Evaluation of protein farnesyltransferase substrate specificity using synthetic peptide libraries.
Ontology highlight
ABSTRACT:
SUBMITTER: Krzysiak AJ
PROVIDER: S-EPMC2077820 | biostudies-literature | 2007 Oct
REPOSITORIES: biostudies-literature
Publications
Evaluation of protein farnesyltransferase substrate specificity using synthetic peptide libraries.
Krzysiak Amanda J AJ Scott Sarah A SA Hicks Katherine A KA Fierke Carol A CA Gibbs Richard A RA
Bioorganic & medicinal chemistry letters 20070816 20
Farnesylation, catalyzed by protein farnesyltransferase (FTase), is an important post-translational modification guiding cellular localization. Recently predictive models for identifying FTase substrates have been reported. Here we evaluate these models through screening of dansylated-GCaaS peptides, which also provides new insights into the protein substrate selectivity of FTase. ...[more]
Similar Datasets
Project description:Protein farnesytransferase (PFTase) catalyzes the farnesylation of proteins with a carboxy-terminal tetrapeptide sequence denoted as a Ca1a2X box. To explore the specificity of this enzyme, an important therapeutic target, solid-phase peptide synthesis in concert with a peptide inversion strategy was used to prepare two libraries, each containing 380 peptides. The libraries were screened using an alkyne-containing isoprenoid analogue followed by click chemistry with biotin azide and subsequent visualization with streptavidin-AP. Screening of the CVa2X and CCa2X libraries with Rattus norvegicus PFTase revealed reaction by many known recognition sequences as well as numerous unknown ones. Some of the latter occur in the genomes of bacteria and viruses and may be important for pathogenesis, suggesting new targets for therapeutic intervention. Screening of the CVa2X library with alkyne-functionalized isoprenoid substrates showed that those prepared from C10 or C15 precursors gave similar results, whereas the analogue synthesized from a C5 unit gave a different pattern of reactivity. Lastly, the substrate specificities of PFTases from three organisms (R. norvegicus, Saccharomyces cerevisiae, and Candida albicans) were compared using CVa2X libraries. R. norvegicus PFTase was found to share more peptide substrates with S. cerevisiae PFTase than with C. albicans PFTase. In general, this method is a highly efficient strategy for rapidly probing the specificity of this important enzyme.
| S-EPMC4136699 | biostudies-literature
Project description:A robust, high-throughput method has been developed to screen one-bead-one-compound peptide libraries to systematically profile the sequence specificity of protein kinases. Its ability to provide individual sequences of the preferred substrates permits the identification of sequence contextual effects and nonpermissive residues. Application of the library method to kinases Pim1, MKK6, and Csk revealed that Pim1 and Csk are highly active toward peptide substrates and recognize specific sequence motifs, whereas MKK6 has little activity or sequence selectivity against peptide substrates. Pim1 recognizes peptide substrates of the consensus RXR(H/R)X(S/T); it accepts essentially any amino acid at the S/T-2 and S/T+1 positions, but strongly disfavors acidic residues (Asp or Glu) at the S/T-2 position and a proline residue at the S/T+1 position. The selected Csk substrates show strong sequence covariance and fall into two classes with the consensus sequences of (D/E)EPIYϕXϕ and (D/E)(E/D)S(E/D/I)YϕXϕ (where X is any amino acid and ϕ is a hydrophobic amino acid). Database searches and in vitro kinase assays identified phosphatase PTP-PEST as a Pim1 substrate and phosphatase SHP-1 as a potential Csk substrate. Our results demonstrate that the sequence specificity of protein kinases is defined not only by favorable interactions between permissive residue(s) on the substrate and their cognate binding site(s) on the kinase but also by repulsive interactions between the kinase and nonpermissive residue(s).
| S-EPMC3773219 | biostudies-literature
Project description:Through processing peptide and protein C termini, carboxypeptidases participate in the regulation of various biological processes. Few tools are however available to study the substrate specificity profiles of these enzymes. We developed a proteome-derived peptide library approach to study the substrate preferences of carboxypeptidases. Our COFRADIC-based approach takes advantage of the distinct chromatographic behavior of intact peptides and the proteolytic products generated by the action of carboxypeptidases, to enrich the latter and facilitate its MS-based identification. Two different peptide libraries, generated either by chymotrypsin or by metalloendopeptidase Lys-N, were used to determine the substrate preferences of human metallocarboxypeptidases A1 (hCPA1), A2 (hCPA2), and A4 (hCPA4). In addition, our approach allowed us to delineate the substrate specificity profile of mouse mast cell carboxypeptidase (MC-CPA or mCPA3), a carboxypeptidase suggested to function in innate immune responses regulation and mast cell granule homeostasis, but which thus far lacked a detailed analysis of its substrate preferences. mCPA3 was here shown to preferentially remove bulky aromatic amino acids, similar to hCPA2. This was also shown by a hierarchical cluster analysis, grouping hCPA1 close to hCPA4 in terms of its P1 primed substrate specificity, whereas hCPA2 and mCPA3 cluster separately. The specificity profile of mCPA3 may further aid to elucidate the function of this mast cell carboxypeptidase and its biological substrate repertoire. Finally, we used this approach to evaluate the substrate preferences of prolylcarboxypeptidase, a serine carboxypeptidase shown to cleave C-terminal amino acids linked to proline and alanine.
| S-EPMC3734572 | biostudies-literature
Project description:Protein prenylation occurs in the protozoan that causes African sleeping sickness (Trypanosoma brucei), and the protein farnesyltransferase appears to be a good target for developing drugs. We have cloned the alpha- and beta-subunits of T. brucei protein farnesyltransferase (TB-PFT) using nucleic acid probes designed from partial amino acid sequences obtained from the enzyme purified from insect stage parasites. TB-PFT is expressed in both bloodstream and insect stage parasites. Enzymatically active TB-PFT was produced by heterologous expression in Escherichia coli. Compared with mammalian protein farnesyltransferases, TB-PFT contains a number of inserts of >25 residues in both subunits that reside on the surface of the enzyme in turns linking adjacent alpha-helices. Substrate specificity studies with a series of 20 peptides SSCALX (where X indicates a naturally occurring amino acid) show that the recombinant enzyme behaves identically to the native enzyme and displays distinct specificity compared with mammalian protein farnesyltransferase. TB-PFT prefers Gln and Met at the X position but not Ser, Thr, or Cys, which are good substrates for mammalian protein farnesyltransferase. A structural homology model of the active site of TB-PFT provides a basis for understanding structure-activity relations among substrates and CAAX mimetic inhibitors.
| S-EPMC2913713 | biostudies-literature
Project description:Proteases comprise the class of enzymes that catalyzes the hydrolysis of peptide bonds, thereby playing a pivotal role in many aspects of life. The amino acids surrounding the scissile bond determine the susceptibility toward protease-mediated hydrolysis. A detailed understanding of the cleavage specificity of a protease can lead to the identification of its endogenous substrates, while it is also essential for the design of inhibitors. Although many methods for protease activity and specificity profiling exist, none of these combine the advantages of combinatorial synthetic libraries, i.e., high diversity, equimolar concentration, custom design regarding peptide length, and randomization, with the sensitivity and detection power of mass spectrometry. Here, we developed such a method and applied it to study a group of bacterial metalloproteases that have the unique specificity to cleave between two prolines, i.e., Pro-Pro endopeptidases (PPEPs). We not only confirmed the prime-side specificity of PPEP-1 and PPEP-2, but also revealed some new unexpected peptide substrates. Moreover, we have characterized a new PPEP (PPEP-3) that has a prime-side specificity that is very different from that of the other two PPEPs. Importantly, the approach that we present in this study is generic and can be extended to investigate the specificity of other proteases.
| S-EPMC10413326 | biostudies-literature
Project description:Characterizing protein kinase substrate specificity motifs represents a powerful step in elucidating kinase-signaling cascades. The protocol described here uses a bacterial system to evaluate kinase specificity motifs in vivo, without the need for radioactive ATP. The human kinase of interest is cloned into a heterologous bacterial expression vector and allowed to phosphorylate E. coli proteins in vivo, consistent with its endogenous substrate preferences. The cells are lysed, and the bacterial proteins are digested into peptides and phosphoenriched using bulk TiO2 . The pooled phosphopeptides are identified by tandem mass spectrometry, and bioinformatically analyzed using the pLogo visualization tool. The ProPeL approach allows for detailed characterization of wildtype kinase specificity motifs, identification of specificity drift due to kinase mutations, and evaluation of kinase residue structure-function relationships. © 2018 by John Wiley & Sons, Inc.
| S-EPMC6014603 | biostudies-literature
Project description: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.
| S-EPMC1456804 | biostudies-literature
Project description:Proteases comprise the class of enzymes that catalyze the hydrolysis of peptide bonds, thereby playing a pivotal role in many aspects of life. The amino acids surrounding the scissile bond determine the susceptibility towards protease-mediated hydrolysis. A detailed understanding of the cleavage specificity of a protease can lead to the identification of its endogenous substrates, while it is also essential for the design of inhibitors. We developed a new method which combines the high diversity of a combinatorial synthetic peptide library with the sensitivity and detection power of mass spectrometry to determine protease cleavage specificity. We applied this method to study a group of bacterial metalloproteases that have the unique specificity to cleave between two prolines, i.e. Pro-Pro endopeptidases (PPEPs). We not only confirmed the prime-side specificity of PPEP-1 and PPEP-2, but also revealed some new unexpected peptide substrates. Moreover, we have characterized a new PPEP (PPEP-3) which has a prime-side specificity that is very different from that of the other two PPEPs. Importantly, the approach that we present in this study is generic and can be extended to investigate the specificity of other proteases.
2023-08-01 | PXD038277 | Pride
Project description:Libraries of randomised peptides displayed on phages or viral particles are essential tools in a wide spectrum of applications. However, there is only limited understanding of a library's fundamental dynamics and the influences of encoding schemes and sizes on their quality. Numeric properties of libraries, such as the expected number of different peptides and the library's coverage, have long been in use as measures of a library's quality. Here, we present a graphical framework of these measures together with a library's relative efficiency to help to describe libraries in enough detail for researchers to plan new experiments in a more informed manner. In particular, these values allow us to answer-in a probabilistic fashion-the question of whether a specific library does indeed contain one of the "best" possible peptides. The framework is implemented in a web-interface based on two packages, discreteRV and peptider, to the statistical software environment R. We further provide a user-friendly web-interface called PeLiCa (Peptide Library Calculator, http://www.pelica.org), allowing scientists to plan and analyse their peptide libraries.
| S-EPMC4456392 | biostudies-literature
Project description:Prenylation is a posttranslational modification whereby C-terminal lipidation leads to protein localization to membranes. A C-terminal "Ca(1)a(2)X" sequence has been proposed as the recognition motif for two prenylation enzymes, protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I. To define the parameters involved in recognition of the a(2) residue, we performed structure-activity analysis which indicates that FTase discriminates between peptide substrates based on both the hydrophobicity and steric volume of the side chain at the a(2) position. For nonpolar side chains, the dependence of the reactivity on side chain volume at this position forms a pyramidal pattern with a maximal activity near the steric volume of valine. This discrimination occurs at a step in the kinetic mechanism that is at or before the farnesylation step. Furthermore, a(2) selectivity is also affected by the identity of the adjacent X residue, leading to context-dependent substrate recognition. Context-dependent a(2) selectivity suggests that FTase recognizes the sequence downstream of the conserved cysteine as a set of two or three cooperative, interconnected recognition elements as opposed to three independent amino acids. These findings expand the pool of proposed FTase substrates in cells. A better understanding of the molecular recognition of substrates performed by FTase will aid in both designing new FTase inhibitors as therapeutic agents and characterizing proteins involved in prenylation-dependent cellular pathways.
| S-EPMC2765569 | biostudies-literature