Towards Complete Sets of Farnesylated and Geranylgeranylated Proteins

Three different prenyltransferases attach isoprenyl anchors to C-terminal motifs in substrate proteins. These lipid anchors serve for membrane attachment or protein–protein interactions in many pathways. Although well-tolerated selective prenyltransferase inhibitors are clinically available, their mode of action remains unclear since the known substrate sets of the various prenyltransferases are incomplete. The Prenylation Prediction Suite (PrePS) has been applied for large-scale predictions of prenylated proteins. To prioritize targets for experimental verification, we rank the predictions by their functional importance estimated by evolutionary conservation of the prenylation motifs within protein families. The ranked lists of predictions are accessible as PRENbase (http://mendel.imp.univie.ac.at/sat/PrePS/PRENbase) and can be queried for verification status, type of modifying enzymes (anchor type), and taxonomic distribution. Our results highlight a large group of plant metal-binding chaperones as well as several newly predicted proteins involved in ubiquitin-mediated protein degradation, enriching the known functional repertoire of prenylated proteins. Furthermore, we identify two possibly prenylated proteins in Mimivirus. The section HumanPRENbase provides complete lists of predicted prenylated human proteins—for example, the list of farnesyltransferase targets that cannot become substrates of geranylgeranyltransferase 1 and, therefore, are especially affected by farnesyltransferase inhibitors (FTIs) used in cancer and anti-parasite therapy. We report direct experimental evidence verifying the prediction of the human proteins Prickle1, Prickle2, the BRO1 domain–containing FLJ32421 (termed BROFTI), and Rab28 (short isoform) as exclusive farnesyltransferase targets. We introduce PRENbase, a database of large-scale predictions of protein prenylation substrates ranked by evolutionary conservation of the motif. Experimental evidence is presented for the selective farnesylation of targets with an evolutionary conserved modification site

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

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

Induction And Regulation Of Autophagy By Novel Prenylation Inhibitors In Sts-26t Malignant Peripheral Nerve Sheath Tumor (mpnst) Cells

Prenylation pathways have been targeted via several different compounds that inhibit farnesyl transferase (FTase) and/or geranylgeranyl transferase (GGTase) enzymes in many cellular and animal models of cancer. Some of these have also been evaluated in clinical trials with limited success. Multiple mechanisms of action have been elucidated for such compounds, including cell cycle arrest, proteasome inhibition, apoptosis and most recently, autophagy. However, there is still an urgent need of effective agents of this class of anti-tumor therapeutics. In this dissertation, I sought to delve into this issue by characterizing our novel prenylation inhibitors and their potential as anti-tumor agents. Novel compounds, GGTI-2Z and FTI-1, were used in combination with lovastatin in STS-26T malignant peripheral nerve sheath tumor (MPNST) cells. We found that GGTI-2Z/lovastatin inhibit proliferation, cause cell cycle arrest in the G1 phase and induce autophagy in STS-26T MPNST cells. FTI-1/lovastatin not only inhibit proliferation and cause cell cycle arrest, but also induce an aborted autophagic program followed by non-apoptotic cell death in STS-26T cells. This distinct phenotype observed with FTI-1/lovastatin is the consequence of their action on the lysosomal trafficking of proteins. The compounds impaired procathepsin trafficking via the endocytic pathway along with degradation of the lysosomal protein, LAMP-2, which is required for autophagosome-lysosome fusion. These effects consequently lead to altered protein turnover and hence non-apoptotic cell death. Our observations identify a novel mechanism of action of GGTIs. We also show that autophagic cell death can be a consequence of an aborted autophagic program versus excessive autophagy. This mechanism also suggests that prenylated proteins may play an important role in a complete autophagic response and blocking their prenylation may interfere with this function of these proteins. Finally, the strategy of combination therapy with low doses of a statin and an FTI or a GGTI compound may serve as a useful tool to develop better therapeutic regimen for many cancers and other Rab-associated trafficking disorders

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

Post-translational modifications and p21ras function

The ras gene family consists of three members that encode highly similar proteins of 21Kd (p21ras/Ras). This protein is plasma membrane associated, binds guanine nucleotides and has intrinsic GTPase activity. Activating point mutations render Ras insensitive to regulation by GAP (GTPase activating protein) and it remains in the active GTP bound state. Membrane association of Ras has been shown to be essential for its biological activity. The plasma membrane targeting of Ras is accomplished by a series of post-translational modifications which occur in 2 steps. Step 1 involves the CAAX motif (C = cysteine, A = aliphatic and X = any amino acid) at the C- terminus. The cysteine is alkylated by C15 farnesyl, the -AAX amino acids are removed and the new C-terminal cysteine undergoes methylesterification. Step 2 involves palmitoylation of cysteine residues near the CAAX motif in the case of H-, N- and K-ras (A). Membrane localisation of K-ras (B) appears to involve electrostatic interaction of the polybasic region (K175-180) with the membrane. Other CAAX containing proteins (rap 1A, G25K) are prenylated with a C20 geranylgeranyl moiety rather than C15 farnesyl. Geranylgeranylation of H- and K-ras (B) also leads to membrane association of the protein but specific targeting to the plasma membrane requires the presence of the polybasic domain or the palmitoylation sites. Another family of proteins (p60src, Gag, cytochrome b5 reductase) is membrane associated by the addition of myristic acid to the N-terminus. Myristoylation can also allow Ras proteins to be membrane associated but specific plasma membrane targeting remains dependent on the presence of palmitoylation sites or a polybasic region. Upstream of the CAAX motif is the hypervariable domain - a region that shows less than 20% homology between the ras genes. The function of this domain is not known and it may simply connect the N- and C-termini. However this region could also confer specificity on the interaction of different Ras proteins with different effector and/or regulatory proteins. Deletions within this region destroy transforming ability and reduce MAP kinase activity suggesting that effector interaction is disrupted. N17 deletion mutants rescue proliferation of NIH 3T3 cells indicating that exchange factor interaction is also influenced by the hypervariable region. This thesis attempts to establish a relationship between the biological activity of Ras, its cellular location and post-translational processing events. A functional role for the hypervariable domain is also examined

Beyond the Mevalonate Pathway: Control of Post-Prenylation Processing by Mutant p53

Missense mutations in the TP53 gene are among the most frequent alterations in human cancer. Consequently, many tumors show high expression of p53 point mutants, which may acquire novel activities that contribute to develop aggressive tumors. An unexpected aspect of mutant p53 function was uncovered by showing that some mutants can increase the malignant phenotype of tumor cells through alteration of the mevalonate pathway. Among metabolites generated through this pathway, isoprenoids are of particular interest, since they participate in a complex process of posttranslational modification known as prenylation. Recent evidence proposes that mutant p53 also enhances this process through transcriptional activation of ICMT, the gene encoding the methyl transferase responsible for the last step of protein prenylation. In this way, mutant p53 may act at different levels to promote prenylation of key proteins in tumorigenesis, including several members of the RAS and RHO families. Instead, wild type p53 acts in the opposite way, downregulating mevalonate pathway genes and ICMT. This oncogenic circuit also allows to establish potential connections with other metabolic pathways. The demand of acetyl-CoA for the mevalonate pathway may pose limitations in cell metabolism. Likewise, the dependence on S-adenosyl methionine for carboxymethylation, may expose cells to methionine stress. The involvement of protein prenylation in tumor progression offers a novel perspective to understand the antitumoral effects of mevalonate pathway inhibitors, such as statins, and to explore novel therapeutic strategies.Fil: Borini Etichetti, Carla Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Arel Zalazar, Evelyn Evangelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; ArgentinaFil: Cocordano, Nabila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; ArgentinaFil: Girardini Brovelli, Javier Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; Argentin

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