Mutations in the gene encoding the amyloid protein precursor (APP) cause autosomal dominant Alzheimer's disease. Cleavage of APP by unidentified proteases, referred to as beta- and gamma-secretases, generates the amyloid beta-peptide, the main component of the amyloid plaques found in Alzheimer's disease patients. The disease-causing mutations flank the protease cleavage sites in APP and facilitate its cleavage. Here we identify a new membrane-bound aspartyl protease (Asp2) with beta-secretase activity. The Asp2 gene is expressed widely in brain and other tissues. Decreasing the expression of Asp2 in cells reduces amyloid beta-peptide production and blocks the accumulation of the carboxy-terminal APP fragment that is created by beta-secretase cleavage. Solubilized Asp2 protein cleaves a synthetic APP peptide substrate at the beta-secretase site, and the rate of cleavage is increased tenfold by a mutation associated with early-onset Alzheimer's disease in Sweden. Thus, Asp2 is a new protein target for drugs that are designed to block the production of amyloid beta-peptide peptide and the consequent formation of amyloid plaque in Alzheimer's disease.

The crystal structure of a complex between chemically synthesized human immunodeficiency virus type 1 (HIV-1) protease and an octapeptide inhibitor has been refined to an R factor of 0.138 at 2.5-A resolution. The substrate-based inhibitor, H-Val-Ser-Gln-Asn-Leu psi [CH(OH)CH2]Val-Ile-Val-OH (U-85548e) contains a hydroxyethylene isostere replacement at the scissile bond that is believed to mimic the tetrahedral transition state of the proteolytic reaction. This potent inhibitor has Ki less than 1 nM and was developed as an active-site titrant of the HIV-1 protease. The inhibitor binds in an extended conformation and is involved in beta-sheet interactions with the active-site floor and flaps of the enzyme, which form the substrate/inhibitor cavity. The inhibitor diastereomer has the S configuration at the chiral carbon atom of the hydroxyethylene insert, and the hydroxyl group is within H-bonding distance of the two active-site carboxyl groups in the enzyme dimer. The two subunits of the enzyme are related by a pseudodyad, which superposes them at a 178 degrees rotation. The main difference between the subunits is in the beta turns of the flaps, which have different conformations in the two monomers. The inhibitor has a clear preferred orientation in the active site and the alternative conformation, if any, is a minor one (occupancy of less than 30%). A new model of the enzymatic mechanism is proposed in which the proteolytic reaction is viewed as a one-step process during which the nucleophile (water molecule) and electrophile (an acidic proton) attack the scissile bond in a concerted manner.

No abstract

The catalytic triad in caspase-8 comprises Cys360, His317 and the backbone carbonyl oxygen atom of Arg258, which points towards the Nepsilon atom of His317. The oxygen atom attached to the tetrahedral carbon in the thiohemiacetal group of the inhibitor is hydrogen bonded to Ndelta of His317, and is not in a region characteristic of a classical 'oxyanion hole'. The N-acetyl group of the inhibitor is in the trans configuration. The caspase-8-inhibitor structure provides the basis for understanding structure/function relationships in this important initiator of the proteolytic cascade that leads to programmed cell death.

No abstract

No abstract

The sequences of five distantly related adenylate kinases have been aligned. The local conservation of amino acids is discussed in the light of the known three-dimensional structure of one of the enzymes, the cytosolic isoenzyme 1 (AKI) from porcine muscle. The similarity profile outlines clearly the active site in the cleft of the spatial structure of AK1. The alignment reveals further that the enzyme family can be subdivided into small and large variants according to the presence or absence of a particular segment of about 30 residues in the middle of the chain. The extra segments of the large variants are strongly conserved. [6] are so similar to each other that only the porcine enzyme is considered here; the three-dimensional structure of porcine AK1 is known [7-91. The sequences of the mammalian isoenzymes AK2 [3, 101 and AK3 [Il, 121, the enzyme from Escherichiu coli [13] and the adenylate kinase of yeast cytosol [14] differ greatly from each other and from that of AK1. Here we report the alignment of five distantly related adenylated kinase sequences. They are compared with each other in the light of the three-dimensional structure of AKI. METHODS Amino acid similarities and alignmentSequence alignments are the more difficult the less related the sequences are. If less than about a third of the amino acid residues have been conserved, it is necessary to take similarities between exchanged amino acids into account. These similarities have to be defined and quantified [15, 161. The most simple criterion for the similarity of two amino acids is the frequency of their exchange at equivalent positions of homologous proteins. These natural exchange frequencies were established by Schwartz and Dayhoff [17] and compiled in the symmetric exchange matrix 'MDM78'. For our alignment we use this empirical matrix because theoretical similarities derived from physicochemical properties of amino acid side chains are prone to be biased one way or the other.Aligning sequences means locating insertions and deletions, which is done by placing gaps in the compared polypeptides. For convenience we use 'gap' for a missing single residue in an alignment, because this notion is better suited for the computational problem than the more general terms 'insertion' and 'deletion'. Even with a few gaps, however, the possible number of alignments is enormous. Allowing for only one gap in each of the five compared AK sequences, for instance, gives rise to about lo1' distinct alignments. Thus, a rigorous alignment of a family of sequences, which would establish absolutely the best fit, surpasses the capabilities of the fastest computers. As a consequence, approximate methods have to be applied. Puirwise sequence comparisonsAs a first step we quantified all ten pairwise comparisons among the five sequences using the McLachlan scheme [IS, 191 with a sampling length of nine residues. This procedure yielded ten maps of homology scores, which are sums of exchange matrix values as taken over the corresponding sampled nonapeptides. The co...

Apoptosis, or programmed cell death, plays a central role in the development and homeostasis of an organism. The breakdown of cellular proteins in apoptosis is mediated by caspases, which comprise a highly conserved family of cysteine proteases with specificity for aspartic acid residues at the P1 positions of their substrates. Multiple lines of evidence show that caspase-9 is critical for an apoptosis pathway mediated via the mitochondria. In this study, the three-dimensional structure of the catalytic domain of caspase-9 and its interaction with the inhibitor acetyl-Asp-Val-Ala-Asp fluoromethyl ketone (Ac-DVAD-fmk) have been predicted by a segment matching modeling procedure. As expected, the predicted caspase-9 structure shows both a high similarity in the overall folding topology and remarkable differences in the surface loop regions as compared to other caspase family members such as caspase-1, -3 and -8, for which crystal structures have been determined. This kind of comparative analysis reflects the convergence^divergence duality among the caspases. Moreover, some subtle differences have been observed between caspase-9 and caspase-3 in the subsite contacts with the covalently linked inhibitor Ac-DVADfmk. Based on the X-ray structural analysis of caspase-8, a main chain carbonyl oxygen appears to be involved in a catalytic triad with the active site Cys and His residues. The corresponding carbonyl oxygen in caspase-9, together with other expected features of the catalytic apparatus, appears in our model. The predicted structure of caspase-9 can serve as a reference for subsite analysis relative to rational design of highly selective caspase inhibitors for therapeutic application.z 2000 Federation of European Biochemical Societies.