The amyloid β-peptide (Aβ), strongly implicated in the pathogenesis of Alzheimer’s disease (AD), is produced from the amyloid β-protein precursor (APP) through consecutive proteolysis by β- and γ-secretases. The latter protease contains presenilin as the catalytic component of a membrane-embedded aspartyl protease complex. Missense mutations in presenilin are associated with early-onset familial AD, and these mutations generally both decrease Aβ production and increase the proportion of the aggregation-prone 42-residue form (Aβ42) over the 40-residue form (Aβ40). The connection between these two effects is not understood. Besides Aβ40 and Aβ42, γ-secretase produces a range of Aβ peptides, the result of initial cutting at the ε site to form Aβ48 or Aβ49 and subsequent trimming every 3–4 residues. Thus, γ-secretase displays both overall proteolytic activity (ε cutting) and processivity (trimming) toward its substrate APP. Here we tested whether a decrease in total activity correlates with decreased processivity using wild type and AD-mutant presenilin-containing protease complexes. Changes in pH, temperature and salt concentration that reduced overall activity of the wild type enzyme did not consistently result in increased proportions of longer Aβ peptides. Low salt concentrations and acidic pH were notable exceptions that subtly alter the proportion of individual Aβ peptides, suggesting that the charged state of certain residues may influence processivity. Five different AD-mutant complexes, representing a broad range of effects on overall activity, Aβ42-to-Aβ40 ratios, and ages of disease onset were also tested, revealing again that changes in total activity and processivity can be dissociated. Factors that control initial proteolysis of APP at the ε site apparently differ significantly from factors affecting subsequent trimming and the distribution of Aβ peptides.

The cell has >60 different farnesylated proteins. Many critically important signal transduction proteins are post-translationally modified with attachment of a farnesyl isoprenoid catalyzed by protein farnesyltransferase (FTase). Recently, it has been shown that farnesyl diphosphate (FPP) analogues can alter the peptide substrate specificity of FTase. We have used combinatorial screening of FPP analogues and peptide substrates to identify patterns in FTase substrate selectivity. Each FPP analogue displays a unique pattern of substrate reactivity with the tested peptides; FTase efficiently catalyzes the transfer of an FPP analogue selectively to one peptide and not another. Furthermore, we have demonstrated that these analogues can enter cells and be incorporated into proteins. These FPP analogues could serve as selective tools to examine the role prenylation plays in individual protein function.Mutant Ras proteins are one of the most important classes of oncogene products and are thus logical targets for cancer chemotherapeutics. Ras, both mutant and normal forms, must be farnesylated by protein farnesyltransferase (FTase) for proper processing, subcellular localization, and thus biological activity (Figure 1). Therefore, significant effort has been focused on the development of small-molecule FTase inhibitors (FTIs) as anticancer, antiRas therapeutics. Two FTIs are in advanced clinical trials (1). The clinical data have demonstrated, however, that FTIs do not function as anti-Ras agents, because K-Ras is alternatively prenylated by geranylgeranyltransferase I upon FTI treatment (23). The cellular (4) and clinical (1) efficacy of FTIs does not correlate with Ras mutational status. The FTI effectiveness observed in non-Ras-positive tumor cells is presumably elicited via inhibition of the farnesylation of other proteins crucial to the growth of tumors. This has led to significant interest in defining the entire set of mammalian prenylated proteins and determining their biological roles (5).Many proteins bearing a Ca 1 a 2 X sequence at their carboxyl terminus are modified by FTase using the C 15 isoprenoid farnesyl diphosphate (FPP) as a co-substrate. Substrate prediction models have estimated that there are >60 farnesylated cellular proteins (67), containing a wide variety of C-terminal sequences, and the inhibition of farnesylation of any individual or a combination of these proteins could be responsible for the antitumor effects of FTI treatment. The investigation of potential "protein-X" FTI targets has uncovered several proteins whose inactivation upon FTI treatment led to profound cellular consequences (8). Correspondingly, the investigation of the role of the farnesyl group on cellular proteins has been aided by the development of FTIs. However, using FTIs to investigate the function of the farnesyl lipid for an individual protein is cumbersome, as they are nonspecific tools. FTIs presumably block the farnesylation of all FTase substrate proteins in mammalian cells. Chemical agents that are capable ...

Proteins bearing a CaaL sequence are typically geranylgeranylated to enable their proper localization and function. We found that many of the dansyl-GCaaL peptides representing mammalian CaaL proteins can be farnesylated by FTase. This result may have important implications for prenylated protein biology.