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

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

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

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

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

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

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

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

Complex Systems Analysis of Cell Cycling Models in Carcinogenesis

A new approach to the modular, complex systems analysis of nonlinear dynamics in cell cycling network transformations involved in carcinogenesis is proposed. Carcinogenesis is a complex process that involves dynamically inter-connected biomolecules in the intercellular, membrane, cytosolic, nuclear and nucleolar compartments that form numerous inter-related pathways referred to as networks.
The variable biotopology of such dynamic networks is highly complex, and has a number of interesting properties that can be formally characterized at one level of organization by mathematical structures called 'biogroupoids'. 
One such family of pathways contains the cell cyclins. Cyclins are proteins that link several critical pro-apoptotic and other cell cycling/ division components, including the tumor suppressor gene TP53 and its product, the Thomsen-Friedenreich antigen (T antigen), Rb, mdm2, c-Myc, p21, p27, Bax, Bad and Bcl-2, which all play major roles in carcinogenesis of many cancers. A novel theoretical analysis is thus possible based on recently published studies of cyclin signaling, with special emphasis placed on the roles of cyclins D1 and E, suggests novel clinical trials and rational therapies of cancer through reestablishment of cell cycling inhibition in metastatic cancer cells

Complex Systems Analysis of Arrested Neural Cell Differentiation during Development and Analogous Cell Cycling Models in Carcinogenesis

A new approach to the modular, complex systems analysis of nonlinear dynamics of arrested neural cell Differentiation--induced cell proliferation during organismic development and the analogous cell cycling network transformations involved in carcinogenesis is proposed. Neural tissue arrested differentiation that induces cell proliferation during perturbed development and Carcinogenesis are complex processes that involve dynamically inter-connected biomolecules in the intercellular, membrane, cytosolic, nuclear and nucleolar compartments. Such 'dynamically inter-connected' biomolecules form numerous inter-related pathways referred to as 'molecular networks'. One such family of signaling pathways contains the cell cyclins. Cyclins are proteins that link several critical pro-apoptotic and other cell cycling/division components, including the tumor suppressor gene TP53 and its product, the Thomsen-Friedenreich antigen (T antigen), Rb, mdm2, c-Myc, p21, p27, Bax, Bad and Bcl-2, which play major roles in various neoplastic transformations of many tissues. The novel theoretical analysis presented here is based on recently published studies of arrested cell differentiation that normally leads to neural system formation during early developmental stages; the perturbed development may involve cyclin signaling and cell cycling responsible for rapidly induced cell proliferation without differentiation into neural cells in such experimental studies; special emphasis in this modular model is placed upon the roles of cyclins D1 and E, and does suggest novel clinical trials as well as rational therapies of cancer through re-establishment of cell cycling inhibition in metastatic cancer cells. Cyclins are proteins that are often over-expressed in cancerous cells (Dobashi et al., 2004). They may also be over-expressed in cells whose differentiation is arrested during the early stages of organismic development, leading to increased cell proliferation instead of differentiation into specialized tissues such as those forming the neural system. Cyclin-dependent kinases (CDK), their respective cyclins, and inhibitors of CDKs (CKIs) were identified as instrumental components of the cell cycle-regulating machinery. In mammalian cells the complexes of cyclins D1, D2, D3, A and E with CDKs are considered motors that drive cells to enter and pass through the “S” phase. Cell cycle regulation is a critical mechanism governing cell division and proliferation, and it is finely regulated by the interaction of cyclins with CDKs and CKIs, among other molecules (Morgan et al., 1995). A categorical and Topos framework for Łukasiewicz Algebraic Logic models of nonlinear dynamics in complex functional genomes and cell interactomes is also proposed. Łukasiewicz Algebraic Logic models of genetic networks and signaling pathways in cells are formulated in terms of nonlinear dynamic systems with n-state components that allow for the generalization of previous logical models of both genetic activities and neural networks. An algebraic formulation of varying 'next-state' functions is extended in a Łukasiewicz-Topos with an n-valued Łukasiewicz Algebraic Logic subobject classifier description that represents non-random and nonlinear network activities as well as their transformations in developmental processes and carcinogenesis. Important aspects of Cell Cycling, the Control of Cell Division,and the Neoplastic Transformation in Carcinogenesis are being considered and subjected to algebraic-logico- relational, and computer-aided investigations. The essential roles of various levels of c-Myc, p27 quasi-complete inhibition/blocking, TP53 and/or p53 inactivation, as well as the perpetual hTERT activation of Telomerase biosynthesis are pointed out as key conditions for Malignant Cell transformations and partial re-differentiation leading to various types of cancer such as lung, breast,skin, prostate and colon. Rational Clinical trials, Individualized Medicine and the potential for optimized Radio-, Chemo-, Gene-, and Immuno- therapies of Cancers are suggested on the basis of integrated complex systems biology modeling of oncogenesis, coupled with extensive genomic/proteomic and interactomic High-throughput/high-sensitivity measurements of identified, sorted cell lines that are being isolated from malignant tumors of patients undergoing clinical trials with adjuvant signaling drug therapies. The implications of the cyclin model for abnormal neural development during early development are being considered in this model that may lead to explanations of subsequent cognitive changes associated with abnormal neural cell differentiation in environmentally-affected embryos. This new model may also be relevant to detecting the onset of senescing neuron transformations in Alzheimer's and related diseases of the human brain in ageing populations at risk

ISOPRENOID ANALOGS AS CHEMICAL GENETIC TOOLS TO PROVIDE INSIGHTS INTO FARNESYL TRANSFERASE TARGET SELECTION AND CELLULAR ACTIVITY

Protein farnesylation is an essential post-translational modification required for the function of numerous cellular proteins including the oncoprotein Ras. The farnesyl transferase (FTase) catalyzed reaction is unique because farnesyl diphosphate (FPP), the farnesyl group donor for the reaction, forms a significant portion of a target protein binding site. The major goal of this research was to exploit this unique property of the FTase reaction and determine if changing the structure of the farnesyl donor group would affect FTase protein targeting. A small library of structural analogues of FPP was synthesized. Michelis-Menten steady-state kinetic analyses and competition reactions were used to determine the effect of these structural modifications on FTase targeting. We found that the analogues did affect FTase protein selectivity and that this could be exploited to induce unnatural target selectivity into the enzyme. The second goal of this research was to determine the effect of FPP analogues on the function of FTase target proteins. To test the effect of these analogues we determined whether the unnatural lipid could ablate oncogenic H-Ras biological function in a Xenopus laevis model system. Several analogues were able to disrupt oncogenic H-Ras function while others mimicked the activity of FPP. These results indicated that some of the FPP analogues may act a prenyl group function inhibitors that could lead to an important new class of anti-cancer therapeutics. Another major goal of this research was to use the FPP analogues as unnatural probes for the endogenous cellular activity of FTase target proteins. We developed antibodies to two of the unnatural FPP analogues to study their activity in cell cultureUtilizing these antibodies we found that alcohol prodrugs of the FPP analogues could be incorporated into cellular proteins in an FTase dependent manner. The ability of cell permeant analogues to be incorporated into live cells enhances the chances that such a molecule could be used to modify oncogenic cellular proteins with a prenyl group function inhibitor

Mutations in Lamin and How It Causes Multiple Tissue-Specific Disorders

Lamins are the major components of the nuclear lamina where they provide a platform for the binding of proteins to the chromatin and confer mechanical stability (Dittmer and Misteli.,2011). Mutations in the human LMNA gene result in at least 15 distinct disorders ranging from muscular dystrophies to neurological disorders to lipodystrophies (Vytopil et al.,2003). Interestingly, some mutant forms of lamin protein aggregate, which may be toxic to the cells. However, it is unknown how specific mutations in lamin give rise to tissue-specific disease. I hypothesize that certain tissues are susceptible to specific lamin mutations due to the inability of tissue-specific quality control mechanisms to degrade those mutant forms, leading to protein aggregation and cellular toxicity. I will be testing if tissue-specific disease mutations in Lam Dm0, one of the fly homologs of LMNA (Gene that codes for Lamin), cause the protein to aggregate in muscles and neurons. Lamin can be post-translationally modified by the addition of a farnesyl group that helps anchor Lamin into the nuclear envelop. We find that the unfarnesylated form (the predominant form) and the farnesylated form of the different Lam Dm0mutant proteins have different expression patterns in the muscle. In addition, we find that the p38 MAPK (p38Kb) interacts with the CASA (Chaperone Assisted Selective Autophagy) complex to regulate the degradation of Lam Dm0. Future experiments will characterize how these mutant forms of Dm0 affect the functionality of the muscles and neurons in flies and if these forms can be targeted for degradation by p38Kb and the CASA complex

Translational Oncogenomics and Human Cancer Interactome Networks

An overview of translational, human oncogenomics, transcriptomics and cancer interactomic networks is presented together with basic concepts and potential, new applications to Oncology and Integrative Cancer Biology. Novel translational oncogenomics research is rapidly expanding through the application of advanced technology, research findings and computational tools/models to both pharmaceutical and clinical problems. A self-contained presentation is adopted that covers both fundamental concepts and the most recent biomedical, as well as clinical, applications. Sample analyses in recent clinical studies have shown that gene expression data can be employed to distinguish between tumor types as well as to predict outcomes. Potentially important applications of such results are individualized human cancer therapies or, in general, ‘personalized medicine’. Several cancer detection techniques are currently under development both in the direction of improved detection sensitivity and increased time resolution of cellular events, with the limits of single molecule detection and picosecond time resolution already reached. The urgency for the complete mapping of a human cancer interactome with the help of such novel, high-efficiency / low-cost and ultra-sensitive techniques is also pointed out

Mechanism of Catalysis and Inhibition of Mammalian Protein Farnesyltransferase.

Mammalian protein farnesyltransferase (FTase) catalyzes the transfer of a 15-carbon prenyl group from farnesyl diphosphate (FPP) to a cysteine residue near the carboxyl terminus of many proteins, including several key molecules involved in signal transduction. Common substrates include oncogenic Ras proteins, and several FTase inhibitors are under development for the treatment of various cancers. FTase is a member of the newest class of zinc metalloenzymes that catalyze sulfur alkylation, and the work described here provides further insight into the mechanism of catalysis for this enzyme, which may lead to an increased understanding of the substrate specificity and inhibition of FTase. The reaction catalyzed by FTase results in two products: diphosphate and farnesylated protein or peptide. To measure the rate constant for diphosphate dissociation, a coupled fluorescent assay was developed. This assay can also be used to measure FTase activity for mechanistic studies and for high throughput screening to identify FTase substrates and inhibitors. The dissociation of the farnesylated product bound to FTase is accelerated by binding FPP. This step is crucial for substrate selectivity, as measured by substrate analog studies, and inhibition studies demonstrate that some FPP-competitive inhibitors function by slowing product dissociation. Together, these studies suggest that the binding of a second substrate molecule to facilitate product release is an important determinant of the substrate specificity, and potentially of the physiological regulation of FTase. To investigate the structure of the chemical transition state of FTase, the primary 14C and α-secondary 3H kinetic isotope effects (KIEs) were measured using transient kinetics. These data suggest that the FTase reaction proceeds via a concerted mechanism with dissociative character, facilitated by the zinc ion which coordinates the thiolate of the peptide substrate. The effects of the Mg2+ concentration and mutations of positively charged residues that interact with the diphosphate leaving group on the α-secondary KIE suggest that Mg2+ and these side chains both stabilize the transition state for farnesylation and facilitate a conformational rearrangement of bound FPP that occurs prior to farnesylation. Finally, the dependence of the α-secondary KIE on peptide structure indicates that this FPP conformational change is important for substrate specificity.Ph.D.Biological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/57672/2/jpais_1.pd