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Use of PEPscreen® Peptides to Investigate Protein Farnesyltransferase Substrate Specificity

Prenylation is an essential post-translational modification for the proper localization and function of many proteins1,2, and is catalyzed by protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase-I). These enzymes catalyze the covalent attachment of a 15-carbon farnesyl group from farnesyl diphosphate (FPP) or a 20-carbon geranylgeranyl group from geranylgeranyl diphosphate (GGPP) to the thiol side chain of a cysteine residue near the C-terminus of a protein substrate 2,3. The attached lipid aids in localization of proteins to cellular membranes and enhances protein-protein interactions4,5, and is required for the proper function of many proteins including members of the Ras and Rho superfamilies of small GTPases 1,6. While many proteins have been experimentally shown to be prenylated in vivo 7-10, the extent of prenylation within the proteome remains unclear. Based on comparison of known prenylated proteins, both FTase and GGTase-I have been proposed to recognize protein or peptide substrates containing a C-terminal “Ca1a2X” sequence 11-17. In this model, “C” refers to a cysteine three residues removed from the C-terminus that is prenylated at the thiol group to form a thioether, “a” refers to any aliphatic amino acid, and “X” refers to a subset of amino acids that are proposed to determine specificity for FTase (methionine, serine, glutamine, alanine) or GGTase-I (leucine, phenylalanine). Expanding upon the “Ca1a2X” box paradigm, bioinformatic analysis and biochemical studies of known substrates and related proteins indicate that sequences immediately upstream of the conserved cysteine residue may also play a role in substrate selectivity 8,18,19.

Prenylation has received significant interest as a target for pharmaceutical development, with FTase inhibitors (FTIs) in development as therapeutics for cancer, parasitic infection, and several other medical conditions 20-22. However, as the total complement of prenylated proteins is unknown, the FTase substrates responsible for FTI efficacy are not yet understood. Defining the prenylation-dependent pathways potentially responsible for the efficacy of these treatments could provide valuable insights for development of novel pharmaceuticals. Understanding the in vivo substrate selectivity of FTase and GGTase-I constitutes an important step towards characterizing the prenylated proteins involved in these pathways. Towards this goal, we have used peptide libraries to study the potential reactivity of human proteins with FTase, as well as to investigate the specific amino acid properties recognized by FTase within the Ca1a2X motif.

To identify potential novel prenylated proteins, we used a PEPscreen peptide library [Sigma Custom Products] to probe the reactivity of naturally occurring Ca1a2X sequences with FTase 23. To generate this library, we searched the human genome database for proteins that contain a cysteine four amino acids from the C-terminus as a minimal specificity requirement. From the resulting list of ~600 proteins, we selected approximately half to screen for activity with FTase. Peptides corresponding to the last four amino acids of these candidate substrates along with N-terminal Thr and Lys residues (TKCxxx) were screened for farnesylation by FTase under both kcat / Km peptide (multiple-turnover-, [Enzyme] << [Substrate]) and single-turnover ([Enzyme] > [Substrate]) reaction conditions. Out of the >300 peptides screened, FTase catalyzed multipleturnover farnesylation of 106 peptides, consistent with the parent proteins serving as FTase substrates in vivo. Surprisingly, of the remaining peptides, 67 % were farnesylated under single-turnover conditions, suggesting that there are two classes of substrates for FTase with distinct reactivity profiles. Analysis of the sequence preferences for these two classes of substrates illustrates a significant difference in FTase selectivity for the multiple- and single-turnover substrates, with the multiple-turnover substrates adhering closely to the current Ca1a2X model for FTase selectivity while the singleturnover substrates show distinct sequence preferences. These different classes of FTase substrates may reflect an unanticipated mechanism for regulating farnesylation, with potential impact on the localization, trafficking, and activity of prenylated proteins within the cell. These results improve the definition of prenyltransferase substrate specificity, test the efficacy of substrate algorithms, and provide valuable information about therapeutic targets. Finally, these data illuminate the potential for in vivo regulation of prenylation through modulation of single- versus multiple-turnover peptide reactivity with FTase.

Biochemical studies of prenyltransferase substrate specificity indicate that recognition of peptide substrates is more complex than originally proposed. For instance, although FTase and GGTase-I specificity is determined predominantly by the X residue 11,13-17,24-27, some substrates react efficiently with both enzymes 11,27. Peptide substrate specificity also depends on interactions of the peptide with the FPP co-substrate 28-30. As these findings reflect, understanding FTase and GGTase-I substrate specificity will require identification and energetic characterization of interactions involved in substrate recognition. In a second study, we used PEPscreen peptides and applied structure-function analysis to define the specific selectivity criteria that FTase employs to recognize the a2 residue of substrate peptides31. We measured the reactivity of FTase with several panels of peptides in which the a2 residue is substituted with all twenty amino acids, while keeping the remainder of the Ca1a2X sequence constant. Correlation of peptide reactivity within each panel against both the amino acid polarity and steric volume of the a2 residue indicates that FTase recognizes both the size and hydrophobicity of the residue at the a2 position, contrasting with the predominantly polarity-based recognition observed at the X residue 19. Furthermore, comparison across peptide panels indicates that a2 selectivity is also affected by the identity of the adjacent X residue, leading to context-dependent substrate recognition. These findings suggest that the current model describing FTase selectivity reflects only a subset of potential FTase substrates, raising the possibility of novel FTase substrates whose C-terminal sequences do not conform to the canonical Ca1a2X motif.

Muliple turn-over sequences

Table 1 & 2.Peptides from the initial library that exhibit multiple turn-over activity with FTase

Peptides are a form of dansyl-TKCxxx, where x represents any amino acid.
Peptides exhibited multiple turn-over reactivity in initial screens using either fluorescence or radioactive detection.

Single turn-over sequences

Table 3.Peptides from the initial and targeted peptide libraries that only exhibit single turn-over activity with FTase
Amino acid compositions

Figure 1.Amino acid compositions at the a2 and X positions of the Ca1a2X sequence in the initial library. (A) Amino acid compositions at the a2 position of the Ca1a2X sequence. The percentages of amino acids at the a2 position, grouped into either canonical (V, I, L, M, and T) or non-canonical residues, are plotted for the initial library, the MTO substrates, the STO substrates, and the non-substrate peptides. Wedges are labeled with the percentage representation of the classes of amino acids within each pool of peptides. An asterisk (*) denotes those percentages that are significantly different in the substrate pools as compared to the library (p<0.02). (B) Amino acid compositions at the X position of the Ca1a2X sequence. The percentages of amino acids at the X position, grouped into canonical (A, S, M, Q, and F), reactive (C, N, and T), and non-canonical residues, are plotted for the initial library, the MTO substrates, the STO substrates, and the non-substrate peptides. Wedges are labeled with the percentage representation of the classes of amino acids within each pool of peptides; percentages may not add to 100% due to rounding. An asterisk (*) denotes those percentages that are significantly different in the substrate pools as compared to the library (p<0.02).

References

1.
Zhang FL, Casey PJ. 1996. Protein Prenylation: Molecular Mechanisms and Functional Consequences. Annu. Rev. Biochem.. 65(1):241-269. http://dx.doi.org/10.1146/annurev.bi.65.070196.001325
2.
Benetka W, Koranda M, Eisenhaber F. 2006. Protein Prenylation: An (Almost) Comprehensive Overview on Discovery History, Enzymology, and Significance in Physiology and Disease. Monatsh. Chem.. 137(10):1241-1281. http://dx.doi.org/10.1007/s00706-006-0534-9
3.
Casey PJ, Seabra MC. 1996. Protein Prenyltransferases. J. Biol. Chem.. 271(10):5289-5292. http://dx.doi.org/10.1074/jbc.271.10.5289
4.
Casey PJ. 1994. Lipid modifications of G proteins. Current Opinion in Cell Biology. 6(2):219-225. http://dx.doi.org/10.1016/0955-0674(94)90139-2
5.
Marshall E. 1993. Response. Science. 259(5091):15-15. http://dx.doi.org/10.1126/science.259.5091.15
6.
Sebti SM, Hamilton AD. 2000. Farnesyltransferase Inhibitors in Cancer Therapy. http://dx.doi.org/10.1385/1592590136
7.
Kho Y, Kim SC, Jiang C, Barma D, Kwon SW, Cheng J, Jaunbergs J, Weinbaum C, Tamanoi F, Falck J, et al. 2004. A tagging-via-substrate technology for detection and proteomics of farnesylated proteins. Proceedings of the National Academy of Sciences. 101(34):12479-12484. http://dx.doi.org/10.1073/pnas.0403413101
8.
Maurer-Stroh S, Koranda M, Benetka W, Schneider G, Sirota FL, Eisenhaber F. Towards Complete Sets of Farnesylated and Geranylgeranylated Proteins. PLoS Comput Biol. 3(4):e66. http://dx.doi.org/10.1371/journal.pcbi.0030066
9.
Maurer-Stroh S, Eisenhaber F. 2005. Genome Biol. 6(6):R55. http://dx.doi.org/10.1186/gb-2005-6-6-r55
10.
Scott Reid T, Terry KL, Casey PJ, Beese LS. 2004. Crystallographic Analysis of CaaX Prenyltransferases Complexed with Substrates Defines Rules of Protein Substrate Selectivity. Journal of Molecular Biology. 343(2):417-433. http://dx.doi.org/10.1016/j.jmb.2004.08.056
11.
Caplin BE, Hettich LA, Marshall MS. 1994. Substrate characterization of the saccharomyces cerevisiae protein farnesyltransferase and type-I protein geranylgeranyltransferase. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 1205(1):39-48. http://dx.doi.org/10.1016/0167-4838(94)90089-2
12.
Omer CA, Kral AM, Diehl RE, Prendergast GC, Powers S, Allen CM, Gibbs JB, Kohl NE. 1993. Characterization of recombinant human farnesyl-protein transferase: Cloning, expression, farnesyl diphosphate binding, and functional homology with yeast prenyl-protein transferases. Biochemistry. 32(19):5167-5176. http://dx.doi.org/10.1021/bi00070a028
13.
Reiss Y, Stradley SJ, Gierasch LM, Brown MS, Goldstein JL. 1991. Sequence requirement for peptide recognition by rat brain p21ras protein farnesyltransferase.. Proceedings of the National Academy of Sciences. 88(3):732-736. http://dx.doi.org/10.1073/pnas.88.3.732
14.
Moores S, Schaber M, Mosser S, Rands E, O'Hara M, Garsky V, Marshall M, Pompliano D, Gibbs J. 1991. Sequence dependence of protein isoprenylation. Journal of Biological Chemistry. 266(22):14603-14610. http://dx.doi.org/10.1016/s0021-9258(18)98729-6
15.
Yokoyama K, Goodwin GW, Ghomashchi F, Glomset JA, Gelb MH. 1991. A protein geranylgeranyltransferase from bovine brain: implications for protein prenylation specificity.. Proceedings of the National Academy of Sciences. 88(12):5302-5306. http://dx.doi.org/10.1073/pnas.88.12.5302
16.
Casey PJ, Thissen JA, Moomaw JF. 1991. Enzymatic modification of proteins with a geranylgeranyl isoprenoid.. Proceedings of the National Academy of Sciences. 88(19):8631-8635. http://dx.doi.org/10.1073/pnas.88.19.8631
17.
Fu HW, Casey PJ. 1999. Enzymology and biology of CaaX protein prenylation. Recent Prog. Horm. Res .. 54315-42.
18.
Maurer-Stroh S, Eisenhaber F. 2005. Genome Biol. 6(6):R55. http://dx.doi.org/10.1186/gb-2005-6-6-r55
19.
Hicks KA, Hartman HL, Fierke CA. 2005. Upstream Polybasic Region in Peptides Enhances Dual Specificity for Prenylation by Both Farnesyltransferase and Geranylgeranyltransferase Type I?. Biochemistry. 44(46):15325-15333. http://dx.doi.org/10.1021/bi050951v
20.
Sebti SM, Hamilton AD. 2000. Farnesyltransferase and geranylgeranyltransferase I inhibitors and cancer therapy: Lessons from mechanism and bench-to-bedside translational studies. Oncogene. 19(56):6584-6593. http://dx.doi.org/10.1038/sj.onc.1204146
21.
Maurer-Stroh S, Washietl S, Eisenhaber F. 2003. Protein Prenyltransferases: Anchor Size, Pseudogenes and Parasites. 384(7): http://dx.doi.org/10.1515/bc.2003.110
22.
Gelb MH, Van Voorhis WC, Buckner FS, Yokoyama K, Eastman R, Carpenter EP, Panethymitaki C, Brown KA, Smith DF. 2003. Protein farnesyl and N-myristoyl transferases: piggy-back medicinal chemistry targets for the development of antitrypanosomatid and antimalarial therapeutics. Molecular and Biochemical Parasitology. 126(2):155-163. http://dx.doi.org/10.1016/s0166-6851(02)00282-7
23.
Hougland JL, Hicks KA, Hartman HL, Kelly RA, Watt TJ, Fierke CA. 2010. Identification of Novel Peptide Substrates for Protein Farnesyltransferase Reveals Two Substrate Classes with Distinct Sequence Selectivities. Journal of Molecular Biology. 395(1):176-190. http://dx.doi.org/10.1016/j.jmb.2009.10.038
24.
Yokoyama K, Gelb M. 1993. Purification of a mammalian protein geranylgeranyltransferase. Formation and catalytic properties of an enzyme-geranylgeranyl pyrophosphate complex.. Journal of Biological Chemistry. 268(6):4055-4060. http://dx.doi.org/10.1016/s0021-9258(18)53579-1
25.
Yokoyama K, McGeady P, Gelb MH. 1995. Mammalian Protein Geranylgeranyltransferase-I: Substrate Specificity, Kinetic Mechanism, Metal Requirements, and Affinity Labeling. Biochemistry. 34(4):1344-1354. http://dx.doi.org/10.1021/bi00004a029
26.
Roskoski R, Ritchie P. 1998. Role of the Carboxyterminal Residue in Peptide Binding to Protein Farnesyltransferase and Protein Geranylgeranyltransferase. Archives of Biochemistry and Biophysics. 356(2):167-176. http://dx.doi.org/10.1006/abbi.1998.0768
27.
Hartman HL, Hicks KA, Fierke CA. 2005. Peptide Specificity of Protein Prenyltransferases Is Determined Mainly by Reactivity Rather than Binding Affinity?. Biochemistry. 44(46):15314-15324. http://dx.doi.org/10.1021/bi0509503
28.
Reigard SA, Zahn TJ, Haworth KB, Hicks KA, Fierke CA, Gibbs RA. 2005. Interplay of Isoprenoid and Peptide Substrate Specificity in Protein Farnesyltransferase?. Biochemistry. 44(33):11214-11223. http://dx.doi.org/10.1021/bi050725l
29.
Krzysiak AJ, Rawat DS, Scott SA, Pais JE, Handley M, Harrison ML, Fierke CA, Gibbs RA. 2007. Combinatorial Modulation of Protein Prenylation. ACS Chem. Biol.. 2(6):385-389. http://dx.doi.org/10.1021/cb700062b
30.
Troutman JM, Subramanian T, Andres DA, Spielmann HP. 2007. Selective Modification of CaaX Peptides withortho-Substituted Anilinogeranyl Lipids by Protein Farnesyl Transferase:  Competitive Substrates and Potent Inhibitors from a Library of Farnesyl Diphosphate Analogues?. Biochemistry. 46(40):11310-11321. http://dx.doi.org/10.1021/bi700516m
31.
Hougland JL, Lamphear CL, Scott SA, Gibbs RA, Fierke CA. 2009. Context-Dependent Substrate Recognition by Protein Farnesyltransferase?. Biochemistry. 48(8):1691-1701. http://dx.doi.org/10.1021/bi801710g