Exploring the putative self‐binding property of the human farnesyltransferase alpha‐subunit

Abstract

Farnesylation is an important post-translational protein modification in eukaryotes. Farnesylation is performed by protein farnesyltransferase, a heterodimer composed of an α- (FTα) and a β-subunit. Recently, homodimerization of truncated rat and yeast FTα has been detected, suggesting a new role for FTα homodimers in signal transduction. We investigated the putative dimerization behaviour of human and rat FTα. Different in vitro and in vivo approaches revealed no self-dimerization and a presumably artificial formation of homotrimers and higher homo-oligomers in vitro. Our study contributes to the clarification of the physiological features of FTase in different species and may be important for the ongoing development of FTase inhibitors.

Impact of a conserved N-terminal proline-rich region of the α-subunit of CAAX-prenyltransferases on their enzyme properties

“…In our previous study, we could show the formation of higher oligomers when incubating IMAC-purified FTα with the crosslinking agent BS 3 [ 15 ]. The same experiment was performed with the truncated versions of human and rat FTα.…”

“…The cloning of the coding regions of full-length as well as truncated human and rat FTα ( FNTA ), FTβ ( FNTB ) and human GGT1β ( PGGT1B ) was performed as previously described [ 15 ]. The vectors used, antibiotic resistances, primer sets and restriction sites are summarized in Tables 1 and 2 .…”

“…To purify His-tagged prey proteins for GST pull-down and in-vitro crosslinking affinity chromatography with Ni-NTA was used as described previously [ 15 ]. In brief, E. coli cells were suspended (5 ml/1 g wet cell weight) in buffer A (50 mM Tris/HCl, pH 7.5, 300 mM KCl, 10 mM MgCl 2 , 10 µM ZnCl 2 , 20 mM imidazole, 5 mM DTT and protease inhibitor cocktail (Roche, Basel, Switzerland), for GST pull-down experiments) or buffer B (100 mM sodium phosphate buffer, 150 mM NaCl, 10 µM ZnCl 2 , for bis(sulfosuccinimidyl)suberate (BS 3 ) crosslinking) and lysed by sonification (3 × 8 min) in an ice ethanol bath.…”

“…In 2015 it was shown that the truncated α-subunit of Rattus norvegicus (FTαΔ1-29) and full length FTα of Saccharomyces cerevisiae are capable of forming homodimers, assuming FTα might be involved in further intracellular signalling pathways [ 14 ]. However, our group was not able to prove homodimerization of native full-length human and rat FTα [ 15 ]. Notably, the artificially removed N-terminal part of rat FTα is a proline-rich region (PRR).…”

Mechanistic insight into impact of phosphorylation on the enzymatic steps of farnesyltransferase

“…The tight interaction between the two subunits has been shown to contribute to the stability of the enzyme, which is crucial for expediting the reaction rate. [2][3][4] The catalytic β-subunit of the heterodimeric FTase is surrounded by a crescent-shaped α-subunit, which is shared between FTase and GGTase-I, another enzyme namely geranylgeranyltransferase type I, is responsible for prenylation of specific proteins. The binding site of FTase is placed at a region where the two subunits intersect each other, thus constituting a funnelshaped cavity therein.…”