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...
Endothelin-converting enzyme 1 (ECE-1) is a membrane-bound metalloprotease that catalyses the conversion of inactive big endothelins into active endothelins. Two different isoforms (ECE-1a and ECE-1b) have previously been identified for human ECE-1. In the present study we have cloned a novel human ECE-1 isoform, termed ECE-1c, and have thus shown for the first time the existence of three distinct ECE-1 isoforms. The three isoforms differ only in their N-terminal regions and are derived from a single gene through the use of alternative promoters. Ribonuclease protection experiments revealed that, although the relative levels of the three isoform mRNA species vary between human tissues, ECE-1c mRNA is generally the predominant isoform messenger. Immunofluorescence microscopy analysis showed distinct subcellular localizations for the three isoforms: whereas ECE-1a and ECE-1c are localized at the cell surface, ECE-1b was found to be intracellular and showed significant co-localization with a marker protein for the trans-Golgi network. We determined that the three isoforms have similar kinetic rate constants (Km, kcat and Vmax) for the processing of big endothelin 1 and that the big endothelin isoforms 1, 2 and 3 are cleaved with similar relative velocities of 1.0:0.1:0.1 by the three isoenzymes.
A region in the human immunodeficiency virus (HIV) env message, with the potential to form a complex secondary structure (designated RRE), interacts with the rev protein (Rev). This interaction is believed to mediate export of HIV structural messenger RNAs from the nucleus to the cytoplasm. In this report the regions essential for Rev interaction with the RRE are further characterized and the functional significance of Rev-RRE interaction in vivo is examined. A single hairpin loop structure within the RRE was found to be a primary determinant for Rev binding in vitro and Rev response in vivo. Maintenance of secondary structure, rather than primary nucleotide sequence alone, appeared to be necessary for Rev-RNA interaction, which distinguishes it from the mechanism for cis-acting elements in DNA. Limited changes within the 200 nucleotides, which preserved the proper RRE conformational structure, were well tolerated for Rev binding and function. Thus, variation among the RRE elements present in the diverse HIV isolates would have little, if any, effect on Rev responsiveness.
The human immunodeficiency virus (HIV) tat protein (Tat) is a positive regulator of virus gene expression and replication. Biotinylated Tat was used as a probe to screen a lambda gt11 fusion protein library, and a complementary DNA encoding a protein that interacts with Tat was cloned. Expression of this protein, designated TBP-1 (for Tat binding protein-1), was observed in a variety of cell lines, with expression being highest in human cells. TBP-1 was localized predominantly in the nucleus, which is consistent with the nuclear localization of Tat. In cotransfection experiments, expression of TBP-1 was able to specifically suppress Tat-mediated transactivation. The strategy described may be useful for direct identification and cloning of genes encoding proteins that associate with other proteins to modulate their activity in a positive or negative fashion.
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