James L. Hougland

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.

Identification of Novel Peptide Substrates for Protein Farnesyltransferase Reveals Two Substrate Classes with Distinct Sequence Selectivities

Prenylation is a posttranslational modification essential for the proper localization and function of many proteins. Farnesylation, the attachment of a 15-carbon farnesyl group near the C-terminus of protein substrates, is catalyzed by protein farnesyltransferase (FTase). Farnesylation has received significant interest as a target for pharmaceutical development, and farnesyltransferase inhibitors are in clinical trials as cancer therapeutics. However, as the total complement of prenylated proteins is unknown, the FTase substrates responsible for farnesyltransferase inhibitor efficacy are not yet understood. Identifying novel prenylated proteins within the human proteome constitutes an important step towards understanding prenylation-dependent cellular processes. Based on sequence preferences for FTase derived from analysis of known farnesylated proteins, we selected and screened a library of small peptides representing the C-termini of 213 human proteins for activity with FTase. We identified 77 novel FTase substrates that exhibit multiple-turnover (MTO) reactivity within this library; our library also contained 85 peptides that can be farnesylated by FTase only under single-turnover (STO) conditions. Based on these results, a second library was designed that yielded an additional 29 novel MTO FTase substrates and 45 STO substrates. The two classes of substrates exhibit different specificity requirements. Efficient MTO reactivity correlates with the presence of a nonpolar amino acid at the a(2) position and a Phe, Met, or Gln at the terminal X residue, consistent with the proposed Ca(1)a(2)X sequence model. In contrast, the sequences of the STO substrates vary significantly more at both the a(2) and the X residues and are not well described by current farnesylation algorithms. 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 STO versus MTO peptide reactivity with FTase.

Context-dependent substrate recognition by protein farnesyltransferase

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.

Synthesis and screening of a CaaL peptide library versus FTase reveals a surprising number of substrates

Proteins bearing a CaaL sequence are typically geranylgeranylated to enable their proper localization and function. We found that many of the dansyl-GCaaL peptides representing mammalian CaaL proteins can be farnesylated by FTase. This result may have important implications for prenylated protein biology.

The 2′-hydroxyl group of the guanosine nucleophile donates a functionally important hydrogen bond in the Tetrahymena ribozyme reaction

In the first step of self-splicing, group I introns utilize an exogenous guanosine nucleophile to attack the 5-splice site. Removal of the 2-hydroxyl of this guanosine results in a 10 (6)-fold loss in activity, indicating that this functional group plays a critical role in catalysis. Biochemical and structural data have shown that this hydroxyl group provides a ligand for one of the catalytic metal ions at the active site. However, whether this hydroxyl group also engages in hydrogen-bonding interactions remains unclear, as attempts to elaborate its function further usually disrupt the interactions with the catalytic metal ion. To address the possibility that this 2-hydroxyl contributes to catalysis by donating a hydrogen bond, we have used an atomic mutation cycle to probe the functional importance of the guanosine 2-hydroxyl hydrogen atom. This analysis indicates that, beyond its role as a ligand for a catalytic metal ion, the guanosine 2-hydroxyl group donates a hydrogen bond in both the ground state and the transition state, thereby contributing to cofactor recognition and catalysis by the intron. Our findings continue an emerging theme in group I intron catalysis: the oxygen atoms at the reaction center form multidentate interactions that function as a cooperative network. The ability to delineate such networks represents a key step in dissecting the complex relationship between RNA structure and catalysis.

Getting a handle on protein prenylation

Protein prenylation plays a key role in the localization and function of many proteins, but the number and identities of prenylated proteins are unknown. A new study uses a multidisciplinary approach to provide a broad yet detailed snapshot of prenylation within the mammalian proteome.

Synthetic Triterpenoid Inhibition of Human Ghrelin O-Acyltransferase: The Involvement of a Functionally Required Cysteine Provides Mechanistic Insight into Ghrelin Acylation

The peptide hormone ghrelin plays a key role in regulating hunger and energy balance within the body. Ghrelin signaling presents a promising and unexploited target for development of small molecule therapeutics for treatment of obesity, diabetes, and other health conditions. Inhibition of ghrelin O-acyltransferase (GOAT), which catalyzes an essential octanoylation step in ghrelin maturation, offers a potential avenue for controlling ghrelin signaling. Through screening a small molecule library, we have identified a class of synthetic triterpenoids that efficiently inhibit ghrelin acylation by the human isoform of GOAT (hGOAT). These compounds function as covalent reversible inhibitors of hGOAT, providing the first evidence of the involvement of a nucleophilic cysteine residue in substrate acylation by a MBOAT family acyltransferase. Surprisingly, the mouse form of GOAT does not exhibit susceptibility to cysteine-modifying electrophiles, revealing an important distinction in the activity and behavior between these closely related GOAT isoforms. This study establishes these compounds as potent small molecule inhibitors of ghrelin acylation and provides a foundation for the development of novel hGOAT inhibitors as therapeutics targeting diabetes and obesity.

Synthesis of Frame-Shifted Farnesyl Diphosphate Analogs

A set of synthetic approaches were developed and applied to the synthesis of eight frame-shifted farnesyl diphosphate (FPP) analogs. These analogs bear increased or decreased methylene units between the double bonds and/or diphosphate moieties of the isoprenoid structure. Evaluation versus mammalian FTase revealed that small structural changes can lead to dramatic changes in substrate ability.

Global Identification of Protein Prenyltransferase Substrates: Defining the Prenylated Proteome

Publisher Summary This chapter discusses the multiple approaches currently used to identify and define protein farnesyltransferase (FTase) and protein geranylgeranyltransferase-I (GGTase-I) substrates. The protein prenyltransferases, FTase and GGTase-I, catalyze the attachment of a 15-carbon farnesyl or 20-carbon geranylgeranyl moiety, respectively, to a cysteine near the C-terminus of a substrate protein targeting it to the membrane. 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 the treatment of diseases such as cancer, parasitic infection, asthma, and progeria. Direct identification approaches involve identifying FTase and GGTase-I substrates one by one or by using lipid donor analogs. A complementary approach to identify the prenylated proteome is to define the modes of FTase and GGTase-I substrate recognition using structure–function studies, peptide library studies, and computational methods. Identification of the entire prenylated proteome appears to be a realistic goal in the next decade, if not sooner. Definition of the prenylated proteome will be essential to better understand the roles of prenylation in cellular signaling, disease processes, and the complex array of posttranslational modifications within the cell.

2'-AMINO-MODIFIED RIBONUCLEOTIDES AS PROBES FOR LOCAL INTERACTIONS WITHIN RNA

The 2-hydroxyl group plays an integral role in RNA structure and catalysis. This ubiquitous component of the RNA backbone can participate in multiple interactions essential for RNA function, such as hydrogen bonding and metal ion coordination, but the multifunctional nature of the 2-hydroxyl renders identification of these interactions a significant challenge. By virtue of their versatile physicochemical properties, such as distinct metal coordination preferences, hydrogen bonding properties, and ability to be protonated, 2-amino-2-deoxyribonucleotides can serve as tools for probing local interactions involving 2-hydroxyl groups within RNA. The 2-amino group can also serve as a chemoselective site for covalent modification, permitting the introduction of probes for investigation of RNA structure and dynamics. In this chapter, we describe the use of 2-aminonucleotides for investigation of local interactions within RNA, focusing on interactions involving 2-hydroxyl groups required for RNA structure, function, and catalysis.