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Available in vitro and in vivo methods for verifying protein substrates for …

Biology Articles » Biochemistry » Protein Biochemistry » Farnesylation or geranylgeranylation? Efficient assays for testing protein prenylation in vitro and in vivo » Background

- Farnesylation or geranylgeranylation? Efficient assays for testing protein prenylation in vitro and in vivo

Prenylation is a lipid posttranslational modification (PTM) of proteins at cysteine residues in the C-terminal region [1-7]. The specific sequence environment recognized by prenyltransferases consists either of the CaaX box for farnesyltransferase (FTase) and geranylgeranyltransferase 1 (GGTase1) or C-terminal cysteines of Rab GTPases in the case of geranylgeranyltransferase 2 (GGTase2). In all instances, the cysteine-containing region must be preceded on the N-terminal side by approximately 10 residues providing a generally polar, flexible, so called linker segment without inherent conformational preferences [7]. The anchor can be of farnesyl (3 isoprenyl units) or of geranylgeranyl (4 isoprenyl units) type [8]. Targeting to cellular membranes [1,9] and mediation of protein-protein interactions [10-16] are well documented biological functions associated with these lipid anchors.

Members of the Ras family of GTPases are of particular medical interest, as their mutational hyperactivation as well as mutations of proteins lying upstream in their signaling pathways are associated with various cancers [17-24]. Several other CaaX proteins from the Rho family of GTPases [25,26] and Rap1A [27] are involved in tumorigenesis as well. Since their lipid modifications are essential for their biological function [10,28-31], inhibitors of prenyltransferases (PTases), especially of FTase [32-34] attracted the interest of pharmaceutical research as anti-cancer drugs. Two such compounds made it to phase III trials [35,36]. Furthermore, there is evidence that inhibitors of prenylation may be useful in the treatment of other diseases such as infestation with protozoa [6,37].

However, we are far from understanding the physiological consequences of inhibiting FTase or GGTase1 in cells since the lists of the respective substrates are essentially not known. Only a few dozen proteins, including several fungal lipopeptide pheromons [38,39] (e.g. a-mating factor of Saccharomyces cerevisiae [40,41]) as well as mammalian proteins of the Ras superfamily of small GTPases [42], the trimeric G proteins [43] and the nuclear lamins of type A [44] and B [45], have been experimentally identified and verified as substrates of specific prenyltransferases yet. Given the critical role of the prenyl anchor for biological function (both with respect to the occurrence of prenylation and to the dependence on anchor type), it is of growing interest to analyze the prenylation status of so far uninvestigated proteins and to enlarge the list of proven prenylated proteins. A recently developed sophisticated in silico method [46] generates a high number of predicted protein candidates for prenylation and, especially for twilight zone predictions, efficient methods for experimental verification of prenylation are necessary.

The standard literature method for in vitro or in vivo analysis of selected candidates involves transcription/translation of a cloned construct and protein prenylation in the presence of 3H-labeled lipid anchor precursors followed by autoradiography/fluorography [47-49]. Necessary controls involve mutations of the C-terminal cysteine expected to be modified, prenyltransferase inhibitor applications and/or exposition to precursors of alternative prenyl anchors during the prenylation reaction. However, the reportedly long exposure times (weeks/months) contradict the need for several repetitions of the experiment. Optimization of protein expression and incubation conditions is typically not avoidable. In our own experience, many attempts with the standard technology ended up without reportable result; i.e., the signals in initial experiments were often below the detection limit. Scientific literature research showed that rarely a lab has studied the prenylation status of more than a single target, apparently as a consequence of the tenacious methodology.

The problem of long exposure times for 3H-autoradiography has prompted us to study a variety of chromatographic and scintillation methods for developing a faster and more sensitive test system. We found a solution using a TLC linear analyzer for testing the prenylation of selected protein targets. N-terminally GST-tagged proteins were in vitro transcribed/translated and incubated with 3H-labeled anchor precursors. Such a quick in vitro screen might also be useful for finding proteins that deserve the effort for detailed in vivo studies. A similar approach can be used in vivo, if HA-tagged target proteins are expressed in cell culture supplemented by radioactive prenyl anchor precursors. This new approach on the detection of weak 3H-signals is expected also to be helpful for monitoring posttranslational modifications with similar 3H-labeled anchors such as myristoyl or palmitoyl.

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