Substrate Library Research Articles

Prediction and assignment of function for a divergent N-succinyl amino acid racemase

The protein databases contain many proteins with unknown function. A computational approach for predicting ligand specificity that requires only the sequence of the unknown protein would be valuable for directing experiment-based assignment of function. We focused on a family of unknown proteins in the mechanistically diverse enolase superfamily and used two approaches to assign function: (i) enzymatic assays using libraries of potential substrates, and (ii) in silico docking of the same libraries using a homology model based on the most similar (35% sequence identity) characterized protein. The results matched closely; an experimentally determined structure confirmed the predicted structure of the substrate-liganded complex. We assigned the N-succinyl arginine/lysine racemase function to the family, correcting the annotation (L-Ala-D/L-Glu epimerase) based on the function of the most similar characterized homolog. These studies establish that ligand docking to a homology model can facilitate functional assignment of unknown proteins by restricting the identities of the possible substrates that must be experimentally tested.

From Peptide to Protein: Comparative Analysis of the Substrate Specificity of N-Linked Glycosylation in C. jejuni

The gram-negative bacterium Campylobacter jejuni was recently discovered to contain a general N-linked protein glycosylation pathway. Central to this pathway is PglB, a homologue of the Stt3p subunit of the eukaryotic oligosaccharyl transferase (OT), which is involved in the transfer of an oligosaccharide from a polyisoprenyl pyrophosphate carrier to the asparagine side chain of proteins within the conserved glycosylation sites D/E-X1-N-X2-S/T, where X1 and X2 can be any amino acids except proline. Using a library of peptide substrates and a quantitative radioactivity-based in vitro assay, we assessed the amino acids at each position of the consensus glycosylation sequence for their impact on glycosylation efficiency, whereby the sequence DQNAT was found to be the optimal acceptor substrate. In the context of a full-length folded protein, the differences between variations of the glycosylation sequences were found to be consistent with the trends observed from their peptidyl counterparts, though less dramatic because of additional influences. In addition to characterizing the acceptor preferences of PglB, we also assessed the selectivity toward the glycan donor. Interestingly, despite recent reports of relaxed selectivity toward the glycan donor, PglB was not found to be capable of utilizing glycosyl donors such as dolichyl-pyrophosphate-chitobiose, which is the minimum substrate for the eukaryotic OT process.

Factors affecting the substrate specificity of histone deacetylases

Histone deacetylases (HDACs) catalyze the deacetylation of ε-acetyl-lysine residues within the N-terminal tail of core histones and thereby mediate changes in the chromatin structure and regulate gene expression in eukaryotic cells. So far, surprisingly little is known about the substrate specificities of different HDACs. Here, we prepared a library of fluorogenic tripeptidic substrates of the general format Ac-P -2-P -1-Lys(Ac)-MCA (P -1, P -2 = all amino acids except cysteine) and measured their HDAC-dependent conversion in a standard fluorogenic HDAC assay. Different HDAC subtypes can be ranked according to their substrate selectivity: HDAH > HDAC8 > HDAC1 > HDAC3 > HDAC6. HDAC1, HDAC3, and HDAC6 exhibit a similar specificity profile, whereas both HDAC8 and HDAH have rather distinct profiles. Furthermore, it was shown that second-site modification (e.g., phosphorylation) of substrate sequences as well as corepressor binding can modulate the selectivity of enzymatic substrate conversion.

Selection of New Chromogenic Substrates of Serine Proteinases Using Combinatorial Chemistry Methods

Chemical synthesis, physicochemical characterization and kinetic investigations of a tetrapeptide library of chromogenic substrates containing the amide of 5-amino-2-nitrobenzoic acid (Anb(5,2)-NH(2)) at their C-termini are reported. Anb(5,2)-NH(2) served as a chromophore released upon enzymatic action. The library consisting of 9567 peptides was synthesized using the portioning-mixing method and was screened against bovine a-chymotrypsin and human leukocyte elastase in solution applying an iterative approach. The selected chromogenic substrates were resynthesized and further modified at their N- and C-termini. Finally, two sequences, Z-Phe-Ala-Thr-Tyr-Anb(5,2)-NH(2) and Z-Phe-Phe-Pro-Val-Anb(5,2)-NH(2), were obtained as highly specific substrates for bovine alpha-chymotrypsin and human leukocyte elastase, respectively. The method of synthesis and selection of chromogenic substrates of serine proteinases described herein is straightforward and can be applied to design substrates for other proteases.

Substrate activity screening (SAS): a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery

Substrate activity screening (SAS) is a fragment-based method for the rapid development of novel substrates and their conversion into non-peptidic inhibitors of Cys and Ser proteases. The method consists of three steps: (i) a library of N-acyl aminocoumarins with diverse, low-molecular-weight N-acyl groups is screened to identify protease substrates using a simple fluorescence-based assay; (ii) the identified N-acyl aminocoumarin substrates are optimized by rapid analog synthesis and evaluation; and (iii) the optimized substrates are converted into inhibitors by direct replacement of the aminocoumarin with known mechanism-based pharmacophores. This protocol describes a general procedure for the solid-phase synthesis of a library of N-acyl aminocoumarin substrates and the screening procedure to identify weak binding substrates.

High-Throughput Screening for Functional Adenosine to Inosine RNA Editing Systems

Deamination of adenosines within messenger RNAs catalyzed by adenosine deaminases that act on RNA (ADAR) enzymes generates inosines at the corresponding nucleotide positions. Because inosine is decoded as guanosine, this reaction can lead to codon changes and the introduction of amino acids into a gene product not encoded in the gene. Translation of the different coding strands created by this process leads to protein structural diversity in the parent organism and is necessary for nervous system function in metazoa. The basis for selective editing of adenosines within certain codons is not well understood at the structural/biochemical level. Here we describe a high-throughput screen for ADAR/substrate combinations capable of RNA editing that can be carried out in the yeast Saccharomyces cerevisiae growing on agar plates. Results from the screening of libraries of human ADAR2 mutants and libraries of RNA substrates shed light on structure-activity relationships in the ADAR-catalyzed adenosine to inosine RNA editing reaction.

Insights to Substrate Binding and Processing by West Nile Virus NS3 Protease through Combined Modeling, Protease Mutagenesis, and Kinetic Studies

West Nile Virus is becoming a widespread pathogen, infecting people on at least four continents with no effective treatment for these infections or many of their associated pathologies. A key enzyme that is essential for viral replication is the viral protease NS2B-NS3, which is highly conserved among all flaviviruses. Using a combination of molecular fitting of substrates to the active site of the crystal structure of NS3, site-directed enzyme and cofactor mutagenesis, and kinetic studies on proteolytic processing of panels of short peptide substrates, we have identified important enzyme-substrate interactions that define substrate specificity for NS3 protease. In addition to better understanding the involvement of S2, S3, and S4 enzyme residues in substrate binding, a residue within cofactor NS2B has been found to strongly influence the preference of flavivirus proteases for lysine or arginine at P2 in substrates. Optimization of tetrapeptide substrates for enhanced protease affinity and processing efficiency has also provided important clues for developing inhibitors of West Nile Virus infection.

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

A large family of UDP-GalNAc:polypeptide alpha-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.

New Efficient Substrates for Semicarbazide-Sensitive Amine Oxidase/VAP-1 Enzyme: Analysis by SARs and Computational Docking

Structure activity relationships for semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1) were studied using a library of arylalkylamine substrates, with the aim of contributing to the discovery of more efficient SSAO substrates. Experimental data were contrasted with computational docking studies, thereby allowing us to examine the mechanism and substrate-binding affinity of SSAO and thus contribute to the discovery of more efficient SSAO substrates and provide a structural basis for their interactions. We also built a model of the mouse SSAO structure, which provides several structural rationales for interspecies differences in SSAO substrate selectivity and reveals new trends in SSAO substrate recognition. In this context, we identified novel efficient substrates for human SSAO that can be used as a lead for the discovery of antidiabetic agents.

Serpin1 of Arabidopsis thaliana is a Suicide Inhibitor for Metacaspase 9

Metacaspases are distant relatives of animal caspases found in plants, fungi and protozoa. We demonstrated previously that two type II metacaspases of Arabidopsis thaliana, AtMC4 and AtMC9 are Arg/Lys-specific cysteine-dependent proteases. We screened a combinatorial tetrapeptide library of 130,321 substrates with AtMC9. Here, we show that AtMC9 is a strict Arg/Lys-specific protease. Based on the position-specific scoring matrix derived from the substrate library results, the tetrapeptide Val-Arg-Pro-Arg was identified as an optimized substrate. AtMC9 had a k cat/ K M of 4.6 × 10 5 M − 1 s − 1 for Ac-Val-Arg-Pro-Arg-amido-4-methyl-coumarin, representing a more than 10-fold improvement over existing fluorogenic substrates. A yeast two-hybrid screen with catalytically inactive AtMC9 as bait identified a serine protease inhibitor, designated AtSerpin1, which was found to be a potent inhibitor of AtMC9 activity in vitro through cleavage of its reactive center loop and covalent binding to AtMC9. On the basis of the substrate profiling of AtMC9 and confirmation through site-directed mutagenesis, the inhibitory P4-P1 cleavage site of AtSerpin1 was determined to be Ile-Lys-Leu-Arg 351. Further mutagenesis of the AtSerpin1 inhibitory cleavage site modulated AtMC9 inhibition positively or negatively. Both AtMC9 and AtSerpin1 were localized in the extracellular space, suggesting an in vivo interaction as well. To our knowledge, this is the first report of plant protease inhibition by a plant serpin.

Specificity Profiling of Seven Human Tissue Kallikreins Reveals Individual Subsite Preferences

Human tissue kallikreins (hKs) form a family of 15 closely related (chymo)trypsin-like serine proteinases. These tissue kallikreins are expressed in a wide range of tissues including the central nervous system, the salivary gland, and endocrine-regulated tissues, such as prostate, breast, or testis, and may have diverse physiological functions. For several tissue kallikreins, a clear correlation has been established between expression and different types of cancer. For example, the prostate-specific antigen (PSA or hK3) serves as tumor marker and is used to monitor therapy response. Using a novel strategy, we have cloned, expressed in Escherichia coli or in insect cells, refolded, activated, and purified the seven human tissue kallikreins hK3/PSA, hK4, hK5, hK6, hK7, hK10, and hK11. Moreover, we have determined their extended substrate specificity for the nonprime side using a positional scanning combinatorial library of tetrapeptide substrates. hK3/PSA and hK7 exhibited a chymotrypsin-like specificity preferring large hydrophobic or polar residues at the P1 position. In contrast, hK4, hK5, and less stringent hK6 displayed a trypsin-like specificity with strong preference for P1-Arg, whereas hK10 and hK11 showed an ambivalent specificity, accepting both basic and large aliphatic P1 residues. The extended substrate specificity profiles are in good agreement with known substrate cleavage sites but also in accord with experimentally solved (hK4, hK6, and hK7) or modeled structures. The specificity profiles may lead to a better understanding of human tissue kallikrein functions and assist in identifying their physiological protein substrates as well as in designing more selective inhibitors.

Mapping coral reef benthic substrates using hyperspectral space-borne images and spectral libraries

The suitability of Hyperion, the first civilian hyperspectral sensor in space, for mapping coral reef benthic substrates has been investigated in this study. An image of Cairns Reef, in the northern section of the Australian Great Barrier Reef (GBR), was acquired during Hyperion Calibration and Validation activities. A field experiment was carried out on Cairns Reef to collect information about the optical properties of the water in the area and to map benthic cover by means of video transects. An approach was used to classify the Hyperion image that allows convenient mapping of benthic substrate type and water depth simultaneously. A hyperspectral library of radiance at Hyperion altitude was simulated using a spectral library of GBR benthic substrates, a Hydrolight 4.1 radiative transfer model, and an in-house atmospheric model similar to Modtran-3.7. The image was then classified using the Hyperion at-sensor radiance data and the Spectral Angle Mapper metric using the simulated at-sensor spectral library. The results suggest that using spectral libraries created with forward modelling from the sea bottom to top of the atmosphere are useful tools for interpretation of reefs and can give better results in image classification than classifying the image after removing atmospheric and water column effects. The results also suggest that bottom type and water depth can be separated and mapped simultaneously provided hyperspectral data is available.

The active essential CFNS3d protein complex

The NS2B-NS3 protease complex is essential for the replication of dengue virus, which is the etiologic agent of dengue and hemorrhagic fevers, diseases that are a burden for the tropical and subtropical areas of the world. The active form of the NS3 protease linked to the 40 residues of the NS2B cofactor shows highly flexible and disordered region(s) that are responsible for its high propensity to aggregate at the concentrations necessary for NMR spectroscopy studies or for crystallization. Limited proteolysis of this active form of the protease enabled us to obtain a folded and new essential form of the NS2B-NS3 protease complex. We found that the region from residues D50 to E80 of NS2B interacts directly and strongly with the NS3 protease domain. The proteolytic activity of the noncovalently binding complex was determined by a rapid and continuous fluorescence resonance energy transfer activity assay using a depsipeptide substrate. The new protein-cofactor complex obtained, encompassing the NS2B fragment (D50-E80) and the NS3 protease, shows proteolytic activity. The (1)H-(15)N-heteronuclear single quantum coherence spectrum of the isotopically enriched protein complex shows good cross-peak dispersion; this is indicative of a stable folded state. Our results significantly complement the X-ray structure of the NS2B-NS3pro complex published recently. Moreover, these results open the way to performing direct structural and interaction studies in solution on a new active NS2B-NS3pro complex with libraries of substrates and inhibitors in order to identify new drugs that prevent viral polyprotein processing.

Approaches to Selective Peptidic Inhibitors of Factor Xa

Inhibitors of procoagulant enzymes, such as factor Xa (fXa) and thrombin, are important for treating thrombosis. Thrombin has complex pro- and anti-coagulant roles and thus fXa is thought to represent an ideal target. Discrete kcat and Km values for cleavage of a library of fluorescence-quenched substrates by fXa were determined. The results highlighted the low selectivity of fXa at its prime sites, and its poor efficiency compared with thrombin, creating a challenge for the design of fXa-specific peptidic inhibitors. We hypothesized that Km rather than kcat/Km values may be better indicators of inhibitor potential for a peptidic sequence, leading us to design peptide sequences for both fXa and thrombin in three forms: fluorescence-quenched substrates, standard alpha-peptides and peptides containing a beta-homoarginine at the cleavage site. Kinetic and competitive inhibition assays with both fXa and thrombin showed the fluorescence-quenched substrates to be the best inhibitors, while the inhibitory effect of the beta-homoarginine peptides varied for the two proteases. Importantly, fXa was inhibited to a much greater extent by the beta-peptides than the corresponding alpha-peptides, resulting in an increased selectivity for fXa inhibition over thrombin for those peptides containing a beta-amino acid at the cleavage site.

Expression and characterization of recombinant γ-tryptase

Tryptases are trypsin-like serine proteases whose expression is restricted to cells of hematopoietic origin, notably mast cells. γ-Tryptase, a recently described member of the family also known as transmembrane tryptase (TMT), is a membrane-bound serine protease found in the secretory granules or on the surface of degranulated mast cells. The 321 amino acid protein contains an 18 amino acid propeptide linked to the catalytic domain (cd), followed by a single-span transmembrane domain. γ-Tryptase is distinguished from other human mast cell tryptases by the presence of two unique cysteine residues, Cys 26 and Cys 145, that are predicted to form an intra-molecular disulfide bond linking the propeptide to the catalytic domain to form the mature, membrane-anchored two-chain enzyme. We expressed γ-tryptase as either a soluble, single-chain enzyme with a C-terminal His tag (cd γ-tryptase) or as a soluble pseudozymogen activated by enterokinase cleavage to form a two-chain protein with an N-terminal His tag (tc γ-tryptase). Both recombinant proteins were expressed at high levels in Pichia pastoris and purified by affinity chromatography. The two forms of γ-tryptase exhibit comparable kinetic parameters, indicating the propeptide does not contribute significantly to the substrate affinity or activity of the protease. Substrate and inhibitor library screening indicate that γ-tryptase possesses a substrate preference and inhibitor profile distinct from that of β-tryptase. Although the role of γ-tryptase in mast cell function is unknown, our results suggest that it is likely to be distinct from that of β-tryptase.

Sequence‐Selective Peptide‐Binding Metallomacrocycles.

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Protease specificity determination by using cellular libraries of peptide substrates (CLiPS)

We report a general combinatorial approach to identify optimal substrates of a given protease by using quantitative kinetic screening of cellular libraries of peptide substrates (CLiPS). A whole-cell protease activity assay was developed by displaying fluorescent reporter substrates on the surface of Escherichia coli as N-terminal fusions. This approach enabled generation of substrate libraries of arbitrary amino acid composition and length that are self-renewing. Substrate hydrolysis by a target protease was measured quantitatively via changes in whole-cell fluorescence by using FACS. FACS enabled efficient screening to identify optimal substrates for a given protease and characterize their cleavage kinetics. The utility of CLiPS was demonstrated by determining the substrate specificity of two unrelated proteases, caspase-3 and enteropeptidase (or enterokinase). CLiPS unambiguously identified the caspase-3 consensus cleavage sequence DXVDG. Enteropeptidase was unexpectedly promiscuous, but exhibited a preference for substrates with the motif (D/E)RM, which were cleaved substantially faster than the canonical DDDDK recognition sequence, widely used for protein purification. CLiPS provides a straightforward and versatile approach to determine protease specificity and discover optimal substrates on the basis of cleavage kinetics.

Hep27, a member of the short-chain dehydrogenase/reductase family, is an NADPH-dependent dicarbonyl reductase expressed in vascular endothelial tissue.

Human Hep27 was originally isolated from growth-arrested HepG2 cells and identified as a member of the superfamily of short-chain dehydrogenases/reductases (SDR). Its substrate specificity has not been determined, but a cross-species comparison suggests that it occurs in widely divergent species, such as human, Cenorhabditis elegans, Drosophila and Arabidopsis thaliana. In this study, Hep27 was expressed as a His(6) fusion protein, and subjected to a substrate screen, using a compound library of SDR substrates, comprising steroids, retinoids, sugars and carbonyl compounds. Whereas no steroid dehydrogenase or retinoid activity was detected, it was found that Hep27 catalyzed the NADPH-dependent reduction of dicarbonyl compounds, like 3,4-hexanedione and 1-phenyl-1,2-propanedione with similar turnover numbers as DCXR (a mitochondrial dicarbonyl reductase/xylulose reductase). In contrast, Hep27 does not convert sugar substrates like xylulose or threose. Based on its substrate specificity and expression in endothelial tissues, it is suggested that Hep27 functions as a dicarbonyl reductase in enzymatic inactivation of reactive carbonyls, involved in covalent modification of cellular components.

Mapping Protease Substrates by Using a Biotinylated Phage Substrate Library

We describe a bacteriophage M13 substrate library encoding the AviTag (BirA substrate) and combinatorial heptamer peptides displayed at the N terminus of the mature form of capsid protein III. Phages are biotinylated efficiently (> or = 50%) when grown in E. coli cells coexpressing BirA, and such viral particles can be immobilized on a streptavidin-coated support and released by protease cleavage within the combinatorial peptide. We have used this library to map the specificity of human Factor Xa and a neuropeptidase, neurolysin (EC3.4.24.16). Validation by analysis of isolated peptide substrates has revealed that neurolysin recognizes the motif hydrophobic-X-Pro-Arg-hydrophobic, where Arg-hydrophobic is the scissile bond.

Functional phenotyping of human plasma using a 361‐fluorogenic substrate biosensing microarray

A microarray presenting glycerol nanodroplets of fluorogenic peptide substrates was used as a biosensor for the detection of multiple enzyme activities within human plasma. Using 10 different plasma proteases (kallikrein, factor XIIa, factor XIa, factor IXa, factor VIIa, factor Xa, thrombin, activated protein C, uPA and plasmin) and a 361-compound fluorogenic substrate library (Ac-Ala-P3-P2-Arg-coumarin for P = all amino acids except Cys), a database was created for deconvoluting the relative activity of each individual enzyme signal in human plasma treated with various activators (calcium, kaolin, or uPA). Three separate deconvolution protocols were tested: searching for "optimal" sensing substrate sequences for a set of 5 enzymes and using these substrates to detect protease signals in plasma; ranking the "optimal" sensing substrates for 10 proteases using local error minimization, resulting in a set of substrates which were bundled via weighted averaging into a super-pixel that had biosensing properties not obtainable by any individual fluorogenic substrate; and treating each 361-element map measured for each plasma preparation as a weighted sum of the 10 maps obtained for the 10 purified enzymes using a global error minimization. The similarity of the results from these latter two protocols indicated that a small subset of <90 substrates contained the majority of biochemical information. The results were consistent with the state of the coagulation cascade expected when treated with the given activators. This method may allow development of future biosensors using minimal and non-specific markers. These substrates can be applied to real-time diagnostic biosensing of complex protease mixtures.