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1
Hao, Yue, Elizabeth Pierce, Daniel Roe, Maho Morita, John A. McIntosh, Vinayak Agarwal, Thomas E. Cheatham, Eric W. Schmidt, and Satish K. Nair. "Molecular basis for the broad substrate selectivity of a peptide prenyltransferase." Proceedings of the National Academy of Sciences 113, no. 49 (November 21, 2016): 14037–42. http://dx.doi.org/10.1073/pnas.1609869113.
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The cyanobactin prenyltransferases catalyze a series of known or unprecedented reactions on millions of different substrates, with no easily observable recognition motif and exquisite regioselectivity. Here we define the basis of broad substrate tolerance for the otherwise uncharacterized TruF family. We determined the structures of the Tyr-prenylating enzyme PagF, in complex with an isoprenoid donor analog and a panel of linear and macrocyclic peptide substrates. Unexpectedly, the structures reveal a truncated barrel fold, wherein binding of large peptide substrates is necessary to complete a solvent-exposed hydrophobic pocket to form the catalytically competent active site. Kinetic, mutational, chemical, and computational analyses revealed the structural basis of selectivity, showing a small motif within peptide substrates that is sufficient for recognition by the enzyme. Attaching this 2-residue motif to two random peptides results in their isoprenylation by PagF, demonstrating utility as a general biocatalytic platform for modifications on any peptide substrate.
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Jabaiah, Abeer M., Jennifer A. Getz, Witold A. Witkowski, Jeanne A. Hardy, and Patrick S. Daugherty. "Identifi cation of protease exosite-interacting peptides that enhance substrate cleavage kinetics." Biological Chemistry 393, no. 9 (September 1, 2012): 933–41. http://dx.doi.org/10.1515/hsz-2012-0162.
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Abstract Many peptidases are thought to require non-active site interaction surfaces, or exosites, to recognize and cleave physiological substrates with high specifi city and catalytic effi ciency. However, the existence and function of protease exosites remain obscure owing to a lack of effective methods to identify and characterize exosite-interacting substrates. To address this need, we modifi ed the cellular libraries of peptide substrates (CLiPS) methodology to enable the discovery of exosite-interacting peptide ligands. Invariant cleavage motifs recognized by the active sites of thrombin and caspase-7 were displayed on the outer surface of bacteria adjacent to a candidate exosite-interacting peptide. Exosite peptide libraries were then screened for ligands that accelerate cleavage of the active site recognition motif using two-color fl ow cytometry. Exosite CLiPS (eCLiPS) identifi ed exosite-binding peptides for thrombin that were highly similar to a critical exosite interaction motif in the thrombin substrate, proteaseactivated receptor 1. Protease activity probes incorporating exosite-binding peptides were cleaved ten-fold faster than substrates without exosite ligands, increasing their sensitivity to thrombin activity in vitro. For comparison, screening with caspase-7 yielded peptides that modestly enhanced (two-fold) substrate cleavage rates. The eCLiPS method provides a new tool to profi le the ligand specifi city of protease exosites and to develop improved substrates.
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Masler, E. P. "Aminopeptidases inCaenorhabditis elegansandPanagrellus redivivus: detection using peptide and non-peptide substrates." Journal of Helminthology 76, no. 1 (March 2002): 45–52. http://dx.doi.org/10.1079/joh200193.
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AbstractAminopeptidase activities were detected in extracts of the free-living nematodesCaenorhabditis elegansandPanagrellus redivivususing the aminoacyl substrate L-alanine-4-nitroanilide. The activities exhibited similarities in Km (C.elegans= 2.22 mM;P.REDIVIVUS= 2.09 Mm) and specific activity (C.elegans=1.38±0.43 mAU min-1 μg-1;P. redivivus, 1.23±0.18 mAU min-1 μg-1). Each is inhibited competitively by amastatin (C. elegansIC50=0.46 μm;P. redivivusIC50=15.90 μm) and non-competitively by leuhistin (C. elegansIC50=3.00 μm;P. redivivusIC50=37.35 μm). The bioactive peptides adipokinetic hormone and substance P decrease the apparent aminopeptidase activities of each extract suggesting that the peptides compete with the Ala-pNA as substrates. With each extract, adipokinetic hormone appeared to be the more effective substrate. Digestion of adipokinetic hormone byC. elegansandP. redivivusextracts in the presence and absence of 1 mm amastatin produced distinct chromatographic profiles that suggest different digestion patterns for the two species. However, amastatin had clear effects on chromatographic profiles from each species indicating that an aminopeptidase is involved in the digestion of the peptide substrates. The data presented indicate that extracts of free-living nematodes are capable of metabolizing peptide hormones, and that this metabolism involves substrate-selective aminopeptidases.
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Rut, Wioletta, and Marcin Drag. "Human 20S proteasome activity towards fluorogenic peptides of various chain lengths." Biological Chemistry 397, no. 9 (September 1, 2016): 921–26. http://dx.doi.org/10.1515/hsz-2016-0176.
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Abstract The proteasome is a multicatalytic protease responsible for the degradation of misfolded proteins. We have synthesized fluorogenic substrates in which the peptide chain was systematically elongated from two to six amino acids and evaluated the effect of peptide length on all three catalytic activities of human 20S proteasome. In the cases of five- and six-membered peptides, we have also synthesized libraries of fluorogenic substrates. Kinetic analysis revealed that six-amino-acid substrates are significantly better for chymotrypsin-like and caspase-like activity than shorter peptidic substrates. In the case of trypsin-like activity, a five-amino-acid substrate was optimal.
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TOOMIK, Reet, and Pia EK. "A potent and highly selective peptide substrate for protein kinase C assay." Biochemical Journal 322, no. 2 (March 1, 1997): 455–60. http://dx.doi.org/10.1042/bj3220455.
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Protein kinases exhibit substrate specificities that are often primarily determined by the amino acids around the phosphorylation sites. Peptides corresponding to protein kinase C phosphorylation sites in several different proteins were synthesized on SPOTs membrane which has recently been found to be applicable for studies of protein kinase specificity. After phosphorylation with protein kinase C, we chose the best phosphorylated peptides for the investigation of the importance of amino acids immediately adjacent to the phosphorylation site. The selectivity of the best protein kinase C substrates from this study was analysed with protein kinases A, CK1 and CK2. According to these tests, the most favourable characteristics of SPOTs-membrane-associated peptides were demonstrated by peptide KRAKRKTAKKR. Kinetic analysis of peptide phosphorylation with protein kinase C revealed an apparent Km of 0.49±0.13 μM and Vmax of 10.0±0.5 nmol/min per mg with soluble peptide KRAKRKTAKKR. In addition, we assayed several other soluble peptides commonly used as protein kinase C substrates. Peptide KRAKRKTAKKR showed the lowest Km and the highest Vmax/Km value in comparison with peptides FKKSFKL, pEKRPSQRSKYL and KRAKRKTTKKR. Furthermore, of the peptides tested, KRAKRKTAKKR was the most selective substrate for protein kinase C. The favourable kinetic parameters combined with the selectivity should make the KRAKRKTAKKR peptide useful as a substrate for protein kinase C in the assays of both purified enzyme and in crude cell extracts.
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Ng, Sandy Y. M., David J. VanDyke, Bonnie Chaban, John Wu, Yoshika Nosaka, Shin-Ichi Aizawa, and Ken F. Jarrell. "Different Minimal Signal Peptide Lengths Recognized by the Archaeal Prepilin-Like Peptidases FlaK and PibD." Journal of Bacteriology 191, no. 21 (August 28, 2008): 6732–40. http://dx.doi.org/10.1128/jb.00673-09.
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ABSTRACT In Archaea, the preflagellin peptidase (a type IV prepilin-like peptidase designated FlaK in Methanococcus voltae and Methanococcus maripaludis) is the enzyme that cleaves the N-terminal signal peptide from preflagellins. In methanogens and several other archaeal species, the typical flagellin signal peptide length is 11 to 12 amino acids, while in other archaea preflagellins possess extremely short signal peptides. A systematic approach to address the signal peptide length requirement for preflagellin processing is presented in this study. M. voltae preflagellin FlaB2 proteins with signal peptides 3 to 12 amino acids in length were generated and used as a substrate in an in vitro assay utilizing M. voltae membranes as an enzyme source. Processing by FlaK was observed in FlaB2 proteins containing signal peptides shortened to 5 amino acids; signal peptides 4 or 3 amino acids in length were unprocessed. In the case of Sulfolobus solfataricus, where the preflagellin peptidase PibD has broader substrate specificity, some predicted substrates have predicted signal peptides as short as 3 amino acids. Interestingly, the shorter signal peptides of the various mutant FlaB2 proteins not processed by FlaK were processed by PibD, suggesting that some archaeal preflagellin peptidases are likely adapted toward cleaving shorter signal peptides. The functional complementation of signal peptidase activity by FlaK and PibD in an M. maripaludis ΔflaK mutant indicated that processing of preflagellins was detected by complementation with either FlaK or PibD, yet only FlaK-complemented cells were flagellated. This suggested that a block in an assembly step subsequent to signal peptide removal occurred in the PibD complementation.
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Balasuriya, Nileeka, Norman E. Davey, Jared L. Johnson, Huadong Liu, Kyle K. Biggar, Lewis C. Cantley, Shawn Shun-Cheng Li, and Patrick O'Donoghue. "Phosphorylation-dependent substrate selectivity of protein kinase B (AKT1)." Journal of Biological Chemistry 295, no. 24 (April 29, 2020): 8120–34. http://dx.doi.org/10.1074/jbc.ra119.012425.
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Protein kinase B (AKT1) is a central node in a signaling pathway that regulates cell survival. The diverse pathways regulated by AKT1 are communicated in the cell via the phosphorylation of perhaps more than 100 cellular substrates. AKT1 is itself activated by phosphorylation at Thr-308 and Ser-473. Despite the fact that these phosphorylation sites are biomarkers for cancers and tumor biology, their individual roles in shaping AKT1 substrate selectivity are unknown. We recently developed a method to produce AKT1 with programmed phosphorylation at either or both of its key regulatory sites. Here, we used both defined and randomized peptide libraries to map the substrate selectivity of site-specific, singly and doubly phosphorylated AKT1 variants. To globally quantitate AKT1 substrate preferences, we synthesized three AKT1 substrate peptide libraries: one based on 84 “known” substrates and two independent and larger oriented peptide array libraries (OPALs) of ∼1011 peptides each. We found that each phospho-form of AKT1 has common and distinct substrate requirements. Compared with pAKT1T308, the addition of Ser-473 phosphorylation increased AKT1 activities on some, but not all of its substrates. This is the first report that Ser-473 phosphorylation can positively or negatively regulate kinase activity in a substrate-dependent fashion. Bioinformatics analysis indicated that the OPAL-activity data effectively discriminate known AKT1 substrates from closely related kinase substrates. Our results also enabled predictions of novel AKT1 substrates that suggest new and expanded roles for AKT1 signaling in regulating cellular processes.
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Srinivasan, J., M. Koszelak, M. Mendelow, Y. G. Kwon, and D. S. Lawrence. "The design of peptide-based substrates for the cdc2 protein kinase." Biochemical Journal 309, no. 3 (August 1, 1995): 927–31. http://dx.doi.org/10.1042/bj3090927.
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The substrate sequence specificity of the cdc2 protein kinase from Pisaster ochraceus has been evaluated. The peptide, Ac-Ser-Pro-Gly-Arg-Arg-Arg-Arg-Lys-amide, serves as an efficient cdc2 kinase substrate with a Km of 1.50 +/- 0.04 microM and a Vmax. of 12.00 +/- 0.18 mumol/min per mg. The amino acid sequence of this peptide is not based on any sequence in a known protein substrate of the cyclin-dependent kinase, but rather was designed from structural attributes that appear to be important in the majority of cdc2 substrates. The cyclin-dependent enzyme is remarkably indiscriminate in its ability to recognize and phosphorylate peptides that contain an assortment of structurally diverse residues at the P-2, P-1 and P+2 positions. However, peptides that contain a free N-terminal serine or lack an arginine at the P+4 position are relatively poor substrates. These aspects of the substrate specificity of the cdc2 protein kinase are compared and contrasted with the previously reported substrate specificity of a cdc2-like protein kinase from bovine brain [Beaudette, Lew and Wang (1993) J. Biol. Chem. 268, 20825-20830].
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Ting, Yi Tian, Paul W. R. Harris, Gaelle Batot, Margaret A. Brimble, Edward N. Baker, and Paul G. Young. "Peptide binding to a bacterial signal peptidase visualized by peptide tethering and carrier-driven crystallization." IUCrJ 3, no. 1 (January 1, 2016): 10–19. http://dx.doi.org/10.1107/s2052252515019971.
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Bacterial type I signal peptidases (SPases) are membrane-anchored serine proteases that process the signal peptides of proteins exported via the Sec and Tat secretion systems. Despite their crucial importance for bacterial virulence and their attractiveness as drug targets, only one such enzyme, LepB from Escherichia coli, has been structurally characterized, and the transient nature of peptide binding has stymied attempts to directly visualize SPase–substrate complexes. Here, the crystal structure of SpsB, the type I signal peptidase from the Gram-positive pathogen Staphylococcus aureus, is reported, and a peptide-tethering strategy that exploits the use of carrier-driven crystallization is described. This enabled the determination of the crystal structures of three SpsB–peptide complexes, both with cleavable substrates and with an inhibitory peptide. SpsB–peptide interactions in these complexes are almost exclusively limited to the canonical signal-peptide motif Ala-X-Ala, for which clear specificity pockets are found. Minimal contacts are made outside this core, with the variable side chains of the peptides accommodated in shallow grooves or exposed faces. These results illustrate how high fidelity is retained despite broad sequence diversity, in a process that is vital for cell survival.
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Stensland, Maria E., Sylvie Pollmann, Øyvind Molberg, Ludvig M. Sollid, and Burkhard Fleckenstein. "Primary sequence, together with other factors, influence peptide deimination by peptidylarginine deiminase-4." Biological Chemistry 390, no. 2 (February 1, 2009): 99–107. http://dx.doi.org/10.1515/bc.2009.019.
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Abstract Enzymes of the peptidylarginine deiminase (PAD) family catalyze the posttranslational deimination of polypeptide-bound arginine residues. Here, we report the selection of peptide substrates by PAD-4, an isoform thought to be involved in the pathogenesis of rheumatoid arthritis. First, we investigated whether PAD-4-mediated deimination is influenced by the nature of amino acid residues flanking the targeted arginine. Using two peptide substrates, residues in positions -2, -1, +1, and +2 relative to the central arginine targeted by PAD-4 were systematically replaced by all natural l-amino acids except cysteine. Each peptide was treated with recombinant human PAD-4 and deimination was analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. In all four flanking positions, amino acids which positively or negatively influenced deimination were identified. We next designed peptides with expected high or low deimination rates and determined their Km and kcat values. These peptides showed PAD-4 substrate behavior as predicted, demonstrating that residues flanking the targeted arginine are important for deimination. Further truncation of peptide substrates suggested additional effects on deimination by residues outside the -2 to +2 region. Finally, we observed that a methylated lysine residue flanking the targeted arginine influences PAD-4-mediated deimination, also suggesting that posttranslational modifications can affect substrate efficiency.
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Gerken, Thomas A., Jayalakshmi Raman, Timothy A. Fritz, and Oliver Jamison. "Identification of Common and Unique Peptide Substrate Preferences for the UDP-GalNAc:Polypeptide α-N-acetylgalactosaminyltransferases T1 and T2 Derived from Oriented Random Peptide Substrates." Journal of Biological Chemistry 281, no. 43 (August 15, 2006): 32403–16. http://dx.doi.org/10.1074/jbc.m605149200.
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A large family of UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAc Ts) catalyzes the first step of mucin-type protein O-glycosylation by transferring GalNAc to serine and threonine residues of acceptor polypeptides. The acceptor peptide substrate specificity and specific protein targets of the individual ppGalNAc T family members remain poorly characterized and poorly understood, despite the fact that mutations in two individual isoforms are deleterious to man and the fly. In this work a series of oriented random peptide substrate libraries, based on the GAGAXXXTXXXAGAGK sequence motif (where X = randomized positions), have been used to obtain the first comprehensive determination of the peptide substrate specificities of the mammalian ppGalNAc T1 and T2 isoforms. ppGalNAc T-glycosylated random peptides were isolated by lectin affinity chromatography, and transferase amino acid preferences were determined by Edman amino acid sequencing. The results reveal common and unique position-sensitive features for both transferases, consistent with previous reports of the preferences of ppGalNAc T1 and T2. The random peptide substrates also reveal additional specific features that have never been described before that are consistent with the x-ray crystal structures of the two transferases and furthermore are reflected in a data base analysis of in vivo O-glycosylation sites. By using the transferase-specific preferences, optimum and selective acceptor peptide substrates have been generated for each transferase. This approach represents a relatively complete, facile, and reproducible method for obtaining ppGalNAc T peptide substrate specificity. Such information will be invaluable for identifying isoform-specific peptide acceptors, creating isoform-specific substrates, and predicting O-glycosylation sites.
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Chen, Di, Lingling Yan, and Weiping Zheng. "Cyclic Peptide-Based Sirtuin Substrates." Molecules 24, no. 3 (January 24, 2019): 424. http://dx.doi.org/10.3390/molecules24030424.
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In the current study, four side chain-to-side chain cyclic peptides (three 5-mers and one 4-mer) harboring Nε-acetyl-lysine or Nε-myristoyl-lysine were found to be in vitro substrates of the human SIRT1/2/3-catalyzed deacylation with good substrate activities, as judged by the kcat/KM ratios.
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Moro, Fernando, Vanesa Fernández-Sáiz, and Arturo Muga. "The Lid Subdomain of DnaK Is Required for the Stabilization of the Substrate-binding Site." Journal of Biological Chemistry 279, no. 19 (February 25, 2004): 19600–19606. http://dx.doi.org/10.1074/jbc.m400921200.
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We examined the effect of deletion of different segments in the helical subdomain (the so-called “lid”) of the DnaK peptide-binding domain on peptide binding and protein stability. At 25 °C, wt DnaK and the deletion mutant proteins are able to stably bind peptides with similar affinity. However, at physiological (37 °C) and stress (42 °C) temperatures, removal of the N-terminal half of αB and the rest of the lid drastically decreases the ability of the protein to bind substrates. Differential scanning calorimetry and infrared spectroscopy show that this behavior is accompanied by destabilization of the peptide-binding domain. Our data suggest that the reversible interaction between the lid and β-sandwich subdomains of DnaK peptide-binding domain is required for the stabilization of the loops that form the peptide-binding site, which in turn modulates the protein affinity for peptide substrates. This interaction might have functional implications because it could prevent rebinding of the peptide substrate, which would be forced to fold.
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Chistov, A. A., A. V. Talanova, M. V. Melnikova, S. S. Kuznetsova, and E. F. Kolesanova. "An Improved Procedure for the Preparation of Thrombin Low Molecular Weight Substrates - Peptide p-Nitroanilides." Biomedical Chemistry: Research and Methods 1, no. 4 (2018): e00057. http://dx.doi.org/10.18097/bmcrm00057.
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Low molecular weight chromogenic thrombin peptide substrates, p-nitroanilides of short peptides protected at their N-terminal amino group, were prepared by solid-phase peptide synthesis on polystyrene-divinylbenzene polymer with trityl groups with preliminary attached p-phenylene diamine moiety. After the cleavage from the resin peptide p-aminoanilides were mildly oxidized to p-nitroanilides with the mixture of potassium sulfate and persulfate. Adsorption onto polymer support Bio-Beads SM-2 with further elution by acetonitrile allowed easy separating peptide p-nitroanilides from the oxidizer and obtaining the thrombin chromogenic substrate preparations with the target substance contents of not less than 95% and yields of 30-40%. Thrombin effectively catalyzed hydrolysis of the prepared substrates with KM and Vmax values of 29-134 mM and 0.03-1/16 mM/s, respectively.
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Matsumi, Rie, Haruyuki Atomi, and Tadayuki Imanaka. "Biochemical Properties of a Putative Signal Peptide Peptidase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1." Journal of Bacteriology 187, no. 20 (October 15, 2005): 7072–80. http://dx.doi.org/10.1128/jb.187.20.7072-7080.2005.
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ABSTRACT We have performed the first biochemical characterization of a putative archaeal signal peptide peptidase (SppATk) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. SppATk, comprised of 334 residues, was much smaller than its counterpart from Escherichia coli (618 residues) and harbored a single predicted transmembrane domain near its N terminus. A truncated mutant protein without the N-terminal 54 amino acid residues (ΔN54SppATk) was found to be stable against autoproteolysis and was examined further. ΔN54SppATk exhibited peptidase activity towards fluorogenic peptide substrates and was found to be highly thermostable. Moreover, the enzyme displayed a remarkable stability and preference for alkaline pH, with optimal activity detected at pH 10. ΔN54SppATk displayed a Km of 240 ± 18 μM and a V max of 27.8 ± 0.7 μmol min−1 mg−1 towards Ala-Ala-Phe-4-methyl-coumaryl-7-amide at 80°C and pH 10. The substrate specificity of the enzyme was examined in detail with a FRETS peptide library. By analyzing the cleavage products with liquid chromatography-mass spectrometry, ΔN54SppATk was found to efficiently cleave peptides with a relatively small side chain at the P-1 position and a hydrophobic or aromatic residue at the P-3 position. The positively charged Arg residue was preferred at the P-4 position, while substrates with negatively charged residues at the P-2, P-3, or P-4 position were not cleaved. When predicted signal sequences from the T. kodakaraensis genome sequence were examined, we found that the substrate specificity of ΔN54SppATk was in good agreement with its presumed role as a signal peptide peptidase in this archaeon.
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Juliano, John Paul, David H. Small, and Marie-Isabel Aguilar. "Peptidomimetic Modulators of BACE1." Australian Journal of Chemistry 73, no. 4 (2020): 366. http://dx.doi.org/10.1071/ch19594.
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The β-site APP Cleaving enzyme 1 (BACE1) is a membrane-associated aspartyl protease which mediates the production of amyloid-β (Aβ), a major component of amyloid plaques in the Alzheimer’s disease brain. We have synthesised and characterised a series of peptidomimetic analogues of BACE substrates that incorporate two distinct stabilising structures. To demonstrate the potential activity of these compounds, a variety of assaying strategies were used to investigate cleavage susceptibility and inhibition potency under competitive and non-competitive conditions. β-Amino acids and scissile site N-methylation were incorporated into peptide substrate templates as transition state isostere (TSI) substitutes by positional scanning to generate series of non-TSI β-peptidomimetics. The amino acid sequences flanking the β-cleavage site within APP carrying the Swedish double mutation (APPSW), Neuregulin, the synthetic hydroxyethylene-based TSI peptide inhibitor OM99-2, and the high affinity peptide sequence SEISYEVEFR, served as the four substrate templates from which over 60 peptides were designed and synthesised by solid phase peptide synthesis. A quenched fluorescent substrate BACE1 assay in conjunction with liquid chromatography–mass spectrometry (LC-MS) analysis was established to investigate cleavage susceptibility and inhibition potency under competitive and non-competitive conditions. It was determined that β-amino acids substituted at the P1 scissile site position within known peptide substrates were resistant to proteolysis, and particular substitutions induced a concentration-dependent stimulation of BACE1, indicating a possible modulatory role of native BACE1 substrates.
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Bageshwar, Umesh K., Antara DattaGupta, and Siegfried M. Musser. "Influence of the TorD signal peptide chaperone on Tat-dependent protein translocation." PLOS ONE 16, no. 9 (September 9, 2021): e0256715. http://dx.doi.org/10.1371/journal.pone.0256715.
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The twin-arginine translocation (Tat) pathway transports folded proteins across energetic membranes. Numerous Tat substrates contain co-factors that are inserted before transport with the assistance of redox enzyme maturation proteins (REMPs), which bind to the signal peptide of precursor proteins. How signal peptides are transferred from a REMP to a binding site on the Tat receptor complex remains unknown. Since the signal peptide mediates both interactions, possibilities include: i) a coordinated hand-off mechanism; or ii) a diffusional search after REMP dissociation. We investigated the binding interaction between substrates containing the TorA signal peptide (spTorA) and its cognate REMP, TorD, and the effect of TorD on the in vitro transport of such substrates. We found that Escherichia coli TorD is predominantly a monomer at low micromolar concentrations (dimerization KD > 50 μM), and this monomer binds reversibly to spTorA (KD ≈ 1 μM). While TorD binds to membranes (KD ≈ 100 nM), it has no apparent affinity for Tat translocons and it inhibits binding of a precursor substrate to the membrane. TorD has a minimal effect on substrate transport by the Tat system, being mildly inhibitory at high concentrations. These data are consistent with a model in which the REMP-bound signal peptide is shielded from recognition by the Tat translocon, and spontaneous dissociation of the REMP allows the substrate to engage the Tat machinery. Thus, the REMP does not assist with targeting to the Tat translocon, but rather temporarily shields the signal peptide.
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Juillard, Vincent, Alain Guillot, Dominique Le Bars, and Jean-Claude Gripon. "Specificity of Milk Peptide Utilization byLactococcus lactis." Applied and Environmental Microbiology 64, no. 4 (April 1, 1998): 1230–36. http://dx.doi.org/10.1128/aem.64.4.1230-1236.1998.
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ABSTRACT To study the substrate specificity of the oligopeptide transport system of Lactococcus lactis for its natural substrates, the growth of L. lactis MG1363 was studied in a chemically defined medium containing milk peptides or a tryptic digest of αs2-casein as the source of amino acids. Peptides were separated into acidic, neutral, and basic pools by solid-phase extraction or by cation-exchange liquid chromatography. Their ability to sustain growth and the time course of their utilization demonstrated the preferential use of hydrophobic basic peptides with molecular masses ranging between 600 and 1,100 Da by L. lactis MG1363 and the inability to use large, acidic peptides. These peptide utilization preferences reflect the substrate specificity of the oligopeptide transport system of the strain, since no significant cell lysis was inferred. Considering the free amino acid content of milk and these findings on peptide utilization, it was demonstrated that the cessation of growth of L. lactis MG1363 in milk was due to deprivation of leucine and methionine.
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Duprez, Kevin, Melissa A. Scranton, Linda L. Walling, and Li Fan. "Structure of tomato wound-induced leucine aminopeptidase sheds light on substrate specificity." Acta Crystallographica Section D Biological Crystallography 70, no. 6 (May 29, 2014): 1649–58. http://dx.doi.org/10.1107/s1399004714006245.
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The acidic leucine aminopeptidase (LAP-A) from tomato is induced in response to wounding and insect feeding. Although LAP-A showsin vitropeptidase activity towards peptides and peptide analogs, it is not clear what kind of substrates LAP-A hydrolyzesin vivo. In the current study, the crystal structure of LAP-A was determined to 2.20 Å resolution. Like other LAPs in the M17 peptidase family, LAP-A is a dimer of trimers containing six monomers of bilobal structure. Each monomer contains two metal ions bridged by a water or a hydroxyl ion at the active site. Modeling of different peptides or peptide analogs in the active site of LAP-A reveals a spacious substrate-binding channel that can bind peptides of five or fewer residues with few geometric restrictions. The sequence specificity of the bound peptide is likely to be selected by the structural and chemical restrictions on the amino acid at the P1 and P1′ positions because these two amino acids have to bind perfectly at the active site for hydrolysis of the first peptide bond to occur. The hexameric assembly results in the merger of the open ends of the six substrate-binding channels from the LAP-A monomers to form a spacious central cavity allowing the hexameric LAP-A enzyme to simultaneously hydrolyze six peptides containing up to six amino acids each. The hexameric LAP-A enzyme may also hydrolyze long peptides or proteins if only one such substrate is bound to the hexamer because the substrate can extend through the central cavity and the two major solvent channels between the two LAP-A trimers.
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Kalb, Suzanne R., Jakub Baudys, Kaitlyn Kiernan, Dongxia Wang, François Becher, and John R. Barr. "Proposed BoNT/A and /B Peptide Substrates Cannot Detect Multiple Subtypes in the Endopep-MS Assay." Journal of Analytical Toxicology 44, no. 2 (July 9, 2019): 173–79. http://dx.doi.org/10.1093/jat/bkz044.
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Abstract Botulinum neurotoxins (BoNTs) are a family of protein toxins consisting of seven known serotypes (BoNT/A—BoNT/G) and multiple subtypes within the serotypes, and all of which cause the disease botulism—a disease of great public health concern. Accurate detection of BoNTs in human clinical samples is therefore an important public health goal. To achieve this goal, our laboratory developed a mass spectrometry-based assay detecting the presence of BoNT via its enzymatic activity on a peptide substrate. Recently, publications reported the use of new peptide substrates to detect BoNT/A and /B with improved results over other peptide substrates. However, the authors did not provide results of their peptide substrate on multiple subtypes of BoNT. In this work, we describe the results of testing the new substrates with multiple BoNT/A and /B subtypes and find that the substrates cannot detect many subtypes of BoNT/A and /B.
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Lamort, Anne-Sophie, Rodolphe Gravier, Anni Laffitte, Luiz Juliano, Marie-Louise Zani, and Thierry Moreau. "New insights into the substrate specificity of macrophage elastase MMP-12." Biological Chemistry 397, no. 5 (May 1, 2016): 469–84. http://dx.doi.org/10.1515/hsz-2015-0254.
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Abstract Macrophage elastase, or MMP-12, is mainly produced by alveolar macrophages and is believed to play a major role in the development of chronic obstructive pulmonary disease (COPD). The catalytic domain of MMP-12 is unique among MMPs in that it is very highly active on numerous substrates including elastin. However, measuring MMP-12 activity in biological fluids has been hampered by the lack of highly selective substrates. We therefore synthesized four series of fluorogenic peptide substrates based on the sequences of MMP-12 cleavage sites in its known substrates. Human MMP-12 efficiently cleaved peptide substrates containing a Pro at P3 in the sequence Pro-X-X↓Leu but lacked selectivity towards these substrates compared to other MMPs, including MMP-2, MMP-7, MMP-9 and MMP-13. On the contrary, the substrate Abz-RNALAVERTAS-EDDnp derived from the CXCR5 chemokine was the most selective substrate for MMP-12 ever reported. All substrates were cleaved more efficiently by full-length MMP-12 than by its catalytic domain alone, indicating that the C-terminal hemopexin domain influences substrate binding and/or catalysis. Docking experiments revealed unexpected interactions between the peptide substrate Abz-RNALAVERTAS-EDDn and MMP-12 residues. Most of our substrates were poorly cleaved by murine MMP-12 suggesting that human and murine MMP-12 have different substrate specificities despite their structural similarity.
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Yoshida, Takuya, Kazuki Yamazaki, Shunta Imai, Akinori Banno, Atsushi Kaneko, Kazuhiro Furukawa, and Yoshiro Chuman. "Identification of a Specific Inhibitor of Human Scp1 Phosphatase Using the Phosphorylation Mimic Phage Display Method." Catalysts 9, no. 10 (October 11, 2019): 842. http://dx.doi.org/10.3390/catal9100842.
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Protein phosphatases are divided into tyrosine (Tyr) phosphatases and serine/threonine (Ser/Thr) phosphatases. While substrate trapping mutants are frequently used to identify substrates of Tyr phosphatases, a rapid and simple method to identify Ser/Thr phosphatase substrates is yet to be developed. The TFIIF-associating component of RNA polymerase II C-terminal domain (CTD) phosphatase/small CTD phosphatase (FCP/SCP) phosphatase family is one of the three types of Ser/Thr protein phosphatases. Defects in these phosphatases are correlated with the occurrence of various diseases such as cancer and neuropathy. Recently, we developed phosphorylation mimic phage display (PMPD) method with AlF4−, a methodology to identify substrates for FCP/SCP type Ser/Thr phosphatase Scp1. Here, we report a PMPD method using BeF3− to identify novel substrate peptides bound to Scp1. After screening peptide phages, we identified peptides that bound to Scp1 in a BeF3−-dependent manner. Synthetic phosphopeptide BeM12-1, the sequence of which was isolated at the highest frequency, directly bound to Scp1. The binding was inhibited by adding BeF3−, indicating that the peptide binds to the active center of catalytic site in Scp1. The phosphorylated BeM12-1 worked as a competitive inhibitor of Scp1. Thus, PMPD method may be applicable for the identification of novel substrates and inhibitors of the FCP/SCP phosphatase family.
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Maffioli, Elisa, Zhenze Jiang, Simona Nonnis, Armando Negri, Valentina Romeo, Christopher B. Lietz, Vivian Hook, et al. "High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma." Molecules 25, no. 18 (September 6, 2020): 4071. http://dx.doi.org/10.3390/molecules25184071.
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Proteomic technologies have identified 234 peptidases in plasma but little quantitative information about the proteolytic activity has been uncovered. In this study, the substrate profile of plasma proteases was evaluated using two nano-LC-ESI-MS/MS methods. Multiplex substrate profiling by mass spectrometry (MSP-MS) quantifies plasma protease activity in vitro using a global and unbiased library of synthetic peptide reporter substrates, and shotgun peptidomics quantifies protein degradation products that have been generated in vivo by proteases. The two approaches gave complementary results since they both highlight key peptidase activities in plasma including amino- and carboxypeptidases with different substrate specificity profiles. These assays provide a significant advantage over traditional approaches, such as fluorogenic peptide reporter substrates, because they can detect active plasma proteases in a global and unbiased manner, in comparison to detecting select proteases using specific reporter substrates. We discovered that plasma proteins are cleaved by endoproteases and these peptide products are subsequently degraded by amino- and carboxypeptidases. The exopeptidases are more active and stable in plasma and therefore were found to be the most active proteases in the in vitro assay. The protocols presented here set the groundwork for studies to evaluate changes in plasma proteolytic activity in shock.
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Iijima, Kazutoshi, Hiroumi Nagahama, Akari Takada, Toshiki Sawada, Takeshi Serizawa, and Mineo Hashizume. "Surface functionalization of polymer substrates with hydroxyapatite using polymer-binding peptides." Journal of Materials Chemistry B 4, no. 21 (2016): 3651–59. http://dx.doi.org/10.1039/c6tb00624h.
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Polymer substrates were modified with hydroxyapatite (HAp) using two bi-functional peptides consists of polymer-binding peptide and triasparate for HAp mineralization in simulated body fluids and HAp-binding peptide for immobilization HAp nanoparticles.
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VARNER, Amanda S., Charles E. DUCKER, Zuping XIA, Yan ZHUANG, Mackenzie L. DE VOS, and Charles D. SMITH. "Characterization of human palmitoyl-acyl transferase activity using peptides that mimic distinct palmitoylation motifs." Biochemical Journal 373, no. 1 (July 1, 2003): 91–99. http://dx.doi.org/10.1042/bj20021598.
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The covalent attachment of palmitate to proteins commonly occurs on cysteine residues near either N-myristoylated glycine residues or C-terminal farnesylated cysteine residues. It therefore seems likely that multiple palmitoyl-acyl transferase (PAT) activities exist to recognize and modify these distinct palmitoylation motifs. To evaluate this possibility, two synthetic peptides representing these palmitoylation motifs, termed MyrGCK(NBD) and FarnCNRas(NBD), were used to characterize PAT activity under a variety of conditions. The human tumour cell lines MCF-7 and Hep-G2 each demonstrated high levels of PAT activity towards both peptides. In contrast, normal mouse fibroblasts (NIH/3T3 cells) demonstrated PAT activity towards the myristoylated substrate peptide but not the farnesylated peptide, while ras-transformed NIH/3T3 cells were able to palmitoylate the FarnCNRas(NBD) peptide. The kinetic parameters for PAT activity were determined using membranes from MCF-7 cells, and indicated that the Km values for palmitoyl-CoA were identical for PAT activity towards the two substrate peptides; however, the Km for MyrGCK(NBD) was 5-fold lower than the Km for FarnCNRas(NBD). PAT activity towards the two substrate peptides was dose-dependently inhibited by 2-bromopalmitate and 3-(1-oxo-hexadecyl)oxiranecarboxamide (16C; IC50 values of approx. 4 and 1.3 μM, respectively); however, 2-bromopalmitate was found to be uncompetitive with respect to palmitoyl-CoA, whereas 16C was competitive. To seek additional evidence for multiple PATs, the effects of altering the assay conditions on the palmitoylation of MyrGCK(NBD) and FarnCNRas(NBD) were compared. PAT activity towards the two peptide substrates was modulated similarly by changing the ionic strength or incubation temperature, or by the addition of dithiothreitol. In contrast, the enzymic palmitoylation of the two peptides was differentially affected by N-ethylmaleimide and thermal denaturation. Overall, these data demonstrate that the enzymic palmitoylation of farnesyl- and myristoyl-containing peptide substrates can be differentiated, suggesting that multiple motif-specific PATs exist.
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Schrul, Bianca, Katja Kapp, Irmgard Sinning, and Bernhard Dobberstein. "Signal peptide peptidase (SPP) assembles with substrates and misfolded membrane proteins into distinct oligomeric complexes." Biochemical Journal 427, no. 3 (April 14, 2010): 523–34. http://dx.doi.org/10.1042/bj20091005.
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SPP (signal peptide peptidase) is an aspartyl intramembrane cleaving protease, which processes a subset of signal peptides, and is linked to the quality control of ER (endoplasmic reticulum) membrane proteins. We analysed SPP interactions with signal peptides and other membrane proteins by co-immunoprecipitation assays. We found that SPP interacts specifically and tightly with a large range of newly synthesized membrane proteins, including signal peptides, preproteins and misfolded membrane proteins, but not with all co-expressed type II membrane proteins. Signal peptides are trapped by the catalytically inactive SPP mutant SPPD/A. Preproteins and misfolded membrane proteins interact with both SPP and the SPPD/A mutant, and are not substrates for SPP-mediated intramembrane proteolysis. Proteins interacting with SPP are found in distinct complexes of different sizes. A signal peptide is mainly trapped in a 200 kDa SPP complex, whereas a preprotein is predominantly found in a 600 kDa SPP complex. A misfolded membrane protein is detected in 200, 400 and 600 kDa SPP complexes. We conclude that SPP not only processes signal peptides, but also collects preproteins and misfolded membrane proteins that are destined for disposal.
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Jedlicka, Sabrina S., Kenneth M. Little, David E. Nivens, Dmitry Zemlyanov, and Jenna L. Rickus. "Peptide ormosils as cellular substrates." Journal of Materials Chemistry 17, no. 48 (2007): 5058. http://dx.doi.org/10.1039/b705393b.
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Beekman, Chapman, Zhenze Jiang, Brian M. Suzuki, Jonathan M. Palmer, Daniel L. Lindner, Anthony J. O’Donoghue, Giselle M. Knudsen, and Richard J. Bennett. "Characterization of PdCP1, a serine carboxypeptidase from Pseudogymnoascus destructans, the causal agent of White-nose Syndrome." Biological Chemistry 399, no. 12 (November 27, 2018): 1375–88. http://dx.doi.org/10.1515/hsz-2018-0240.
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Abstract Pseudogymnoascus destructans is a pathogenic fungus responsible for White-nose Syndrome (WNS), a disease afflicting multiple species of North American bats. Pseudogymnoascus destructans infects susceptible bats during hibernation, invading dermal tissue and causing extensive tissue damage. In contrast, other Pseudogymnoascus species are non-pathogenic and cross-species comparisons may therefore reveal factors that contribute to virulence. In this study, we compared the secretome of P. destructans with that from several closely related Pseudogymnoascus species. A diverse set of hydrolytic enzymes were identified, including a putative serine peptidase, PdCP1, that was unique to the P. destructans secretome. A recombinant form of PdCP1 was purified and substrate preference determined using a multiplexed-substrate profiling method based on enzymatic degradation of a synthetic peptide library and analysis by mass spectrometry. Most peptide substrates were sequentially truncated from the carboxyl-terminus revealing that this enzyme is a bona fide carboxypeptidase. Peptides with arginine located close to the carboxyl-terminus were rapidly cleaved, and a fluorescent substrate containing arginine was therefore used to characterize PdCP1 activity and to screen a selection of peptidase inhibitors. Antipain and leupeptin were found to be the most potent inhibitors of PdCP1 activity.
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Stachowiak, Krystyna, Monika Tokmina, Anna Karpińska, Renata Sosnowska, and Wiesław Wiczk. "Fluorogenic peptide substrates for carboxydipeptidase activity of cathepsin B." Acta Biochimica Polonica 51, no. 1 (March 31, 2004): 81–92. http://dx.doi.org/10.18388/abp.2004_3599.
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Cathepsin B is a lysosomal cysteine protease exhibiting mainly dipeptidyl carboxypeptidase activity, which decreases dramatically above pH 5.5, when the enzyme starts acting as an endopeptidase. Since the common cathepsin B assays are performed at pH 6 and do not distinguish between these activities, we synthesized a series of peptide substrates specifically designed for the carboxydipeptidase activity of cathepsin B. The amino-acid sequences of the P(5)-P(1) part of these substrates were based on the binding fragments of cystatin C and cystatin SA, the natural reversible inhibitors of papain-like cysteine protease. The sequences of the P'(1)-P'(2) dipeptide fragments of the substrates were chosen on the basis of the specificity of the S'(1)-S'(2) sites of the cathepsin B catalytic cleft. The rates of hydrolysis by cathepsin B and papain, the archetypal cysteine protease, were monitored by a continuous fluorescence assay based on internal resonance energy transfer from an Edans to a Dabcyl group. The fluorescence energy donor and acceptor were attached to the C- and the N-terminal amino-acid residues, respectively. The kinetics of hydrolysis followed the Michaelis-Menten model. Out of all the examined peptides Dabcyl-R-L-V-G-F- E(Edans) turned out to be a very good substrate for both papain and cathepsin B at both pH 6 and pH 5. The replacement of Glu by Asp turned this peptide into an exclusive substrate for cathepsin B not hydrolyzed by papain. The substitution of Phe by Nal in the original substrate caused an increase of the specificity constant for cathepsin B at pH 5, and a significant decrease at pH 6. The results of kinetic studies also suggest that Arg in position P(4) is not important for the exopeptidase activity of cathepsin B, and that introducing Glu in place of Val in position P(2) causes an increase of the substrate preference towards this activity.
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ISAAC, R. Elwyn, A. Tracy WILLIAMS, Mohammed SAJID, Pierre CORVOL, and David COATES. "Cleavage of arginyl-arginine and lysyl-arginine from the C-terminus ofpro-hormone peptides by human germinal angiotensin I-converting enzyme (ACE) and the C-domain of human somatic ACE." Biochemical Journal 328, no. 2 (December 1, 1997): 587–91. http://dx.doi.org/10.1042/bj3280587.
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Mammalian germinal angiotensin I-converting enzyme (gACE) is a single-domain dipeptidyl carboxypeptidase found exclusively in male germ cells, which has almost identical sequence and enzymic properties with the C-domain of the two-domain somatic ACE. Mutant mice that do not express gACE are infertile, suggesting a role for the enzyme in the processing of undefined peptides involved in fertilization. A number of spermatid peptides [e.g. cholecystokinin (CCK) and gastrin] are processed from pro-hormones by endo- and exo-proteolytic cleavages which might generate substrates for gACE. We have shown that peptide hormone intermediates with Lys/Arg-Arg at the C-terminus are high-affinity substrates for human gACE. gACE from human sperm cleaved Arg-Arg from the C-terminus of the CCK5-GRR (GWMDFGRR), a peptide corresponding to the C-terminus of a CCK-gastrin prohormone intermediate. Hydrolysis of CCK5-GRR by recombinant human C-domain ACE was Cl- dependent, with maximal activity achieved in 5-10 mM NaCl at pH 6.4. C-Domain ACE cleaved Lys/Arg-Arg from the C-terminus of dynorphin-(1-7), a pro-TRH peptide KRQHPGKR, and two insect peptides FSPRLGKR and FSPRLGRR. C-Domain ACE displayed high affinity towards all these substrates with Vmax/Km values between 14 and 113 times greater than the Vmax/Km for the conversion of the best known ACE substrate, angiotensin I, into angiotensin II. In conclusion, we have identified a new class of substrates for human gACE, and we suggest that gACE might be an alternative to carboxypeptidase E for the trimming of basic dipeptides from the C-terminus of intermediates generated from pro-hormones by subtilisin-like convertases in human male germ cells.
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Verri, T., M. Maffia, A. Danieli, M. Herget, U. Wenzel, H. Daniel, and C. Storelli. "Characterisation of the H(+)/peptide cotransporter of eel intestinal brush-border membranes." Journal of Experimental Biology 203, no. 19 (October 1, 2000): 2991–3001. http://dx.doi.org/10.1242/jeb.203.19.2991.
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H(+)/peptide cotransport in brush-border membrane vesicles (BBMVs) from eel (Anguilla anguilla) intestine was studied by measuring d-[(3)H]-phenylalanyl-l-alanine uptake and by monitoring peptide-dependent intravesicular acidification using the pH-sensitive dye Acridine Orange. d-[(3)H]-phenylalanyl-l-alanine influx was greatly stimulated by an inside-negative membrane potential and enhanced by an inwardly directed H(+) gradient. In parallel, vesicular H(+) influx was significantly increased in the presence of extravesicular d-phenylalanyl-l-alanine or a series of glycyl and l-prolyl peptides. H(+)/peptide cotransport displayed saturable kinetics involving a single carrier system with apparent substrate affinities of 0.9-2.6 mmol l(−1) depending on the particular peptide. All substrates tested competed with this system. Pre-incubation of BBMVs with dipeptides prevented diethylpyrocarbonate inhibition of transport activity, suggesting that the substrates mask histidine residues involved in the catalytic function of the transporter. Using human PepT1-specific primers, a reverse transcription-polymerase chain reaction (RT-PCR) signal was detected in eel intestine. Our results suggest that, in eel intestine, a brush-border membrane ‘low-affinity’-type H(+)/peptide cotransport system is present that shares kinetic features with the mammalian intestinal PepT1-type transporters.
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SHAROM, Frances J., Xiaohong YU, Giulio DiDIODATO, and Joseph W. K. CHU. "Synthetic hydrophobic peptides are substrates for P-glycoprotein and stimulate drug transport." Biochemical Journal 320, no. 2 (December 1, 1996): 421–28. http://dx.doi.org/10.1042/bj3200421.
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P-Glycoprotein functions as an ATP-driven active efflux pump for many natural products and chemotherapeutic drugs. Hydrophobic peptides have been shown to block drug uptake by P-glycoprotein, indicating that they might be transport substrates. The present study examines the interaction of the synthetic peptide series NAc-LnY-amide with the multidrug transporter. Several peptides in this series caused up to 3.5-fold enhancement of colchicine accumulation in membrane vesicles from multidrug resistant (MDR) cells, which suggests the existence of novel interactions between the binding sites for peptides and drug. Peptides did not stimulate vinblastine transport, which was inhibited as expected for competing substrates. These peptides displayed modest stimulatory effects on the ATPase activity of P-glycoprotein. None blocked azidopine photoaffinity labelling, showing that they probably occupy a binding site separate from that for the drug. Studies with 125I-labelled NAc-LLY-amide showed that it was transported by P-glycoprotein in both membrane vesicles and reconstituted proteoliposomes. Uptake of the peptide was rapid, saturable, osmotically sensitive and occurred against a concentration gradient. The enhancing effect of NAc-LLY-amide on colchicine transport was reciprocated, i.e. colchicine greatly increased the transport of labelled peptide by P-glycoprotein. Peptide transport was also modulated, both positively and negatively, by other MDR spectrum drugs. It is concluded that linear hydrophobic peptides are indeed transported by P-glycoprotein, and some have interactions with drug substrates that result in mutual stimulation of transport.
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Chon, John H., and Elliot L. Chaikof. "Soluble heparin-binding peptides regulate chemokinesis and cell adhesive forces." American Journal of Physiology-Cell Physiology 280, no. 6 (June 1, 2001): C1394—C1402. http://dx.doi.org/10.1152/ajpcell.2001.280.6.c1394.
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The ability of a soluble heparin-binding peptide sequence derived from fibronectin to modulate the adhesion and chemokinetic migration behavior of arterial smooth muscle cells was assessed using a novel glass microsphere centrifugation assay and automated time-lapse fluorescence videomicroscopy, respectively. Treatment of cells grown on fibronectin-coated substrates with the soluble heparin-binding peptide resulted in the disassembly of focal adhesions, as assessed by immunohistochemical staining. These observations were consistent with an observed dose-dependent two- to fivefold reduction in cell-substrate adhesive strength ( P < 0.001) and a biphasic effect on migration speed ( P < 0.05). Moreover, heparin-binding peptides induced a twofold reduction ( P < 0.01) in two-dimensional cell dispersion in the presence of a non-heparin-binding growth factor, platelet-derived growth factor-AB (PDGF-AB). Heparin-binding peptides were unable to mediate these effects when cells were grown on substrates lacking a heparin-binding domain. These data support the notion that competitive interactions between cell surface heparan sulfates with heparin-binding peptides may modulate chemokinetic cell migration behavior and other adhesion-related processes.
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Hwang, Bum-Yeol, Navin Varadarajan, Haixin Li, Sarah Rodriguez, Brent L. Iverson, and George Georgiou. "Substrate Specificity of the Escherichia coli Outer Membrane Protease OmpP." Journal of Bacteriology 189, no. 2 (November 3, 2006): 522–30. http://dx.doi.org/10.1128/jb.01493-06.
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ABSTRACT Escherichia coli OmpP is an F episome-encoded outer membrane protease that exhibits 71% amino acid sequence identity with OmpT. These two enzymes cleave substrate polypeptides primarily between pairs of basic amino acids. We found that, like OmpT, purified OmpP is active only in the presence of lipopolysaccharide. With optimal peptide substrates, OmpP exhibits high catalytic efficiency (k cat/Km = 3.0 × 106 M−1s−1). Analysis of the extended amino acid specificity of OmpP by substrate phage revealed that both Arg and Lys are strongly preferred at the P1 and P1′ sites of the enzyme. In addition, Thr, Arg, or Ala is preferred at P2; Leu, Ala, or Glu is preferred at P4; and Arg is preferred at P3′. Notable differences in OmpP and OmpT specificities include the greater ability of OmpP to accept Lys at the P1 or P1′, site as well as the prominence of Ser at P3 in OmpP substrates. Likewise, the OmpP P1 site could better accommodate Ser; as a result, OmpP was able to cleave a peptide substrate between Ser-Arg about 120 times more efficiently than was OmpT. Interestingly, OmpP and OmpT cleave peptides with three consecutive Arg residues at different sites, a difference in specificity that might be important in the inactivation of cationic antimicrobial peptides. Accordingly, we show that the presence of an F′ episome results in increased resistance to the antimicrobial peptide protamine both in ompT mutants and in wild-type E. coli cells.
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Haverstick, DM, JF Cowan, KM Yamada, and SA Santoro. "Inhibition of platelet adhesion to fibronectin, fibrinogen, and von Willebrand factor substrates by a synthetic tetrapeptide derived from the cell-binding domain of fibronectin." Blood 66, no. 4 (October 1, 1985): 946–52. http://dx.doi.org/10.1182/blood.v66.4.946.946.
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Abstract The role in platelet function of the cell-binding region of fibronectin was explored by the use of synthetic peptides. The prototypical peptide gly-arg-gly-asp-ser was capable of inhibiting thrombin-induced platelet aggregation without altering the degree of platelet activation as judged by the secretion of 14C-serotonin. The peptide also effectively inhibited, in a concentration-dependent manner, the binding of radiolabeled fibronectin to platelets and the adhesion of platelets to fibronectin substrates. The smallest peptide from the cell-binding region of fibronectin which retained full activity was arg-gly-asp-ser. Transposition of amino acids or conservative substitutions of amino acids within this short sequence resulted in inactive peptides. Peptides containing the arg-gly-asp-ser sequence were also capable of inhibiting the adhesion of platelets to fibrinogen and von Willebrand factor substrates. Examination of the entire panel of synthetic peptides for ability to inhibit adhesion to fibrinogen or von Willebrand factor substrates revealed the same structure-function relationships that had been determined in the studies with fibronectin.
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Haverstick, DM, JF Cowan, KM Yamada, and SA Santoro. "Inhibition of platelet adhesion to fibronectin, fibrinogen, and von Willebrand factor substrates by a synthetic tetrapeptide derived from the cell-binding domain of fibronectin." Blood 66, no. 4 (October 1, 1985): 946–52. http://dx.doi.org/10.1182/blood.v66.4.946.bloodjournal664946.
Full textAbstract:
The role in platelet function of the cell-binding region of fibronectin was explored by the use of synthetic peptides. The prototypical peptide gly-arg-gly-asp-ser was capable of inhibiting thrombin-induced platelet aggregation without altering the degree of platelet activation as judged by the secretion of 14C-serotonin. The peptide also effectively inhibited, in a concentration-dependent manner, the binding of radiolabeled fibronectin to platelets and the adhesion of platelets to fibronectin substrates. The smallest peptide from the cell-binding region of fibronectin which retained full activity was arg-gly-asp-ser. Transposition of amino acids or conservative substitutions of amino acids within this short sequence resulted in inactive peptides. Peptides containing the arg-gly-asp-ser sequence were also capable of inhibiting the adhesion of platelets to fibrinogen and von Willebrand factor substrates. Examination of the entire panel of synthetic peptides for ability to inhibit adhesion to fibrinogen or von Willebrand factor substrates revealed the same structure-function relationships that had been determined in the studies with fibronectin.
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Hughes, S. J., H. Smith, and S. J. H. Ashcroft. "Characterization of Ca2+/calmodulin-dependent protein kinase in rat pancreatic islets." Biochemical Journal 289, no. 3 (February 1, 1993): 795–800. http://dx.doi.org/10.1042/bj2890795.
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We have attempted to identify islet Ca2+/calmodulin-dependent protein kinase (CaM kinase) by comparing its activity with purified brain CaM kinase II. Islet CaM kinase, in the presence of calmodulin and Ca2+, phosphorylated major endogenous substrates of 102, 57 and 53 kDa and also exogenous glycogen synthase; brain CaM kinase II phosphorylated glycogen synthase and peptides of 57 and 53 kDa. Alloxan (1 mM) inhibited the phosphorylation of glycogen synthase and the 102, 57 and 53 kDa islet peptides by islet CaM kinase; the phosphorylation of glycogen synthase and the 57 and 53 kDa substrates by brain CaM kinase II was also inhibited by alloxan. The Ca2+ and calmodulin-dependencies of phosphorylation of the endogenous islet substrates differed. In the presence of 400 nM calmodulin, half-maximal phosphorylation was attained at Ca2+ concentrations of 80 +/- 9, 401 +/- 61 and 459 +/- 59 nM for the 102, 57 and 53 kDa substrates respectively. In the presence of 10 microM Ca2+, half-maximal phosphorylation was attained at calmodulin concentrations of 9 +/- 2, 38 +/- 2.5 and 37 +/- 2 nM for the 102, 57 and 53 kDa substrates respectively. Differential centrifugation located the 102 kDa substrate in the post-100,000 g supernatant and the 57 and 53 kDa substrates in the particulate fraction. These data suggest that islet CaM kinase is similar to, if not identical with, brain CaM kinase II, but that phosphorylation of the endogenous 102 kDa substrate occurs by a distinct kinase which shows different sensitivities to Ca2+ and calmodulin. This kinase probably corresponds to CaM kinase III and the 102 kDa peptide to elongation factor 2 (EF-2), since the 102 kDa peptide was shown to undergo ADP-ribosylation in the presence of diphtheria toxin and NAD+.
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Steffansen, Bente, Carsten Uhd Nielsen, and Sven Frokjaer. "Delivery aspects of small peptides and substrates for peptide transporters." European Journal of Pharmaceutics and Biopharmaceutics 60, no. 2 (July 2005): 241–45. http://dx.doi.org/10.1016/j.ejpb.2005.01.004.
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Temple, Catherine S., Andrew K. Stewart, David Meredith, Norma A. Lister, Keith M. Morgan, Ian D. Collier, Richard D. Vaughan-Jones, C. A. R. Boyd, Patrick D. Bailey, and J. Ramsey Bronk. "Peptide Mimics as Substrates for the Intestinal Peptide Transporter." Journal of Biological Chemistry 273, no. 1 (January 2, 1998): 20–22. http://dx.doi.org/10.1074/jbc.273.1.20.
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Wilkin, J. M., M. Jamin, B. Joris, and J. M. Frere. "Mechanism of action of dd-peptidases: role of asparagine-161 in the Streptomyces R61 dd-peptidase." Biochemical Journal 293, no. 1 (July 1, 1993): 195–201. http://dx.doi.org/10.1042/bj2930195.
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The role of residue Asn-161 in the interaction between the Streptomyces R61 DD-peptidase and various substrates or beta-lactam inactivators was probed by site-directed mutagenesis. The residue was successively replaced by serine and alanine. In the first case, acylation rates were mainly affected with the peptide and ester substrates but not with the thiol-ester substrates and beta-lactams. However, the deacylation rates were decreased 10-30-fold with the substrates yielding benzoylglycyl and benzoylalanyl adducts. The Asn161Ala mutant was more generally affected, although the acylation rates with cefuroxime and cefotaxime remained similar to those observed with the wild-type enzyme. Surprisingly, the deacylation rates of the benzoylglycyl and benzoylalanyl adducts were very close to those observed with the wild-type enzyme. The results also indicate that the interaction with the peptide substrate and the transpeptidation reaction were more sensitive to the mutations than the other reactions studied. The results are discussed and compared with those obtained with the Asn-132 mutants of a class A beta-lactamase.
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Engel, Kate, Tomoaki Sasaki, Qi Wang, and John Kuriyan. "A highly efficient peptide substrate for EGFR activates the kinase by inducing aggregation." Biochemical Journal 453, no. 3 (July 12, 2013): 337–44. http://dx.doi.org/10.1042/bj20130537.
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Formation of an asymmetric dimer by the EGFR (epidermal growth factor receptor) kinase domains results in allosteric activation. Since this dimer does not readily form in solution, the EGFR kinase domain phosphorylates most peptide substrates with a relatively low catalytic efficiency. Peptide C is a synthetic peptide substrate of EGFR developed by others that is phosphorylated with a significantly higher catalytic efficiency, and we sought to understand the basis for this. Peptide C was found to increase EGFR kinase activity by promoting formation of the EGFR kinase domain asymmetric dimer. Activation of the kinase domain by Peptide C also enhances phosphorylation of other substrates. Aggregation of the EGFR kinase domain by Peptide C probably underlies activation, and Peptide C precipitates several other proteins. Peptide C was found to form fibrils independent of the presence of EGFR, and these fibrils may facilitate aggregation and activation of the kinase domain. These results establish that a peptide substrate of EGFR may increase catalytic activity by promoting kinase domain dimerization by an aggregation-mediated mechanism.
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Dunn, B. M., M. Jimenez, B. F. Parten, M. J. Valler, C. E. Rolph, and J. Kay. "A systematic series of synthetic chromophoric substrates for aspartic proteinases." Biochemical Journal 237, no. 3 (August 1, 1986): 899–906. http://dx.doi.org/10.1042/bj2370899.
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The hydrolysis of the chromogenic peptide Pro-Thr-Glu-Phe-Phe(4-NO2)-Arg-Leu at the Phe-Phe(4-NO2) bond by nine aspartic proteinases of animal origin and seven enzymes from micro-organisms is described [Phe(4-NO2) is p-nitro-L-phenylalanine]. A further series of six peptides was synthesized in which the residue in the P3 position was systematically varied from hydrophobic to hydrophilic. The Phe-Phe(4-NO2) bond was established as the only peptide bond cleaved, and kinetic constants were obtained for the hydrolysis of these peptide substrates by a representative selection of aspartic proteinases of animal and microbial origin. The value of these water-soluble substrates for structure-function investigations is discussed.
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George, Jeanette, Michelle L. Teear, Christopher G. Norey, and D. Dougal Burns. "Evaluation of an Imaging Platform during the Development of a FRET Protease Assay." Journal of Biomolecular Screening 8, no. 1 (January 2003): 72–80. http://dx.doi.org/10.1177/1087057102239778.
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Synthetic peptide substrates labeled with a fluorescent donor and quenching moiety flanking an enzyme cleavage site provide a reliable method for monitoring enzyme activity. The dye pair Mca/Dnp has been widely used for this purpose, but poor solubility characteristics, combined with fluorescence emission in the region of the spectrum associated with interference from bi-ologicals and library compounds, can limit the usefulness of Mca/Dnp substrates in a high-throughput screening (HTS) environment. Peptide Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2 is a matrix-metalloproteinase 3 (MMP-3) enzyme substrate that the authors have labeled with a CyDye pair, Cy3/Cy5Q. The Mca/Dnp- and CyDye-labeled substrates were compared during the development of an MMP-3 inhibitor assay. The results obtained showed that although the peptide substrates behaved similarly throughout the development of the MMP-3 assay, during a test screen of 934 compounds randomly selected from a collection of more than 70,000 compounds, the CyDye substrate was considerably more reliable. Screen Z factor values of 0.84 and 0.15 were obtained using the CyDye and Mca/Dnp peptides respectively, and the authors found that although < 1% of the test compounds were auto-fluorescent at Cy3 wavelengths, > 10% could not be screened using the Mca/Dnp substrate because of compound auto-fluorescence and interference. During this study, the authors used a PMTbased fluorescence plate reader and at the same time evaluated a charged couple device (CCD)—based imaging platform specifically optimized for use with CyDye reagents. The imaging platform gave improved read accuracy and faster plate processing times compared with the PMT reader. Overall, the results presented here highlight the potential benefit of employing the red-shifted CyDye reagents and imaging technology during the development and execution of HTS protease screens. (Journal of Biomolecular Screening 2003:72-80)
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Pfund, Christine, Peggy Huang, Nelson Lopez-Hoyo, and Elizabeth A. Craig. "Divergent Functional Properties of the Ribosome-Associated Molecular Chaperone Ssb Compared with Other Hsp70s." Molecular Biology of the Cell 12, no. 12 (December 2001): 3773–82. http://dx.doi.org/10.1091/mbc.12.12.3773.
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Ssbs of Saccharomyces cerevisiae are ribosome-associated molecular chaperones, which can be cross-linked to nascent polypeptide chains. Because Ssbs are members of a divergent subclass of Hsp70s found thus far only in fungi, we asked if the structural requirements for in vivo function were similar to those of “classic” Hsp70s. An intact peptide-binding domain is essential and an alteration of a conserved residue in the peptide-binding cleft (V442) affects function. However, Ssb tolerates a number of alterations in the peptide-binding cleft, revealing a high degree of flexibility in its functional requirements. Because binding of Ssb to peptide substrates in vitro was undetectable, we assessed the importance of substrate binding using the chimera BAB, in which the peptide binding domain of Ssb is exchanged for the analogous domain of the more “classical” Hsp70, Ssa. BAB, which binds peptide substrates in vitro, can substitute for Ssb in vivo. Alteration of a residue in the peptide-binding cleft of BAB creates a protein with a reduced affinity for peptide and altered ribosome binding that is unable to substitute for Ssb in vivo. These results indicate that Ssb's ability to bind unfolded polypeptides is likely critical for its function. This binding accounts, in part, for its stable interaction with translating ribosomes, even although it has a low affinity for peptides that detectably bind to other Hsp70s in vitro. These unusual properties may allow Ssb to function efficiently as a chaperone for ribosome-bound nascent chains.
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Berkovich, A., M. C. O'Keefe, P. Hensley, and L. Caporale. "Effect of N-methylation on the modulation by synthetic peptides of the activity of the complement-factor-B-derived serine proteinase CVFBb." Biochemical Journal 270, no. 2 (September 1, 1990): 531–37. http://dx.doi.org/10.1042/bj2700531.
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Although they share the active-site catalytic triad of less-specific enzymes such as trypsin and chymotrypsin, the serine proteinases of the complement and coagulation cascades each cleave a highly restricted set of substrates. Peptides with sequences similar to that at which C3 is cleaved by the alternative-pathway complement proteinase CVFBb were synthesized by solid-phase methodology and examined for their effects on the activity of this enzyme as measured by three different types of assays. It was found that a peptide methylated at the scissile bond was a far more effective inhibitor of the cleavage of the protein substrate C5 and of the lysis of guinea-pig erythrocytes by the alternative pathway than was the equivalent unmethylated peptide. Whereas the unmethylated peptide inhibited cleavage of the peptide substrate, the methylated peptide actually stimulated cleavage in this assay. This stimulation was found to be due to a 2.8-fold increase in kcat; the dissociation constant for the substrate was not altered significantly. One model consistent with this behaviour is that the binding of the activator peptide in the extended substrate-recognition region stabilizes a catalytically more active conformation of the active site. A small peptide substrate may have access to such an activated active site, whereas the larger substrate, C5, may be excluded from the site. These results demonstrate that the observed effect of a given compound on activity of an enzyme with an extended substrate-recognition region may depend upon the substrate.
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Gan, Weiqiong, Feng Gao, Keke Xing, Minze Jia, Haiping Liu, and Weimin Gong. "Structural basis of the substrate specificity of the FPOD/FAOD family revealed by fructosyl peptide oxidase fromEupenicillium terrenum." Acta Crystallographica Section F Structural Biology Communications 71, no. 4 (March 20, 2015): 381–87. http://dx.doi.org/10.1107/s2053230x15003921.
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The FAOD/FPOD family of proteins has the potential to be useful for the longterm detection of blood glucose levels in diabetes patients. A bottleneck for this application is to find or engineer a FAOD/FPOD family enzyme that is specifically active towards α-fructosyl peptides but is inactive towards other types of glycated peptides. Here, the crystal structure of fructosyl peptide oxidase fromEupenicillium terrenum(EtFPOX) is reported at 1.9 Å resolution. In contrast to the previously reported structure of amadoriase II, EtFPOX has an open substrate entrance to accommodate the large peptide substrate. The functions of residues critical for substrate selection are discussed based on structure comparison and sequence alignment. This study reveals the first structural details of group I FPODs that prefer α-fructosyl substrates and could provide significant useful information for uncovering the mechanism of substrate specificity of FAOD/FPODs and guidance towards future enzyme engineering for diagnostic purposes.
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Wilson, Claire H., Hui Emma Zhang, Mark D. Gorrell, and Catherine A. Abbott. "Dipeptidyl peptidase 9 substrates and their discovery: current progress and the application of mass spectrometry-based approaches." Biological Chemistry 397, no. 9 (September 1, 2016): 837–56. http://dx.doi.org/10.1515/hsz-2016-0174.
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Abstract The enzyme members of the dipeptidyl peptidase 4 (DPP4) gene family have the very unusual capacity to cleave the post-proline bond to release dipeptides from the N-terminus of peptide/protein substrates. DPP4 and related enzymes are current and potential therapeutic targets in the treatment of type II diabetes, inflammatory conditions and cancer. Despite this, the precise biological function of individual dipeptidyl peptidases (DPPs), other than DPP4, and knowledge of their in vivo substrates remains largely unknown. For many years, identification of physiological DPP substrates has been difficult due to limitations in the available tools. Now, with advances in mass spectrometry based approaches, we can discover DPP substrates on a system wide-scale. Application of these approaches has helped reveal some of the in vivo natural substrates of DPP8 and DPP9 and their unique biological roles. In this review, we provide a general overview of some tools and approaches available for protease substrate discovery and their applicability to the DPPs with a specific focus on DPP9 substrates. This review provides comment upon potential approaches for future substrate elucidation.
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Butler, Randall, Nicholas Ferrell, Derek Hansford, and Rajesh Naik. "Soft lithography-mediated microscale patterning of silica on diverse substrates." Journal of Materials Research 24, no. 5 (May 2009): 1632–38. http://dx.doi.org/10.1557/jmr.2009.0200.
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We have developed a soft lithography-based process to create microscale patterns of silica on a diverse array of substrates. A sacrificial polymer layer was first patterned using a micromolding technique. A peptide was adsorbed on the substrate and the sacrificial layer was removed. The patterned peptide template then catalyzed the deposition of silica from a silicic acid solution. With this procedure, we have created both continuous and discontinuous silica patterns on metallic, ceramic, and polymer substrates.
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Tagore, Debarati M., Whitney M. Nolte, John M. Neveu, Roberto Rangel, Liliana Guzman-Rojas, Renata Pasqualini, Wadih Arap, William S. Lane, and Alan Saghatelian. "Peptidase substrates via global peptide profiling." Nature Chemical Biology 5, no. 1 (November 16, 2008): 23–25. http://dx.doi.org/10.1038/nchembio.126.
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MCMURRAY, JOHN S., RAYMOND J. A. BUDDE, and DOUGLAS F. DYCKES. "Cyclic peptide substrates of pp60c-src." International Journal of Peptide and Protein Research 42, no. 3 (January 12, 2009): 209–15. http://dx.doi.org/10.1111/j.1399-3011.1993.tb00134.x.
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