Tumor necrosis factor (TNF)␣amyloid ͉ apoptosis

Protein farnesyltransferase (FTase), a heterodimer enzyme consisting of alpha and beta subunits, catalyzes the addition of farnesyl groups to the C termini of proteins such as Ras. In this paper, we report that the protein substrate specificity of yeast FTase can be switched to that of a closely related enzyme, geranylgeranyltransferase type I (GGTase I) by a single amino acid change at one of the three residues: Ser-159, Tyr-362, or Tyr-366 of its beta-subunit, Dpr1. All three Dpr1 mutants can function as either FTase or GGTase I beta subunit in vivo, although some differences in efficiency were observed. These results point to the importance of two distinct regions (one at 159 and the other at 362 and 366) of Dpr1 for the recognition of the protein substrate. Analysis of the protein, after site directed mutagenesis was used to change Ser-159 to all possible amino acids, showed that either asparagine or aspartic acid at this position allowed FTase beta to function as GGTase I beta. A similar site-directed mutagenesis study on Tyr-362 showed that leucine, methionine, or isoleucine at this position also resulted in the ability of mutant FTase beta to function as GGTase I beta. Interestingly, in both position 159 and 362 substitutions, amino acids that could change the protein substrate specificity had similar van der Waals volumes. Biochemical characterization of the S159N and Y362L mutant proteins showed that their kcat/Km values for GGTase I substrate are increased about 20-fold compared with that of the wild type protein. These results demonstrate that the conversion of the protein substrate specificity of FTase to that of GGTase I can be accomplished by introducing a distinct size amino acid at either of the two residues, 159 and 362.

Protein farnesyltransferase (FTase) is a key enzyme responsible for the lipid modification of a large and important number of proteins including Ras. Recent demonstrations that inhibitors of this enzyme block the growth of a variety of human tumors point to the importance of this enzyme in human tumor formation. In this paper, we report that a mutant form of human FTase, Y361L, exhibits increased resistance to farnesyltransferase inhibitors, particularly a tricyclic compound, SCH56582, which is a competitive inhibitor of FTase with respect to the CAAX (where C is cysteine, A is an aliphatic amino acid, and X is the C-terminal residue that is preferentially serine, cysteine, methionine, glutamine or alanine) substrates. The Y361L mutant maintains FTase activity toward substrates ending with CIIS. However, the mutant also exhibits an increased affinity for peptides terminating with CIIL, a motif that is recognized by geranylgeranyltransferase I (GGTase I). The Y361L mutant also demonstrates activity with Ha-Ras and Cdc42Hs proteins, substrates of FTase and GGTase I, respectively. In addition, the Y361L mutant shows a marked sensitivity to a zinc chelator HPH-5 suggesting that the mutant has altered zinc coordination. These results demonstrate that a single amino acid change at a residue at the active site can lead to the generation of a mutant resistant to FTase inhibitors. Such a mutant may be valuable for the study of the effects of FTase inhibitors on tumor cells.Protein farnesyltransferase (FTase) 1 catalyzes the transfer of a farnesyl group onto a conserved cysteine residue four amino acids from the C terminus of a number of proteins, such as Ras, that are involved in cell growth and morphogenesis (1-4). This modification is critical for membrane association and subsequent protein-protein interactions of these proteins. FTase is a heterodimeric enzyme consisting of ␣-and ␤-subunits. The ␣-subunit of FTase is shared with the related prenyltransferase, protein geranylgeranyltransferase type I (GGTase I), whereas the ␤-subunits of FTase and GGTase I are approximately 30% homologous. The FTase enzyme recognizes the CAAX motif (C is cysteine, A is an aliphatic amino acid, and X is the C-terminal residue that is preferentially serine, cysteine, methionine, glutamine or alanine) that is found at the C termini of the substrate proteins. GGTase I enzyme also recognizes a CAAX motif; however, the terminal X amino acid is predominantly leucine or phenylalanine. This motif is referred to as the CAAL motif.The three-dimensional structures of the rat FTase enzyme have recently been resolved without substrate bound (5, 6), with bound substrate farnesyl pyrophosphate (FPP) (7), and with bound FPP and CAAX peptide analogs (8). In the structure without bound substrate, a non-cognate peptide provided by an adjacent ␤-subunit was modeled into the active site of the enzyme. The structure showed that the ␣-and ␤-subunits are largely composed of ␣-helices. The ␤-subunit forms a barrel-like structure, and one side of this barrel i...

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