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.

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Farnesylation, catalyzed by protein farnesyltransferase (FTase), is an important posttranslational modification guiding cellular localization. Recently predictive models for identifying FTase substrates have been reported. Here we evaluate these models through screening of dansylatedGCaaS peptides, which also provides new insights into the protein substrate selectivity of FTase.Farnesylation is a posttranslation modification that tags proteins with a farnesyl (C 15 ) isoprenoid supplied by farnesyl diphosphate (FPP) for the purpose of locating the protein to cellular membranes. These lipidated protein substrates are modified by farnesyltransferase (FTase) on their C-terminal FTase recognition sequence called the CaaX box. When it was found that farnesylation occurs on oncogenic Ras proteins 1 , FTase became a chemotherapeutic drug target. 2 Although initially developed based on a simple paradigm where FTIs would target Ras-driven tumors, FTIs have proven to work via a complex mechanism, and their activity is now attributed to the perturbation of a number of cellular proteins. 2, 3The complex and unexpected biology observed with FTIs has made a precise definition of the set of farnesylated proteins in a human cell critically important. It is not known how many proteins in the cell are farnesylated or what are the critical targets of FTIs. Early biochemical studies of Brown and Goldstein 4 and the Merck group 5 demonstrated that tetrapeptides bearing a cysteine, two amino acids, and the appropriate X residue are farnesylated and serve as the minimum substrate for FTase recognition. Recent modelling studies have provided predictions for FTase Ca 1 a 2 X box specificity and thus its protein substrates. 6, 7 These models are only predictive and require additional investigation 8, 9 to determine cellular protein farnesylation. Using traditional biological tools (radiolabeling and/or western blot analysis), it would be timeconsuming to confirm the cellular farnesylation of these hypothetical FTase substrates. Therefore, a screening approach to validate that FTase accepts and modifies the minimal substrate Ca 1 a 2 X boxes of a select group of these proteins would be useful ( Figure 1).As part of our laboratory's investigation into FTase specificity, we have synthesized a library of Dansyl-GCa 1 a 2 S pentapeptides representing FTase substrate candidates. The sequences were identified from a Swissprot database search for carboxyl-terminal Ca 1 a 2 S boxes. Sequences were chosen to represent a) biologically important farnesylated proteins, and b) † These authors contributed equally to this work.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the...

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Farnesyl diphosphate (FPP) analogues have proven to be both potent inhibitors of protein-farnesyltransferase (FTase) and valuable probes for the investigation of the function of prenylated proteins. Previously, we have demonstrated that certain 3-substituted and 7-substituted FPP analogues can act as inhibitors of FTase, while others are effective alternative substrates. We have now utilized our vinyl triflate-mediated route to synthesize the first seven FPP variants bearing substituents in both the 3- and 7-positions of the isoprene unit. Despite their exceptional steric bulk with respect to FPP itself, six of the seven analogues bind to FTase. Two of the analogues are potent inhibitors of the enzyme, but a more striking finding is that three FPP variants (4a, 4b, and 4f) are efficient alternative substrates for FTase.

Batten disease is a fatal neurodegenerative disease affecting children between the ages of five and ten years with an incidence of 2 to 4:100 000 live births in the United States and Canada. It is a type of lysosomal storage disease that causes an accumulation of lipofuscin in neuronal cells eventually leading to cellular degradation by normal physiological factors. The cause of Batten disease has been determined to be a 1.02 kb genomic deletion that mutates a gene known as CLN3, producing a mutant protein responsible for the pathology. The function this CLN3 protein has not been determined, although it has been predicted to be a transmembrane protein involved with lysosome trafficking. To aid future investigations pertaining to the pathology of Batten disease and in the development of therapies, we investigated the normal function of CLN3. Knockdown of CLN3 in HeLa cells was used to determine its role in autophagy. We determined that CLN3 has a role in cellular autophagy.