated, the strongest antimelanogenic activity was seen with hexapeptide B6 (FSHHLG-NH 2 ). Hexapeptide B6 decreased melanin content and levels of melanogenic enzymes in B16-F10 cells stimulated with α-MSH. Additionally, it decreased melanin content in human epidermal melanocytes stimulated with L-tyrosine. The present study identified novel antimelanogenic hexapeptides that might be useful for controlling skin pigmentation.

A novel brain-derived neurotrophic factor-modulating peptide attenuates Aβ1-42-induced neurotoxicity in vitro

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family, which plays important roles in learning and memory formation and in protecting neurons from diverse neurotoxic insults, such as amyloid-beta (Aβ). Since BDNF expression is decreased in patients with Alzheimer’s disease, various strategies have attempted to increase BDNF levels. In a previous study, we screened and identified a novel BDNF-modulating peptide (consisting of methionine–valine–glycine, named Neuropep-1) by a positional scanning–synthetic peptide combinatorial library (PS–SPCL). Neuropep-1 exhibited neuroprotective effects against in vitro and in vivo Alzheimer’s disease models. Based on the previous PS–SPCL data, we modified the amino acid sequence of Neuropep-1 in this study to identify a more potent novel BDNF-modulating peptide. By replacing the valine in the second position with aspartic acid, the resulting Neuropep-4 was found to be highly effective in inducing BDNF expression even at concentrations of 1pM in the SH–SY5Y cell line and rat primary cortical neurons. In addition, among the tested peptides, Neuropep-4 provided neurons with the strongest protection against oligomeric and/or fibrillar Aβ1-42-induced cell death through BDNF upregulation. These results suggest the potential of Neuropep-4 as a therapeutic candidate for treating neurodegenerative diseases, such as AD.

Identification of Antimicrobial Peptide Hexamers against Oral Pathogens through Rapid Screening of a Synthetic Combinatorial Peptide Library

Identification of Antimicrobial Peptide Hexamers against Oral Pathogens through Rapid Screening of a Synthetic Combinatorial Peptide Library

Screening of a synthetic peptide combinatorial library to identify inhibitors of the appressorium formation in Magnaporthe oryzae

The rice blast disease caused by Magnaporthe oryzae is one of the most devastating diseases of cultivated rice. One of the most important stages in the infective cycle of M. oryzae is the formation of the dome-shaped structure called appressorium. The purpose of the present study was to identify novel peptides to control the rice blast disease by blocking the appressorium formation through screening of a synthetic peptide combinatorial library. As result of the screening, a set of 29 putative bioactive peptides were identified, synthesized and assayed in comparison with the previously identified peptide PAF104. The peptides MgAPI24, MgAPI40 and MgAPI47 showed improved inhibitory activity on the M. oryzae appressorium formation. Our data show that these peptides have a differential effect on two developmental structures: appressoria and appressorium-like structures. Antimicrobial assays against M. oryzae and other non-target microorganisms showed a weak or no toxicity of these peptides, demonstrating their specific activity blocking the appressorium formation. Therefore, the outcome of this research would be useful in the development of novel target-oriented peptides to use in plant protection.

Methodological Approach to Identify and Isolate Disease Relevant Brain-infiltrating T Cell Clones in Multiple Sclerosis Patients

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system considered to be mediated by T cells. Two main approaches have been employed to study pathogenic T cells in MS in the past. The first approach focused on the functional phenotype of peripheral blood T cells, which were generated with myelin proteins, but for which a pathogenic role was otherwise unclear. Based on the ‘skewed’ TCR repertoire of brain infiltrating T cells, the second approach focused on clonally expanded T cells identified by expressing particular TCRs. This second approach is not biased by assumptions about autoantigens and considers that clonally expanded T cells are probably disease relevant. However, the main limitation in the few reported studies was the inability to further characterize the functional phenotype and specificity of infiltrating T cells since only frozen autopsy tissue was available. At least one group currently attempts to “revive” brain-infiltrating T cells by expressing TCR alpha and beta chains in recombinant systems that may allow to dissect the specificity but most likely not the functional phenotype of the original T cell clones (TCC). Here we present a new strategy consisting in a) identifying clonally expanded, brain-infiltrating T cells from autopsy tissue of a MS patient, who died after very aggressive disease course and b) subsequent isolation of the relevant TCC from the patient’s cerebrospinal fluid (CSF). An actively demyelinating lesion based on reduced density of myelinated fibers and myelin degradation products within infiltrating macrophages was identified. Using deep sequencing approaches, we sequenced the TCR variable beta (V-beta) chains expressed by T cells infiltrating this lesion. We identified 312 different lesion-infiltrating TCC. Both CD4 (32%) and CD8 (68%) TCC were present and clonally expanded with frequencies up to 30%. In a following step, we were able to isolate 14 of the most frequent CD4+ and CD8+ TCC using as a cell source in vitro PHA-expanded CSF T cells. T cells expressing V-beta families of interest were sorted and cloned by limiting dilution. These TCC are currently being characterized for functional phenotype and antigen specificity by using a combination of two complementary unbiased methods, synthetic combinatorial peptide libraries and an autologous brain-derived cDNA library, which is expressed in suitable antigen presenting cells.

Neuropep-1 ameliorates learning and memory deficits in an Alzheimer's disease mouse model, increases brain-derived neurotrophic factor expression in the brain, and causes reduction of amyloid beta plaques

Alzheimer's disease (AD) is a neurodegenerative disease characterized by amyloid beta (Aβ) deposits, hyperphosphorylated tau deposition, and cognitive dysfunction. Abnormalities in the expression of brain-derived neurotrophic factor (BDNF), which plays an important role in learning and memory formation, have been reported in the brains of AD patients. A BDNF modulating peptide (Neuropep-1) was previously identified by positional-scanning synthetic peptide combinatorial library. Here we examine the neuroprotective effects of Neuropep-1 on several in vitro neurotoxic insults, and triple-transgenic AD mouse model (3xTg-AD). Neuropep-1 protects cultured neurons against oligomeric Aβ1–42, 1-methyl-4-phenylpyridinium, and glutamate-induced neuronal cell death. Neuropep-1 injection also significantly rescues the spatial learning and memory deficits of 3xTg-AD mice compared with vehicle-treated control group. Neuropep-1 treatment markedly increases hippocampal and cortical BDNF levels. Furthermore, we found that Neuropep-1-injected 3xTg-AD mice exhibit dramatically reduced Aβ plaque deposition and Aβ levels without affecting tau pathology. Neuropep-1 treatment does not alter the expression or activity of full-length amyloid precursor protein, α-, β-, or γ-secretase, but levels of insulin degrading enzyme, an Aβ degrading enzyme, were increased. These findings suggest Neuropep-1 may be a therapeutic candidate for the treatment of AD.

138 Use of CEACAM5 Synthetic Peptide Combinatorial Library to Characterize Binding of CEACAM5 to CD8α

138 Use of CEACAM5 Synthetic Peptide Combinatorial Library to Characterize Binding of CEACAM5 to CD8α

Identification of B cell and T cell epitopes using synthetic peptide combinatorial libraries.

This unit presents a combinatorial library method that consists of the synthesis and screening of mixture-based synthetic combinatorial libraries of peptide molecules. The protocols employ peptide libraries to identify peptides recognized by MAbs and T cells. The first protocol uses a positional scanning peptide library made up of hexapeptides to identify antigenic determinants recognized by MAbs. The 120 mixtures in the hexapeptide library are tested for their inhibitory activity in a competitive ELISA. The second protocol uses a decapeptide library to identify T cell peptide ligands. The 200 mixtures of the decapeptide library are tested for their ability to induce T cell activation. Support protocols cover optimization of the assay conditions for each MAb or T cell, to achieve the best level of sensitivity and reproducibility, and preparation of a hexapeptide library, along with deconvolution approaches.

The Safety Evaluation of a Potent Angiogenic Activator, Synthetic Peptide (SFKLRY-NH2) for the Skin Application

A novel synthetic hexapeptide (SFKLRY-NH2) that displays angiogenic activity has been identified by positional scanning of a synthetic peptide combinatorial library (PS-SPCL). This study was carried out to investigate the irritation of the SFKLRY-NH2 on the skin. The tests were performed on the basis of Korea Food and Drug Administration (KFDA) guidelines. In results, cell toxicity is not appeared for SFKLRY-NH2 in HaCaT cells and B16F10 cells. SFKLRY-NH2 induced no skin irritation at low concentration (10 μM), mild irritation at high concentration (10mM). We consider that this result is helpful for saying about the safety of SFKLRY-NH2 in clinical use.

Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides

BackgroundHemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins.ResultsAn aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus) microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1'.ConclusionsBmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.

Pericellular activation of hepatocyte growth factor by the transmembrane serine proteases matriptase and hepsin, but not by the membrane-associated protease uPA

HGF (hepatocyte growth factor) is a pleiotropic cytokine homologous to the serine protease zymogen plasminogen that requires canonical proteolytic cleavage to gain functional activity. The activating proteases are key components of its regulation, but controversy surrounds their identity. Using quantitative analysis we found no evidence for activation by uPA (urokinase plasminogen activator), despite reports that this is a principal activator of pro-HGF. This was unaffected by a wide range of experimental conditions, including the use of various molecular forms of both HGF and uPA, and the presence of uPAR (uPA receptor) or heparin. In contrast the catalytic domains of the TTSPs (type-II transmembrane serine proteases) matriptase and hepsin were highly efficient activators (50% activation at 0.1 and 3.4 nM respectively), at least four orders of magnitude more efficient than uPA. PS-SCL (positional-scanning synthetic combinatorial peptide libraries) were used to identify consensus sequences for the TTSPs, which in the case of hepsin corresponded to the pro-HGF activation sequence, demonstrating a high specificity for this reaction. Both TTSPs were also found to be efficient activators at the cell surface. Activation of pro-HGF by PC3 prostate carcinoma cells was abolished by both protease inhibition and matriptase-targeting siRNA (small interfering RNA), and scattering of MDCK (Madin-Darby canine kidney) cells in the presence of pro-HGF was abolished by inhibition of matriptase. Hepsin-transfected HEK (human embryonic kidney)-293 cells also activated pro-HGF. These observations demonstrate that, in contrast with the uPA/uPAR system, the TTSPs matriptase and hepsin are direct pericellular activators of pro-HGF, and that together these proteins may form a pathway contributing to their involvement in pathological situations, including cancer.