Icmt (Isoprenylcysteine Carboxyl Methyltransferase)
Isoprenylcysteine carboxyl methyltransferase (Icmt) is the only known prenylcysteine protein-dependent methyltransferase and plays a critical role in the posttranslational modification of prenylated proteins (Bergo et al. 2000). The Icmt ortholog from Saccharomyces cerevisiae, STE14p, was the first prenylcysteine carboxyl methyltransferase to be cloned and sequenced, and it is considered the founding member of this eukaryote protein methyltransferase family (Anderson et al. 2005). STE14p was originally recognized in a screen of mutant yeast, deficient of STE14, which rendered the yeast sterile due to their failure to methylate the mating pheromone a-factor, a protein crucial for fertility. STE14p is a 26 kDa integral membrane protein with multiple transmembrane (TM)-spanning domains and is localized to the endoplasmic reticulum (ER) (Romano and Michaelis 2001). Human Icmt cDNA possess notable homology to STE14, and upon its expression in STE14-deficient yeast, the sterile phenotype is reversed (Svensson et al. 2006). Icmt orthologs such as Schizosaccharomyces pombe (mam4p), and Xenopus laevis (Xmam4p), also exhibit considerable amino acid homology with STE14p and behave as functionally complimentary prenylcysteine carboxyl methyltransferases (Wright et al. 2009). STE14p and its orthologs lack significant amino acid conservation to other nucleotide, DNA, or protein methyltransferases, including the absence of the commonly observed consensus S-adenosylmethionine (SAM or AdoMet)-binding motifs (Wright et al. 2009). The novelty and specificity of its biologic role and its unique evolutionary conservation, along with the Icmt knockout mouse being embryonic lethal, suggest that the function of Icmt is critical (Bergo et al. 2001).
Posttranslational modification of eukaryotic polypeptides is critical for proper function and localization of mature proteins. Many of these proteins are modified by a three-step enzymatic process which is dictated by the succession of amino acids at the C-terminus. These proteins, termed CaaX proteins, contain a specialized carboxyl-terminal amino acid sequence that orchestrates modification of nascent proteins. Icmt is responsible for the final and only potentially reversible enzymatic step in CaaX protein processing, which involves methylation of the carboxylic acid of the protein terminal isoprenylated cysteine residue. Ras, and other critical signaling proteins, including Rho GTPases, undergo this posttranslational modification via the CaaX motif (Anderson et al. 2005).
The CaaX sequence is represented by a cysteine, followed by two residues that are typically aliphatic (aa), and finally an amino acid which dictates which of two prenyl groups will be covalently attached to the cysteine in the motif by the enzymes protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase-I). Note there is a second class of prenylated proteins, the Rab CXC proteins, which include a CXC or CC sequence motif. CXC proteins are prenylated by protein geranylgeranyltransferase type II (GGTase-II). Icmt is capable of identifying substrates from both classes of proteins (Anderson et al. 2005). Despite the substrate prenylation specificity of the two CaaX prenyl transferases, it has been documented that alternate utilization of protein substrates does occur (Wright et al. 2009).
Following prenylation, the second step of CaaX protein modification involves Ras-converting enzyme I (RceI). This ER integral membrane protease cleaves the terminal three -aaX amino acid residues of the CaaX motif rendering the prenylated cysteine residue the new carboxy-terminus. Upon cleavage by RceI, CaaX proteins are specifically methylated on the alpha carboxyl group of the prenylated cysteine by Icmt. Methylation removes the negative charge from the carboxylic acid of the prenylated cysteine, and is therefore the final crucial step to modify proteins in order to increase their hydrophobicity, by leaving the protein with a terminal isoprenylcysteine methyl ester residue. The major observed effect of the isoprene lipid and methylation of the CaaX protein is a high degree of association with cell membranes.
Structure, Interactions, and Regulation
Icmt differs from other methyltransferases due to its unique ability to selectively bind and methylate prenylated and proteolyzed CaaX proteins. Icmt also appears to be the sole enzyme responsible for methyl-esterification of the Rab CXC proteins (Bergo et al. 2000). Icmt is restricted to the ER, and hydropathy-based topology predictions from sequence studies show STE14p likely navigates through the ER membrane with six TM segments (Wright et al. 2009). Functional evidence suggests STE14p is active as a homodimer and may even be able to further oligomerize, and the enzyme contains a GXXGXXG motif that is responsible for dimerization of several other TM proteins (Griggs et al. 2010). The human Icmt protein contains an additional N-terminal domain predicted to contain two additional TM segments for a total of eight TM segments (Wright et al. 2009). The evolution of this additional sequence grants consideration that Icmt has acquired a unique regulatory role in mammals (Wright et al. 2009). The structure of a prokaryotic ortholog of ICMT was solved and demonstrated that ICMT is a five alpha-helical transmembrane bundle. The catalytic mechanism takes advantage of lipid access to the plasma membrane using a channel which opens between helix one and two and extends into the transmembrane region. The aqueous exposed intracellular domains (extraER) and C-terminal domain provide a mechanism for interaction with the SAM cofactor with the space between the second intracellular loop and the C-terminal domain providing an entry/exit site for the soluble methyl donor (Yang et al. 2011). Along with the X-ray data, mutagenesis studies have aided in the identification of two conserved SAM-binding motifs unique to the ICMT family of methyltransferases (1:HxVxxxxYxxxRHPxY and 2:RxxxEExxLxxxFxxxYxxxY) (Diver and Long 2014).
Based on the well-characterized substrates of Icmt (farnesyl- and geranylgeranylcysteine), synthetic prenylcysteine analogs were designed to act as efficient Icmt substrates and as classical competitive inhibitors. Establishment of an ordered sequential kinetic mechanism determined that Icmt binds first to the methyl donor SAM (Km ∼2 μM) followed by the binding of a CaaX protein, thereby allowing methylation to occur (Baron and Casey 2004). The Icmt-catalyzed reaction leaves a newly methylated protein and S-adenosylhomocysteine (SAH or AdoHcy).
Currently, Icmt activity regulation appears to be mostly substrate availability-dependent. The active guanine triphosphate (GTP)-bound state of guanine nucleotide binding CaaX proteins, compared to the inactive GDP-bound state, displays a characteristic increase in methylation proficiency (Kowluru et al. 1996). Despite apparent constitutive expression and activation in most cell types, there is evidence indicating Icmt is responsible for increased methylation of CaaX proteins as a consequence of particular ligands in specific cell types and that its expression can be regulated. Icmt is responsible for the methylation of a large number of Ras family GTPases and a variety of non-Ras-signaling proteins such as the nuclear lamins, heterotrimeric G-protein gamma subunits, the prostacyclin and prostaglandin E2 receptors, and some phosphatases and kinases (Reid et al. 2004). Methylation of CaaX proteins is also reversible through the action of proteins like carboxyesterase, creating a dynamic and likely regulated equilibrium between methylated and un- or demethylated protein (Cushman et al. 2013). Certain disease states such as Parkinson’s disease, or physiologic conditions resulting from pharmacological treatment as seen with antifolate drugs like the dihydrofolate reductase inhibitor methotrexate, can elevate the Icmt reaction end product SAH (Winter-Vann et al. 2003). Like with many enzymes and their end products, SAH inhibits Icmt leading to cellular apoptosis and provides a partial mechanism for the therapeutic effects of drugs like methotrexate in cancer, arthritis, and psoriasis (Winter-Vann et al. 2003). Evidence supports a mechanistic role of Icmt in neuroblastoma differentiation due to a decrease in the methylation of key proteins induced by retinoic acid (Van Dessel et al. 2002). Detectable increase in methylation of CaaX and CXC proteins is observed in such cases as Cdc42 upon glucose treatment and Rap1 upon potassium treatment in beta cells (Kowluru et al. 1996).
Expression and Phenotype
Icmt is ubiquitously expressed but displays enhanced expression in testes, liver, and brain tissues, with expression of up to three splice variants being possible (Bergo et al. 2001). An alteration of Icmt expression has only been observed during mouse development, where detectable mRNA levels are observed by postnatal day 11 and increase to reach a maximum expression by week four. Icmt is regulated posttranscriptionally by miR-100, with binds Icmt 3′ untranslated region of the RNA and prevents its translation (Zhou et al. 2014). In hepatocarcinoma cells this leads to deceased mobility through decreased metalloproteinase function and lamellipodia formation. The inactivation of one Icmt allele produces no apparent phenotype, whereas the deletion of Icmt locus in mice by homologous recombination results in embryonic lethality (Bergo et al. 2001). Icmt-deficient mice embryos die from anemia and cellular apoptosis at mid-gestation by day 11.5 with the primary afflicted tissue being testes, brain, and liver, with additional disruption of skeletal muscle development (Bergo et al. 2001).
More details of Icmt function were demonstrated through genetic ablation. Tissue-specific knockout of Icmt in the retina prevents an effective association between the visual G-protein transducin and phosphodiesterase, stopping this critical signaling cascade and a resulting impairment of vision (Christiansen et al. 2016). Oncogenic Kras-driven pancreatic ductal adenocarcinoma is accelerated with the loss of Icmt through increased Wnt signaling and decreased CDK activity (Court et al. 2013). Knockdown of Icmt decreased cellular respiration by inhibiting the function of mitochondrial complexes leading to suppression of oxidative phosphorylation, which in turn decreased available ATP and subsequent metabolic processes. By decreasing cellular respiration, Icmt knockdown suppressed proliferation and sensitized cells to autophagy (Teh et al. 2015). Partial loss of Icmt prevents the appropriate localization of prelamin A and causes deregulated activation of the AKT-mTOR pathway leading to amelioration of the prelamin A accumulation in the nucleus associated with aggressive progeria (Ibrahim et al. 2013).
Mutations of the Ras proto-oncogene are frequent aberrancies observed in human cancer, and consequently, Ras proteins are the most studied CaaX protein. Icmt is critical for the proper plasma membrane targeting of H-, N-, and K-Ras, and localization is essential for their correct function. Icmt-deficient cells exhibit mislocation and cytoplasmic accumulation of Ras proteins (Michaelson et al. 2005). The demonstration of a reduction in K-Ras methylation being linked to slowed cell growth and attenuation of oncogenic transformation has lead to the consideration of Icmt inhibition as an attractive therapeutic target. Given the important role of oncogenic Ras protein in human tumorigenesis, it has been hypothesized that the inhibition of Icmt could be an efficacious strategy to block Ras-induced oncogenic transformation. A large effort has been undertaken in targeting FTase in CaaX processing, leading to the development of highly selective and potent FTase inhibitors, but these compounds were found to lack clinical efficacy. Icmt has been the favored alternate target due to the inability of Ras to promote transformation if it is unmethylated, irrespective if prenylation and proteolysis occurs (Svensson et al. 2006).
With mounting evidence indicating inhibiting Icmt would provide a unique therapeutic effect, several compound series have been developed and used in cell and animal models. Design of potent and selective compounds has proved challenging considering that recognition by Icmt is driven by an isoprenylated cysteine. These compounds include cysmethynil, an alkanated phenylindole, and its more recent improved deviates as well as the prenylcysteine mimetics based on farnesyl-thiopropionic acid triazole (Go et al. 2010; Bergman et al. 2011, 2012; Majmudar et al. 2011, 2012; Ramanujulu et al. 2013; Lau et al. 2014). Attempts to prevent Ras-induced cellular transformation by inhibiting Icmt with substrate analogs displayed limitations, due to collateral inhibition of other isoprenylated proteins and/or other methyltransferases. The necessity for a specific pharmacologic agent against Icmt led to the identification of cysmethynil, an Icmt-specific inhibitor that has no observable effects on Icmt-deficient cells (Winter-Vann et al. 2005; Svensson et al. 2006). Icmt inhibition by cysmethynil results in decreased cell proliferation and a reduction in Ras-induced oncogenic transformation (Svensson et al. 2006). Studies show the treatment of cysmethynil on human colon cancer cells results in the mislocation of Ras, a decline in anchorage-independent growth, and a reduction in the response to treatment with epidermal growth factor (EGF) (Winter-Vann et al. 2005; Svensson et al. 2006). The effects of cysmethynil are reversible upon rescue by overexpression of Icmt. Recently, evidence suggests a role for Icmt in cellular migration by impacting the function of Rho GTPases through methylation (Fig. 1). Many Rho GTPases are involved in cell migration by synchronizing changes in actin cytoskeleton from external stimuli and therefore have implications in metastasis in oncogenic states. Cysmethynil inhibits migration in a highly metastatic breast cancer cell line, MDA-MB-231, with RhoA and Rac1 activity significantly diminished (Cushman and Casey 2009). This decrease in activity is attributed to the aforementioned observed increase in RhoGDI binding affinity to Rac and Rho; however, overexpression of Rho GTPases rescues cell migration repressed by inhibition of Icmt (Cushman and Casey 2011). The culmination of these data supports Icmt as a promising target in cancers commonly afflicted with aberrancies associated with prenylated proteins.
Icmt is a posttranslational modifying enzyme that specifically methylates prenylated proteins. Further evaluation of its structure and oligomerization state is critical for more in-depth analyses of its mechanistic properties and function. Additional domain within human Icmt is still not understood despite suggestions it may play a regulatory role. The unique biologic function of methylation raises the question about the existence and regulation of a potential prenylcysteine-dependent methylesterase. Addition of dynamic methylation/demethylation into RhoGDI cycling model will further characterize the impact of Icmt-induced methylation in cell signaling and protein degradation. Regardless of the future outcome of Icmt inhibition as an effective cancer treatment, investigation of Icmt-catalyzed methylation of prenylated proteins is pivotal for a complete understanding of the consequences it plays in protein interactions.
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