10 research outputs found

    Identification of a Novel Class of Farnesylation Targets by Structure-Based Modeling of Binding Specificity

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    Farnesylation is an important post-translational modification catalyzed by farnesyltransferase (FTase). Until recently it was believed that a C-terminal CaaX motif is required for farnesylation, but recent experiments have revealed larger substrate diversity. In this study, we propose a general structural modeling scheme to account for peptide binding specificity and recapitulate the experimentally derived selectivity profile of FTase in vitro. In addition to highly accurate recovery of known FTase targets, we also identify a range of novel potential targets in the human genome, including a new substrate class with an acidic C-terminal residue (CxxD/E). In vitro experiments verified farnesylation of 26/29 tested peptides, including both novel human targets, as well as peptides predicted to tightly bind FTase. This study extends the putative range of biological farnesylation substrates. Moreover, it suggests that the ability of a peptide to bind FTase is a main determinant for the farnesylation reaction. Finally, simple adaptation of our approach can contribute to more accurate and complete elucidation of peptide-mediated interactions and modifications in the cell

    Enriching Peptide Libraries for Binding Affinity and Specificity Through Computationally Directed Library Design

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    Peptide reagents with high affinity or specificity for their target protein interaction partner are of utility for many important applications. Optimization of peptide binding by screening large libraries is a proven and powerful approach. Libraries designed to be enriched in peptide sequences that are predicted to have desired affinity or specificity characteristics are more likely to yield success than random mutagenesis. We present a library optimization method in which the choice of amino acids to encode at each peptide position can be guided by available experimental data or structure-based predictions. We discuss how to use analysis of predicted library performance to inform rounds of library design. Finally, we include protocols for more complex library design procedures that consider the chemical diversity of the amino acids at each peptide position and optimize a library score based on a user-specified input model.National Institute of General Medical Sciences (U.S.) (Award R01 GM110048

    Molecular Recognition of Substrates by Protein Farnesyltransferase and Geranylgeranyltransferase-I.

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    Prenylation is an important post-translational modification that targets proteins to the cellular membrane. Farnesyltransferase (FTase) catalyzes the attachment of the 15-carbon farnesyl moiety from farnesyldiphosphate to a cysteine near the C-terminus of a protein, while geranylgeranyltransferase-I (GGTase-I) catalyzes the analogous attachment of the 20-carbon geranylgeranyl group from geranylgeranyldiphosphate. Substrates of the prenyltransferases are involved in a myriad of signaling pathways and processes within the cell, therefore inhibitors targeting FTase and GGTase-I are being developed as therapeutics for treatment of diseases such as cancer, parasitic infection, and progeria. FTase and GGTase-I were proposed to recognize a Ca1a2X motif, where C is the cysteine where the prenyl group is attached, a1 and a2 are aliphatic amino acids, and X confers specificity between FTase and GGTase-I with X being methionine, serine, glutamine, and alanine for FTase and leucine or phenylalanine for GGTase-I. Recent work indicates that the Ca1a2X paradigm should be expanded; therefore, further studies are needed to define the prenylated proteome, to understand normal cellular processes, and to determine the targets of prenyltransferase inhibitors. In this study, we probed the molecular recognition of GGTase-I by testing a 400 peptide library for activity with GGTase-I. The enzyme modifies two classes of substrates: multiple turnover substrates (MTO) and single turnover-only (STO) which undergo chemistry but not product release. Statistical analysis was used to determine that MTO substrates typically follow the Ca1a2X definition, but the STO sequences are more diverse, further indicating GGTase-I recognizes a broader range of substrates. Additionally, with collaborators at the Hebrew University of Jerusalem, a computational program that predicts FTase substrates was developed, FlexPepBind. This novel method successfully predicted new peptide substrates with FTase and identified a new class of substrates containing a positively charged X residue. Lastly, to examine prenylation in vivo, we created a library of GFP-Ca1a2X fusion proteins and measured protein localization using fluorescence microscopy. The identity of the C-terminal sequence caused the proteins to localize to different cellular compartments presumably due to modification status. Together, these studies provide insight into the in vivo specificity of prenyltransferases and the involvement of prenylation in various cellular processes.Ph.D.Biological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/91519/1/lamphear_1.pd

    EXPANDING THE POTENTIAL PRENYLOME: PRENYLATION OF SHORTENED TARGET SUBSTRATES BY FTASE AND DEVELOPMENT OF FRET-BASED SYSTEM FOR DETECTING POTENTIALLY “SHUNTED” PROTEINS

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    Protein prenylation is a posttranslational modification involving the attachment of a C15 or C20 isoprenoid group to a cysteine residue near the C-terminus of the target substrate by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I), respectively. Both of these protein prenyltransferases recognize a C-terminal CaaX sequence in their protein substrates, but recent studies in yeast- and mammalian-based systems have demonstrated FTase can also accept sequences that diverge in length from the canonical four-amino acid motif, such as the recently reported five-amino acid C(x)3X motif. In this work, we further expand the substrate scope of FTase by demonstrating sequence-dependent farnesylation of shorter three-amino acid Cxx C-terminal sequences using both genetic and biochemical assays. Surprisingly, biochemical assays utilizing purified mammalian FTase and Cxx substrates reveal prenyl donor promiscuity leading to both farnesylation and geranylgeranylation of these sequences. The work herein expands the substrate pool of sequences that can be potentially prenylated, further refines our understanding of substrate recognition by FTase and GGTase-I and suggests the possibility of a new class of prenylated proteins within proteomes. To identify potential new Cxx substrates in human proteomes, we explored a FRET-based system using phosphodiesterase delta subunit (PDE) as the acceptor protein for potentially prenylated Cxx sequences. While not conclusive, this work lays the foundation for an assay not dependent on membrane localization as a signal for prenylation inside cells and suggests future studies to improve upon the utility of this assay. Lastly, this work demonstrates FTase’s flexibility in accepting a prenyl donor analogue with an azobenzene moiety that can be modulated with light. This establishes a potential new avenue for mediating membrane localization behavior of prenylated proteins

    REDEFINING THE SCOPE OF PRENYLATION: DISCOVERY OF “FORBIDDEN” SUBSTRATE RECOGNITION AND DEVELOPMENT OF METHODS UTILIZING PRENYLATED PROTEINS

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    Post-translational modifications play a central role in controlling biological function and cell behavior through changes in protein structure, activity, and localization. Prenylation is one such modification wherein a 15- or 20-carbon isoprenoid group is attached to a cysteine residue near the C-terminus of a substrate protein by one of three enzymes: protein farnesyltransferase (FTase), protein geranylgeranyltransferase type I (GGTase-I) or protein geranylgeranyltransferase type II (GGTase-II, also known as Rab GGTase). These covalent modifications can aid in protein association with cellular membranes, with this localization necessary for function of many prenylated proteins. FTase and GGTase-I have been proposed to recognize a four amino acid “Ca1a2X” C-terminal sequence based on biochemical, structural, and computational studies of these enzymes. However, recent genetic screening studies in yeast suggest the potential for FTase to prenylate sequences of the form -C(x)3X, with four amino acids downstream of the cysteine residue to be prenylated. The work herein begins to define the sequence scope for this -C(x)3X motif, establishes the biological relevance of this new class of prenyltransferase substrates in cells, and supports future investigation of the impact of these non-canonical prenylated proteins on cell behavior and biological function. With the discovery of new -C(x)3X recognition motifs in prenylation, new methods with which to identify proteins capable of being prenylated are required. To this end, we have explored the use of engineered FTase variants, specifically RL FTase, selected for the ability to prenylate substrate sequences that are unreactive with WT FTase. Combining this engineered FTase variant with functionalized FPP analogues yields a bioorthogonal selective technique for isolating target proteins, even in the presence of other prenyltransferase substrate proteins in cell lysates. The value of this method is demonstrated by selective pulldown of model fluorescent proteins in bacterial lysates in the presence of competitor proteins. The selectivity of FTase-catalyzed prenylation and the minimal size of the C-terminal FTase recognition motif render this approach applicable to a wide range of target proteins. A second quantitative method introduced here is Protein-Lipidation Quantitation (PLQ); a new method that can simultaneously measure the amounts of a non-lipidated substrate protein and its lipidated product in a cellular context. In PLQ, use of a fluorescent protein fused to the substrate under investigation allows for quantitative detection of both the non-lipidated substrate and the lipidated product. Upon prenylation in cells, the substrate and the product in these cell lysates are separated by surfactant-mediated capillary electrophoresis (CE) and quantitated by integrating fluorescence intensity over respective CE peaks. This work demonstrates the usefulness of PLQ both in principle and in application with its ability to confirm a link between a mutation in the p53 tumor suppressor gene and cellular prenylation activity. The quantitative capabilities of PLQ will allow researchers to address previously unanswered hypotheses regarding protein lipidation and its roles in cellular regulation and biological function

    Amélioration des propriétés pharmacocinétiques de peptides par différentes alkylations N-terminales

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    Modélisations moléculaires réalisés avec le logiciel HyperChem 8.L’effet de différentes alkylations sur l’activité biologique et la stabilité enzymatique d’un peptide linéaire L, énantiomère du modulateur allostérique des récepteurs prostaglandine F2α ont été étudiés. Dans une étude antérieure, le peptide D PDC-31 avait montré un potentiel d’inhibition des contractions du myomètre et permettait de retarder l’accouchement dans des modèles animaux et humains. Il est possible que le peptide L possède une activité semblable, mais les protéases, abondantes dans le tissu myométrial, le dégradent probablement avant qu’il ne puisse atteindre le site actif. La synthèse peptidique sur support solide suivie d’une amination réductive a permis d’obtenir différents peptides portant différentes chaines alkyle et PEG N-terminales. La protection de l’amine terminale par un groupement ortho-nitrobenzène sulfonyle suivie par une réaction de Mitsunobu a permis l’obtention d’un analogue portant une chaine farnesyle. Malgré le fait que ni l’analogue PEGylé, ni l’analogue farnesylé n’aient montrés la moindre activité, certains analogues alkylés se sont avérés actifs dans l’essai tissulaire de contractions myométriales. Le peptide L portant une chaine dodecyle s’est avéré posséder une activité statistiquement significative et reproductible. Qui plus est, l’analogue D du peptide possédant une chaine de 12 carbones s’est avéré posséder une activité inférieure à l’analogue L portant la même chaine, ce qui représente une perte d’activité significative par rapport au peptide D nonmodifié (PDC-31).The application of hydrophobic grafts to prolong the biological activity of rapidly metabolized peptides has been explored by modification of the L-peptide of the prostaglandin F2α receptor modulator PDC-31. The all-D peptide PDC-31 has previously been shown to inhibit myometrial contractions and delay labour in various animal models as well as in humans. The L-peptide may have activity; however, proteases, which are abundant in myometrial tissue, may likely degrade the peptide before it is capable of showing activity. Solid-phase peptide synthesis followed by Nterminal modification by reductive aminations with different aldehydes provided linear aliphatic alkyl and PEG-grafted peptide analogs. Alternatively, ortho-nitrobenzensulfonylation of the peptide followed by Mitsunobu alkylation with farnesol and deprotection gave a farnesylated analog. Although the PEG and fanesylated analogs exhibited no activity, certain N-alkyl analogs exhibited inhibitory activity on myometrial contractions, with the most active analog possessing a dodecyl chain. Moreover, the N-dodecyl analog of PDC-31, exhibited lower activity than its L-counterpart in the myometrial contraction assay, and with reduced potency relative to its unmodified structure

    Statins improve wound healing through inhibition of activation of the glucocorticoid receptor by farnesyl pirophosphate

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    Uvod: Glukokortikoidni hormoni (GH) su mimo svog anti-inflamatornog dejstva i jedni od najpoznatijih inhibitora zarastanja rana. Nedavno je pokazano da farnezil pirofosfat (FPP), ključni međuprodukt mevalonatskog puta sinteze holesterola i farnezilacije proteina može delovati i kao agonista za nekoliko nuklearnih hormonskih receptora uključujući glukokortikodni receptor (GR). Interesantno je da statini, lekovi koji se koriste u terapiji ateroskleroze, svoje dejstvo ostvaruju upravo inhibirajući ovaj sintetski put. Međutim, statini imaju i dodatne plejotropne efekte, koji se ostvaruju nezavisno od sniženja koncentracije holesterola u plazmi i značajno doprinose njihovom korisnom dejstvu u različitim bolestima. Jedan od dobro dokumentovanih plejotropnih efekata je poboljšana reparacija tkiva uključujući i zarastanje rana. Zanimljivo je da se upravo inhibicija farnezilacije signalnih molekula, usled sniženja nivo ćelijskog FPP-a, smatrala do sada ključnim mehanizmom kojim statini ostvaruju ove svoje dodatne efekte. Međutim otkriće da FPP može delovati kao ligand za GR otvara vrata za drugačiju interpretaciju mehanizma dejstva plejotropnih efekata statina i novu primenu ovih lekova. Ciljevi: U ovoj disertaciji dokazivano je postojanje novog mehanizma dejstva statina, kojim oni mogu pospešiti zarastanje rana. Ovaj efekat se zasniva na sniženju koncentracije endogenog FPP-a u ćelijama kože, putem inhibicije HMG-CoA reduktaze Osnovna hipoteza je da, s obzirom da statini svoje dejstvo ostvaruju upravo inhibirajući ovaj sintetski put, modifikuju FPP-GR signalizaciju i tako učestvuju u stimulaciji zarastanje rana. Prvi cilj istraživanja je bio aktivacije glukokortikoidnog signalnog puta i stimulacije transkripcije gena. Potom je ispitatano da li povećanje nivoa endogenog FPP-a putem inhibicije enzima skvalen sintetaze i farnezil transferaze ima isti efekat na aktivaciju glukokortikoidnog receptora kao i dodavanje egzogenog FPP-a...Introduction: Glucocorticoid hormones, in addition to their anti-immflamatory effect, are well known inhibitors of wound healing. Recent studies have shown that farnesyl pyrophosphate (FPP), a key intermediate in the mevalonate pathway of the cholesterol synthesis and protein farnesylation, can act as an agonist for several nuclear hormone receptors including glucocorticoid receptor (GR). Interestingly, mechanism of action of statins, drugs used in therapy of atherosclerosis, is based on inhibition of mevalonath pathway. However, statins might exert also additional pleiotropic, noncholesterol lowering effects which significantly contribute to their therapeutic action. One of the well known pleiotropic effects of the statins is stimulation of tissue repair, including wound healing. Interestingly, mechanism of pleiotropic effects is traditionally explained by statin mediated inhibition of the farensylation of signaling molecules. By inhibiting the enzime HMG-CoA reductase, statins prevent the synthesis of substrate for reaction, farnesylpyrophosphate. Given that is recently shown that FPP can act as a ligand for GR, it is not surprising that statins might have additional mechanisms of action beyond inhibition of the farnesylation. Better understanding of this mechanism may contribute to novel therapeutic roles for this drugs. Objectives: In this study we are exploring new mechanism of action of statins that can contribute to improvement of wound healing. This effect is based on decreasing the levels of endogenus FPP in keratinocytes by inhibition of the HMG-CoA reductase. We postulate that statins, acting as an inhibitors of mevalonate pathway, stimulate wound healing through modulation of FPP-GR signal. The first step in in our study is to prove that the exogenous FPP, acting as an agonist for GR, can activate the glucocorticoid signaling pathway and regulate the transcription of the target genes. Second, will explore the effects of ZGA and B581, which both can increase the level of endogenous FPP, on activation of glucocorticoid receptors. ZGA and B581 acomplish their effects through inhibition of squalene synthetase and farnesyl transferase respectively..

    Regulation of Activity and Selectivity of Histone Deacetylases

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    Acetylation is an important post-translational modification (PTM). Lysine acetylation is a reversible PTM, where deacetylation is catalyzed by histone deacetylases (HDACs). Histone deacetylase function is crucial for a correctly functioning cell as aberrant acetylation, or deacetylation, has been linked to cancer, diabetes, neurodegeneration, and auto-immune disorders. Yet information about proper regulation of these enzymes is limited. Regulation of HDAC activity and selectivity has been proposed to include: the identity of the divalent active site metal ion, post-translational modifications, and protein interactions to form stable multi-protein complexes. HDAC activity and selectivity is further influenced by substrate amino acid sequence. This thesis explores how these different regulatory measures impact HDAC activity and selectivity. The biochemically well-characterized HDAC8 was used to investigate novel HDAC inhibitors and it was found that the identity of the active site divalent metal ion plays an important role in determining inhibitor selectivity. The identification and characterization of inhibitors with selective metal-binding groups, particularly Fe(II)-HDAC8 selective inhibitors, demonstrates structural differences between different HDAC8 metalloforms. This work also identified that the tropolone metal binding group potently inhibits HDAC8. To examine the impact of post-translational modifications and protein interactions on deacetylase activity and selectivity, a simplified CoREST complex including HDAC1 was reconstituted. In vitro HDAC1 complex formation significantly increases deacetylase activity (>10-fold) in comparison to HDAC1 in isolation. The presence of post-translational modifications, specifically phosphorylation, was found to impact substrate selectivity with the identification of a phosphorylation-specific acetylation site, without preventing complex formation. Finally, to explore the sequence-level substrate selectivity of HDAC6, we successfully constructed a structure-based model of the catalytic domain of HDAC6. This model was used to predict novel substrates that were then validated using peptide mimics. These data demonstrated that the substrate selectivity of HDAC6 is more promiscuous than HDAC8. The comparison of the activity of the single catalytic domain of HDAC6 with HDAC6 containing both catalytic domains demonstrates that the different structural components influence the activity and substrate selectivity profile of the enzyme. The findings discussed within this thesis illustrate several regulatory factors impart a sizeable contribution to deacetylase activity and selectivity. Such factors include structural components, including cofactors and post-translational modifications, in addition to protein interactions. The contribution of this thesis to the growing knowledge of how HDACs are regulated provides insight into the enzymes’ biological function to lead to the development of more effective therapeutic interventions.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/167952/1/kdiffley_1.pd

    Machine Learning based Protein Sequence to (un)Structure Mapping and Interaction Prediction

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    Proteins are the fundamental macromolecules within a cell that carry out most of the biological functions. The computational study of protein structure and its functions, using machine learning and data analytics, is elemental in advancing the life-science research due to the fast-growing biological data and the extensive complexities involved in their analyses towards discovering meaningful insights. Mapping of protein’s primary sequence is not only limited to its structure, we extend that to its disordered component known as Intrinsically Disordered Proteins or Regions in proteins (IDPs/IDRs), and hence the involved dynamics, which help us explain complex interaction within a cell that is otherwise obscured. The objective of this dissertation is to develop machine learning based effective tools to predict disordered protein, its properties and dynamics, and interaction paradigm by systematically mining and analyzing large-scale biological data. In this dissertation, we propose a robust framework to predict disordered proteins given only sequence information, using an optimized SVM with RBF kernel. Through appropriate reasoning, we highlight the structure-like behavior of IDPs in disease-associated complexes. Further, we develop a fast and effective predictor of Accessible Surface Area (ASA) of protein residues, a useful structural property that defines protein’s exposure to partners, using regularized regression with 3rd-degree polynomial kernel function and genetic algorithm. As a key outcome of this research, we then introduce a novel method to extract position specific energy (PSEE) of protein residues by modeling the pairwise thermodynamic interactions and hydrophobic effect. PSEE is found to be an effective feature in identifying the enthalpy-gain of the folded state of a protein and otherwise the neutral state of the unstructured proteins. Moreover, we study the peptide-protein transient interactions that involve the induced folding of short peptides through disorder-to-order conformational changes to bind to an appropriate partner. A suite of predictors is developed to identify the residue-patterns of Peptide-Recognition Domains from protein sequence that can recognize and bind to the peptide-motifs and phospho-peptides with post-translational-modifications (PTMs) of amino acid, responsible for critical human diseases, using the stacked generalization ensemble technique. The involved biologically relevant case-studies demonstrate possibilities of discovering new knowledge using the developed tools

    Determining HDAC8 Substrate Specificity.

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    Histone deacetylases (HDACs) are a group of 18 enzymes that catalyze the deacetylation of acetyl lysine residues in proteins. Acetyl lysine residues are present within thousands of proteins, and acetylation/deacetylation has been shown to affect protein properties integral to cellular homeostasis and disease states. Determining which protein is deacetylated by which HDAC isozyme is central to understanding biological regulation. To better identify HDAC substrates, I developed an assay to measure the acetate product formed by deacetylation catalyzed by metal-dependent HDACs for the evaluation of peptide substrates. Using this assay, I advanced a computational algorithm that predicts HDAC8 peptide substrates based on short range interactions. This algorithm accurately predicts the catalytic efficiency of 7-mer peptide substrates based on the sequence of the peptide. Using the deacetylation assay, I also demonstrated the reactivity of HDAC8 with peptide substrates derived from proteins with increased acetylation in vivo upon treatment with HDAC8 specific inhibitors. These experiments suggest that a subset of in vivo HDAC8 substrates can be predicted based on the six amino acids flanking the acetyl lysine. I have also utilized singly acetylated histone tetramers to establish that HDAC8 has enhanced activity in comparison to corresponding peptide substrates. Combined with the peptide work, these results suggest that molecular recognition by HDAC8 is determined by a combination of short and long-range interactions and acetyl lysine accessibility of in vivo substrates. Additionally, I have identified slow product dissociation as a novel regulatory method for HDAC activity. In the future we will expand these methods to identify substrates for other HDACs.PhDBiological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/108746/1/noahw_1.pd
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