All vertebrates display a characteristic asymmetry of internal organs with the cardiac apex, stomach and spleen towards the left, and the liver and gall bladder on the right. Left-right (L-R) axis abnormalities or laterality defects are common in humans (1 in 8,500 live births). Several genes (such as Nodal, Ebaf and Pitx2) have been implicated in L-R organ positioning in model organisms. In humans, relatively few genes have been associated with a small percentage of human situs defects. These include ZIC3 (ref. 5), LEFTB (formerly LEFTY2; ref. 6) and ACVR2B (encoding activin receptor IIB; ref. 7). The EGF-CFC genes, mouse Cfc1 (encoding the Cryptic protein; ref. 9) and zebrafish one-eyed pinhead (oep; refs 10, 11) are essential for the establishment of the L-R axis. EGF-CFC proteins act as co-factors for Nodal-related signals, which have also been implicated in L-R axis development. Here we identify loss-of-function mutations in human CFC1 (encoding the CRYPTIC protein) in patients with heterotaxic phenotypes (randomized organ positioning). The mutant proteins have aberrant cellular localization in transfected cells and are functionally defective in a zebrafish oep-mutant rescue assay. Our findings indicate that the essential role of EGF-CFC genes and Nodal signalling in left-right axis formation is conserved from fish to humans. Moreover, our results support a role for environmental and/or genetic modifiers in determining the ultimate phenotype in humans.
Many key regulatory proteins, including members of the Ras family of GTPases, are modified at their C terminus by a process termed prenylation. This processing is initiated by the addition of an isoprenoid lipid, and the proteins are further modified by a proteolytic event and methylation of the C-terminal prenylcysteine. Although the biological consequences of prenylation have been characterized extensively, the contributions of prenylcysteine methylation to the functions of the modified proteins are not well understood. This reaction is catalyzed by the enzyme isoprenylcysteine carboxyl methyltransferase (Icmt). Recent genetic disruption studies have provided strong evidence that blocking Icmt activity has profound consequences on oncogenic transformation. Here, we report the identification of a selective small-molecule inhibitor of Icmt, 2-[5-(3-methylphenyl)-1-octyl-1H-indol-3-yl]acetamide (cysmethynil). Cysmethynil treatment results in inhibition of cell growth in an Icmt-dependent fashion, demonstrating mechanism-based activity of the compound. Treatment of cancer cells with cysmethynil results in mislocalization of Ras and impaired epidermal growth factor signaling. In a human colon cancer cell line, cysmethynil treatment blocks anchorage-independent growth, and this effect is reversed by overexpression of Icmt. These findings provide a compelling rationale for development of Icmt inhibitors as another approach to anticancer drug development.cell transformation ͉ protein isoprenylation ͉ protein methylation ͉ Ras signaling A C-terminal CaaX motif, where C is cysteine, the a's are aliphatic amino acids, and X can be any of a number of amino acids, targets a variety of eukaryotic proteins to a series of posttranslational modifications important for their localization and function (1, 2). This processing is initiated by the covalent attachment of a 15-carbon farnesyl or a 20-carbon geranylgeranyl lipid to the cysteine of the CaaX motif, a reaction catalyzed by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (3). After prenylation, the C-terminal three amino acids (i.e., the -aaX) are removed by a specific CaaX protease termed Rce1 (4, 5) and the now Cterminal prenylcysteine is methylated by isoprenylcysteine carboxyl methyltransferase (Icmt; refs. 6-8). As polytopic membrane proteins localized to the endoplasmic reticulum, both Rce1 and Icmt are unusual in their respective classes (9).Proteins that terminate in a -CaaX motif regulate a number of pathways important in oncogenesis. The best studied example is the central role of the Ras family of proteins in growth factor activation of the MAP kinase signaling cascade (10, 11). Constitutive activation of this pathway is transforming in a wide variety of cell types, and activating mutations in Ras have been found in almost 30% of all cancers, including 50% of colon cancers and up to 90% of pancreatic cancers (12). In addition, many cancers contain alterations upstream of Ras, and the resultant hyperactivation of Ras is thought to...
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