The virion cores of the replication competent type 1 human immunodeficiency virus (HIV‐1), a retrovirus, contain and RNA genome associated with nucleocapsid (NC) and reverse transcriptase (RT p66/p51) molecules. In vitro reconstructions of these complexes with purified components show that NC is required for efficient annealing of the primer tRNALys,3. In the absence of NC, HIV‐1 RT is unable to retrotranscribe the viral RNA template from the tRNA primer. We demonstrate that the HIV‐1 RT p66/p51 specifically binds to its cognate primer tRNALys,3 even in the presence of a 100‐fold molar excess of other tRNAs. Cross‐linking analysis of this interaction locates the contact site to a region within the heavily modified anti‐codon domain of tRNALys,3.

We have modified an Escherichia coli vector expressing 66-kDa HIV-1 reverse transcriptase (p66) so that it simultaneously expresses this and the pol-coded protease. The twin expression cassette yields high quantities of both reverse transcriptase and protease ; however, under these conditions, 50% of the over-expressed p66 reverse transcriptase is processed, resulting in accumulation of large quantities of p66/p51 enzyme. Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse-transcriptase-coding sequence (His-p66) permits a simple, rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography. Purified His-p66 and His-p66/His-p51 reverse transcriptase exhibit both reverse transcriptase and RNase H activity. Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer.Reverse transcriptase (deoxynucleoside-triphosphate: DNA deoxynucleotidyltransferase, RNA-directed) of human immunodeficiency virus, type 1 (HIV-1) is presently under extensive study as a target for antiviral drugs. Reverse transcriptase constitutes one component of the HIV-1 pol open reading frame [l] and is processed, in either virions or heterologous systems expressing this polyprotein, to polypeptides of 66 kDa and 51 kDa, through the action of the polcodcd protease [2 -61. Although expression systems for highlevel synthesis of the enzymatically active 66-kDa reverse transcriptase (p66) have been documented [7, 81, the occurrence, nevertheless, of a 51 -kDa polypeptide (p51) suggests that a p66/p51 heterodimer may be the relevant enzyme form. To elucidate this, large quantities of the p66/p51 enzyme are required to determine whether its enzymatic or physical properties differ from those of p66. Since p51 reverse transcriptase is produced from p66 via C-terminal processing [9], and its C-terminus (and hence p51 /p66 cleavage recognition site) is not clearly defined, it is impossible by conventional cloning technology to produce a p.51 polypeptide with an authentic C-terminus. We have addressed this problem by over-expressing p66 reverse transcriptase and the pol-coded protease as separate proteins on the same vector, under the control of similar transcription and translation signals. In the absence of protease, p66 reverse transcriptase accumulates to approximately 10% of the total cellular protein. In the dual expression system, a 17-kDa precursor HIV-1 protease (p17) is cleaved to the enzymatically active 10-kDa molecule, which Correspondence to S. F. J. Le Grice, Central Research Units, F. Hoffmann-La Roche & Co. Ltd, CH-4002 Basel, SwitzerlandAhhreviutians. HIV-1, human immunodeficiency virus, type 1 ; p66, 66-kDa HIV-1 reverse transcriptase polypeptide; p51, 51-kDa proteolytic product of p66; p17, 17-kDa precursor polypeptide of HIV-1 protease; His-p66, p66 with (His), added at the amino terminus; His-p51, p51 with (His)6 ...

The full-length and ectodomain forms of ␤-site APP cleavage enzyme (BACE) have been cloned, expressed in Sf9 cells, and purified to homogeneity. This aspartic protease cleaves the amyloid precursor protein at the ␤-secretase site, a critical step in the Alzheimer's disease pathogenesis. Comparison of BACE to other aspartic proteases such as cathepsin D and E, napsin A, pepsin, and renin revealed little similarity with respect to the substrate preference and inhibitor profile. On the other hand, these parameters are all very similar for the homologous enzyme BACE2. Based on a collection of decameric substrates, it was found that BACE has a loose substrate specificity and that the substrate recognition site in BACE extends over several amino acids. In common with the aspartic proteases mentioned above, BACE prefers a leucine residue at position P1. Unlike cathepsin D etc., BACE accepts polar or acidic residues at positions P2 and P1 but prefers bulky hydrophobic residues at position P3. BACE displays poor kinetic constants toward its known substrates (wild-type substrate, SEVKM2DAEFR, K m ‫؍‬ 7 M, K cat ‫؍‬ 0.002 s ؊1 ; Swedish mutant, SEVNL2DAEFR, K m ‫؍‬ 9 M, K cat ‫؍‬ 0.02 s ؊1 ). A new substrate (VVEVDA2AVTP, K m ‫؍‬ 1 M, K cat ‫؍‬ 0.004) was identified by serendipity.Alzheimer's disease is characterized by the extracellular deposition of insoluble amyloid plaques. The main component of amyloid plaques is the 39 -43-amino acid ␤-amyloid peptide (A␤), 1 which derives from a larger protein precursor (amyloid precursor protein, APP). A␤ is excised from APP by the sequential action of two proteases known, respectively, as ␤-secretase, which cuts amino-terminal to A␤, and ␥-secretase, which cleaves at the carboxyl terminus. Several reports appeared recently describing the cloning and characterization of ␤-secretase (1-5). This protein, designated Asp-2, BACE, or memapsin 2, according to the laboratory in which it was discovered, is a novel transmembrane aspartic protease that cleaves APP at the ␤-secretase site. BACE possesses all the characteristics expected for ␤-secretase in terms of substrate preference, pH optimum for activity, tissue distribution, and subcellular localization. In addition, two recent reports indicate that A␤ levels in the brains of BACE knockout mice are reduced by more than 90% compared with control mice (6, 7). In addition to cleaving APP at the ␤-secretase site, BACE cuts APP further downstream within the amyloid region (between Tyr-10 and Glu-11 of A␤), generating a truncated form of A␤ that is probably still amyloidogenic (3,8). Parallel to the discovery of BACE, a second, homologous transmembrane aspartic protease termed Asp-1, BACE2, memapsin 1, or down region aspartic protease was reported (4, 5, 9, 10). Preliminary analysis of BACE2 indicated that it can also function as a ␤-secretase in vitro (8,9).In seeking to develop a disease-modifying therapy for Alzheimer's Disease, BACE presents itself as an ideal drug target. It belongs to a well understood class of protease where inhibitors...

Two single site substitutions (E478 → Q and H539 → F) were introduced into the C‐terminal RNase H domain of HIV‐1 reverse transcriptase. These mutant proteins were expressed in Escherichia coli and purified by Ni2+‐nitrilotriacetic acid affinity chromatography. Both enzymes are clearly defective in RNase H function, but exhibit wild type reverse transcriptase activity.

Phytase is a high molecular weight acid phosphatase. The structure has an alpha/beta-domain similar to that of rat acid phosphatase and an alpha-domain with a new fold.

The Alzheimer ␤-amyloid peptide (A␤) and a fragment of the prion protein have the capacity of forming amyloid-like fibrils when incubated under physiological conditions in vitro. Here we show that a small amyloid ligand, RO-47-1816/001, enhances this process severalfold by binding to amyloid molecules and apparently promote formation of the peptide-to-peptide bonds that join the monomers of the amyloid fibrils. This effect could be antagonized by other ligands, including analogues of RO-47-1816/001, as well as the structurally unrelated ligand Congo red. Analogues of RO-47-1816/001 with low affinity for amyloid did not display any antagonistic effect. In conclusion, these data suggest that synthetic molecules, and possibly also small natural substances present in the brain, may act in a chaperone-like fashion, promoting A␤ polymerization and growth of amyloid fibrils in vitro and possibly also in vivo. Furthermore, we demonstrate that small organic molecules can be used to inhibit the action of amyloid-enhancing compounds.The amyloid deposits in brain vasculature and parenchyma that are the main histopathological hallmarks of Alzheimer's disease (1) are composed of large polymers of Alzheimer ␤-amyloid peptide (A␤). 1 This peptide is present in two major forms, one being 40 amino acids long, and the other, more aggregable form being 42 amino acids long (2-4). The A␤ peptide is secreted by numerous cell types in the body but the amyloid deposits are only present in the central nervous system.The amyloid deposits are formed by the aggregation of individual monomers of A␤ peptide into very large polymers which have a fibril-like appearance when observed in an electron microscope (5). These polymers can also be observed by light microscopy following staining with certain histological dyes such as Congo red and thioflavine T (6).Soluble A␤ peptide can be detected in blood and cerebrospinal fluid. The levels are, however, very low, usually in the low nano-to picomolar range and at these concentrations the peptide polymerizes at a very slow rate (7). A number of recent studies on the mechanisms of amyloid formation have conclusively shown that the presence of preformed oligo-or polymers of the amyloid peptide in the reaction mixture increases the polymerization rate dramatically (7). These multimers serve as templates for the reaction and, as a result, the initial, slow phase of primary nucleation is eliminated.It has also been proposed that charged molecules, such as gangliosides (8) and metal ions, including Zn 2ϩ and Cu 2ϩ (9), may enhance formation of amyloid in vitro and in vivo. Apolipoprotein E4, a well established risk factor for Alzheimer's disease (10) has also been suggested to enhance amyloid formation by serving as a "pathological chaperone" (11-13). In the case of apolipoprotein E, there are conflicting data suggesting that the protein can enhance as well as inhibit amyloidogenesis (14, 15).The existence of putative co-factors capable of enhancing amyloid formation potentially offers new targets for pharm...

We describe an assay system for the identification of site-specific proteases. The assay is based on a protein substrate that is immobilized on ceramic beads. After incubation with cell homogenates, the beads are washed and digested with endoproteinase Lys-C to liberate a defined set of peptides. The peptide fragments are identified by mass spectrometry. The assay was used to screen for beta-secretase, the protease that cleaves amyloid precursor protein (APP) at the beta-site. Cathepsin D was identified as the enzyme responsible for beta-secretase-like activity in two cell lines. Subsequent analysis of the related aspartic protease, cathepsin E, revealed almost identical cleavage specificity. Both enzymes are efficient in cleaving Swedish mutant APP at the beta-site but show almost no reactivity with wild-type APP. Treatment of cell lines with pepstatin inhibited the production of amyloid peptide (Abeta) when they were transfected with a construct bearing the Swedish APP mutant. However, when the cells were transfected with wild-type APP, the generation of Abeta was increased. This suggests that more than one enzyme is capable of generating Abeta in vivo and that an aspartic protease is involved in the processing of Swedish mutant APP.

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