Protein prenylation specificity

Protein prenylation leads to an increased hydrophobicity of proteins, typically resulting in an increased affinity for membranes. In 2004 studies on the cellular location of prenylated RhoB proteins showed that RhoB can undergo farnesylation (RhoB-F) as well as geranylgeranylation (RhoB-GG). With the aid of specific prenyl transferase inhibitors, it was revealed that RhoB-GG is localized to multivesicular late endosomes. 

We confirmed that the Insect Cell Extract has the ability to perform eukaryote-specific protein modifications, such as AT-myristoylation (3) and prenylation (4). To obtain such modified proteins effectively, specific substrates for each protein modification should be added to the translation reaction mixture. 

Prenylated proteins have characteristic C-terminal sequences. For example, the three allelic Ras proteins (H-Ras, K-Ras, and N-Ras) expressed in mammalian tissues contain a C-terminal tetrapeptide which begins with cysteine, and ends with either methionine or serine. This part of the molecule is referred to as the CaaX box where C = cysteine, a = an aliphatic amino acid, and X = a prenylation specificity residue. The first step in the posttranslational processing of Ras proteins utilizes FTase and farnesyl diphosphate (FPP) to covalently attach a farnesyl group to the cysteine thiol of the CaaX box. While subsequent processing events involve proteolytic removal of the aaX tripeptide and methylation of the resulting C-termi-nal carboxylate group, only the farnesyl modification is required for mutant Ras proteins to associate with the cell membrane and transform a cell.2-6... 

Yokoyama, K., Zimmerman, K., Scholten, J., and Gelb, M.H. (1997). Differential prenyl pyrophosphate binding to mammalian protein geranylgeranyltransferase-I and protein farnesyltransferase and its consequence on the specificity of protein prenylation. J Biol Chem 272 3944-3952. 

Synthetic derivatives and analogs of prenyl diphosphates have historically played a key role in defining key featnres of the mechanism of enzymes that ntilize these key intermediates in the isoprenoid pathway. This has also been the case with the investigation of the protein prenyl-transferases. A brief introduction to the protein prenyltransferase enzymes is given along with outlines on the previous use of prenyl diphosphate tools and key aspects of their synthesis. The development of prenyl diphosphate-based FTase inhibitors is described. The use of prenyl diphosphate derivatives as mechanistic and structural probes is next discussed. In particular, the use of fluorinated, isotopically labeled, and photoaffinity derivatives is presented. An overview of the extensive work on the determination of FTase isoprenoid substrate specificity is then given, and the chapter concludes with a section on the development of prenyl diphosphate tools for proteomic studies. 

Dursina, B., ct al. (2006). Identification and specificity profihng of protein prenyltransfer-ase inhibitors using New flnorescent phosphoisoprenoids./Am Chem Soc 128 2822-2835. Liu, X., and Prestwich, G.D. (2002). Didehydrogeranylgeranyl (DDGG) a flnorescent probe for protein prenylation. J Am Chem Soc 124 20-21. 

S-geranylgeranylation. They are similar in their behavior, but they differ in specificity with regards to the C-terminal sequence of proteins. Prenylation can be performed via covalent attachment of either Cis famesyl or C20 geranylgeranyl moiety to the cysteine residue of CAAX motif present in the protein to be modified (A aliphatic amino acid, X variable residue) [L. A. Beck et al.. Cell Biol. 1988,107,1307 F. L. Zhang, P. J. Casey, Annu. Rev. Biochem. 1996, 65, 241 D. M. Leonard, J. Med. Chem. 1997, 40, 2971]. 

The farnesylation and subsequent processing of the Ras protein. Following farnesylation by the FTase, the carboxy-terminal VLS peptide is removed by a prenyl protein-specific endoprotease (PPSEP) in the ER, and then a prenylprotein-specific methyltransferase (PPSMT) donates a methyl group from S-adenosylmethionine (SAM) to the carboxy-terminal S-farnesylated cysteine. Einally, palmitates are added to cysteine residues near the C-terminus of the protein. 

In another application of coupling proteins to surfaces using click chemistry, Duckworth et al. (2006) carried out prenylation of a protein using a farnesyl azide derivative and the enzyme farnesyl transferase for subsequent chemoselective ligation to alkyne-functionalized agarose beads. The result is a highly discrete, site-specific attachment of the protein to the solid phase at a single location. 

MICHAELIS-MENTEN KINETICS PREEXPONENTIAL FACTOR ARRHENIUS EQUATION COLLISION THEORY TRANSITION-STATE THEORY ENTROPY OF ACTIVATION PRENYL-PROTEIN-SPECIFIC ENDOPEP-TIDASE... 

We have demonstrated that this insect cell-free protein synthesis system is one of the most effective protein synthesis systems among those based on animal extracts (2). Furthermore, it has the potential to perform eukaryote-specific protein modifications such as protein W-myristoylation and prenylation (3, 4). Thus, we expect that the insect cell-free protein synthesis system will be a useful method for target protein production in the reverse chemical genetics era, as well as for postgenomic studies. In this chapter, we describe standard protocols to synthesize proteins of interest using the insect cell-free protein synthesis system. 

A third lipid anchor is provided by the polyprenyl farnesyl (15-carbon) and geranylgeranyl (20-carbon) groups in thioether linkage to cysteine residues. These must be present in specific recognition sequences at the C termini of proteins, most often with the sequence CAAX.211-215 The prenylation (also called isoprenyla-tion) reaction is followed by proteolytic removal of the last three residues (AAX) and methylation of the new C-terminal carboxyl group as is discussed in Chapter 11, Section D,3. See also Chapter 22, Section A,4. 

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