A protein geranylgeranyltransferase (PGT) that catalyzes the transfer of a 20-carbon prenyl group from geranylgeranyl pyrophosphate to a cysteine residue in protein and peptide acceptors was detected in bovine brain cytosol and partially purified. The enzyme was shown to be distinct from a previously characterized protein farnesyltransferase (PFT). The PGT selectively geranylgeranylated a synthetic peptide corresponding to the C terminus of the y6 subunit of bovine brain G proteins, which have previously been shown to contain a 20-carbon prenyl modification. Likewise, a peptide corresponding to the C terminus of human lamin B, a known farnesylated protein, selectively served as a substrate for farnesylation by the PFT. However, with high concentrations of peptide acceptors, both prenyl transferases were able to use either peptide as substrates and the PGT was able to catalyze farnesyl transfer. Among the prenyl acceptors tested, peptides and proteins with leucine or phenylalanine at their C termini served as geranylgeranyl acceptors, whereas those with C-terminal serine were preferentially farnesylated. These results suggest that the C-terminal amino acid is an important structural determinant in controlling the specificity of protein prenylation.

New therapeutics to combat malaria are desperately needed. Here we show that the enzyme protein farnesyltransferase (PFT) from the malaria parasite Plasmodium falciparum (P. falciparum) is an ideal drug target. PFT inhibitors (PFTIs) are well tolerated in man, but are highly cytotoxic to P. falciparum. Because of their anticancer properties, PFTIs comprise a highly developed class of compounds. PFTIs are ideal for the rapid development of antimalarials, allowing "piggy-backing" on previously garnered information. Low nanomolar concentrations of tetrahydroquinoline (THQ)-based PFTIs inhibit P. falciparum PFT and are cytotoxic to cultured parasites. Biochemical studies suggest inhibition of parasite PFT as the mode of THQ cytotoxicity. Studies with malaria-infected mice show that THQ PFTIs dramatically reduce parasitemia and lead to parasite eradication in the majority of animals. These studies validate P. falciparum PFT as a target for the development of antimalarials and describe a potent new class of THQ PFTIs with antimalaria activity.

Ipomoea is the largest genus in the family Convolvulaceae. Ipomoea nil (Japanese morning glory) has been utilized as a model plant to study the genetic basis of floricultural traits, with over 1,500 mutant lines. In the present study, we have utilized second- and third-generation-sequencing platforms, and have reported a draft genome of I. nil with a scaffold N50 of 2.88 Mb (contig N50 of 1.87 Mb), covering 98% of the 750 Mb genome. Scaffolds covering 91.42% of the assembly are anchored to 15 pseudo-chromosomes. The draft genome has enabled the identification and cataloguing of the Tpn1 family transposons, known as the major mutagen of I. nil, and analysing the dwarf gene, CONTRACTED, located on the genetic map published in 1956. Comparative genomics has suggested that a whole genome duplication in Convolvulaceae, distinct from the recent Solanaceae event, has occurred after the divergence of the two sister families.

Protein geranylgeranyltransferase-I (PGGT-I) catalyzes the transfer of the 20-carbon prenyl group from geranylgeranyl pyrophosphate to the cysteine residue near the C-termini of a variety of eukaryotic proteins. Kinetic analysis of homogenous PGGT-I from bovine brain reveals that the reaction follows a sequential pathway in which either prenyl donor or acceptor can bind first to the enzyme and that the reaction operates at steady-state rather than at rapid equilibrium. Substrate inhibition by prenyl acceptor but not by prenyl donor suggests that geranylgeranyl pyrophosphate binding first to free enzyme is the kinetically preferred pathway. This is supported by isotope trapping experiments which show that the ternary complex goes on to products faster than the release of geranylgeranyl pyrophosphate from the complex. The KM for the interaction of geranylgeranyl pyrophosphate with PGGT-I is markedly affected by the structure of the prenyl acceptor bound to the enzyme. A detailed analysis of the substrate specificity of PGGT-I reveals that peptides which contain a C-terminal leucine are preferred (kcat/KM = 1-5 x 10(5) M-1 s-1) to those that end in serine (kcat/KM = 2-4 x 10(3) M-1 s-1) or phenylalanine (kcat/KM = 0.5 x 10(3) M-1 s-1). PGGT-I also catalyzes the farnesylation of peptides that have a C-terminal leucine; kcat for farnesylation and KM for farnesyl pyrophosphate are similar to those for geranylgeranylation, but the KM for the peptide is 30-fold higher. Geranyl pyrophosphate is utilized by PGGT-I but is a poor substrate. Optimal activity of PGGT-I is obtained in the presence of micromolar amounts of Zn2+ and mM amounts of Mg2+. Mn2+ or Cd2+ but not Co2+ can substitute for Zn2+ and for Mg2+. Metals are not required for tight-binding of geranylgeranyl pyrophosphate to PGGT-I, and the measured dissociation equilibrium constant for this binary complex is 16 nM. Photoaffinity analogues of geranylgeranyl pyrophosphate and farnesyl pyrophosphate were prepared and shown to exclusively label the beta-subunit. The implication of the results for the substrate specificity of protein prenylation in cells is briefly discussed.

Mutations in patatin-like phospholipase domain-containing 1 (PNPLA1) cause autosomal recessive congenital ichthyosis, but the mechanism involved remains unclear. Here we show that PNPLA1, an enzyme expressed in differentiated keratinocytes, plays a crucial role in the biosynthesis of ω-O-acylceramide, a lipid component essential for skin barrier. Global or keratinocyte-specific Pnpla1-deficient neonates die due to epidermal permeability barrier defects with severe transepidermal water loss, decreased intercellular lipid lamellae in the stratum corneum, and aberrant keratinocyte differentiation. In Pnpla1−/− epidermis, unique linoleate-containing lipids including acylceramides, acylglucosylceramides and (O-acyl)-ω-hydroxy fatty acids are almost absent with reciprocal increases in their putative precursors, indicating that PNPLA1 catalyses the ω-O-esterification with linoleic acid to form acylceramides. Moreover, acylceramide supplementation partially rescues the altered differentiation of Pnpla1−/− keratinocytes. Our findings provide valuable insight into the skin barrier formation and ichthyosis development, and may contribute to novel therapeutic strategies for treatment of epidermal barrier defects.