Structural Features of Human Memapsin 2 （β-Secretase） and Their Biological and Pathological Implications

Alzheimer’s disease (AD), which is characterized bythe progressive destruction of brain functions in olderpeople, was first recognized in the early 20th century.Since then, modern medicine has further increased thenumber of people living to old age. AD h…     

Specificity of memapsin 1 and its implications on the design of memapsin 2 (beta-secretase) inhibitor selectivity

Memapsin 1 is closely homologous to memapsin 2 (BACE), or beta-secretase, whose action on beta-amyloid precursor protein (APP) leads to the production of beta-amyloid (A beta) peptide and the progression of Alzheimer's disease. Memapsin 2 is a current target for the development of inhibitor drugs to treat Alzheimer's disease. Although memapsin 1 hydrolyzes the beta-secretase site of APP, it is not significantly present in the brain, and no direct evidence links it to Alzheimer's disease. We report here the residue specificity of eight memapsin 1 subsites. In substrate positions P(4), P(3), P(2), P(1), P(1)', P(2)', P(3)', and P(4)', the most preferred residues are Glu, Leu, Asn, Phe, Met, Ile, Phe, and Trp, respectively, while the second preferred residues are Gln, Ile, Asp, Leu, Leu, Val, Trp, and Phe, respectively. Other less preferred residues can also be accommodated in these subsites of memapsin 1. Despite the broad specificity, these residue preferences are strikingly similar to those of human memapsin 2 [Turner et al. (2001) Biochemistry 40, 10001-10006] and thus pose a serious problem to the design of differentially selective inhibitors capable of inhibiting memapsin 2. This difficulty was confirmed by the finding that several potent memapsin 2 inhibitors effectively inhibited memapsin 1 as well. Several possible approaches to overcome this problem are discussed.     

In vivo inhibition of Abeta production by memapsin 2 (beta-secretase) inhibitors

We have previously reported structure-based design of memapsin 2 (beta-secretase) inhibitors with high potency. Here we show that two such inhibitors covalently linked to a "carrier peptide" penetrated the plasma membrane in cultured cells and inhibited the production of beta-amyloid (Abeta). Intraperitoneal injection of the conjugated inhibitors in transgenic Alzheimer's mice (Tg2576) resulted in a significant decrease of Abeta level in the plasma and brain. These observations verified that memapsin 2 is a therapeutic target for Abeta reduction and also establish that transgenic mice are suitable in vivo models for the study of memapsin 2 inhibition.     

Subsite specificity of memapsin 2 (beta-secretase): implications for inhibitor design

Memapsin 2 is the protease known as beta-secretase whose action on beta-amyloid precursor protein leads to the production of the beta-amyloid (Abeta) peptide. Since the accumulation of Abeta in the brain is a key event in the pathogenesis of Alzheimer's disease, memapsin 2 is an important target for the design of inhibitory drugs. Here we describe the residue preference for the subsites of memapsin 2. The relative k(cat)/K(M) values of residues in each of the eight subsites were determined by the relative initial cleavage rates of substrate mixtures as quantified by MALDI-TOF mass spectrometry. We found that each subsite can accommodate multiple residues. The S(1) subsite is the most stringent, preferring residues in the order of Leu > Phe > Met > Tyr. The preferences of other subsites are the following: S(2), Asp > Asn > Met; S(3), Ile > Val > Leu; S(4), Glu > Gln > Asp; S(1)', Met > Glu > Gln > Ala; S(2)', Val > Ile > Ala; S(3)', Leu > Trp > Ala; S(4)', Asp > Glu > Trp. In general, S subsites are more specific than the S' subsites. A peptide comprising the eight most favored residues (Glu-Ile-Asp-Leu-Met-Val-Leu-Asp) was found to be hydrolyzed with the highest k(cat)/K(M) value so far observed for memapsin 2. Residue preferences at four subsites were also studied by binding of memapsin 2 to a combinatorial inhibitor library. From 10 tight binding inhibitors, the consensus preferences were as follows: S(2), Asp and Glu; S(3), Leu and Ile; S(2)', Val; and S(3)', Glu and Gln. An inhibitor, OM00-3, Glu-Leu-Asp-LeuAla-Val-Glu-Phe (where the asterisk represents the hydroxyethylene tansition-state isostere), designed from the consensus residues, was found to be the most potent inhibitor of memapsin 2 so far reported (K(i) of 3.1 x 10(-10) M). A molecular model of OM00-3 binding to memapsin 2 revealed critical improvement of the interactions between inhibitor side chains with enzyme over a previous inhibitor, OM99-2 [Ghosh, A. K., et al. (2000) J. Am. Chem. Soc. 14, 3522-3523].     

Structure-based design of cycloamide-urethane-derived novel inhibitors of human brain memapsin 2 (beta-secretase)

A series of novel macrocyclic amide-urethanes was designed and synthesized based upon the X-ray crystal structure of our lead inhibitor (1, OM99-2 with eight residues) bound to memapsin 2. Ring size and substituent effects have been investigated. Cycloamide-urethanes containing 14- to 16-membered rings exhibited low nanomolar inhibitory potencies against human brain memapsin 2 (beta-secretase).     

Potent memapsin 2 (beta-secretase) inhibitors: design, synthesis, protein-ligand X-ray structure, and in vivo evaluation

Structure-based design, synthesis, and biological evaluation of a series of peptidomimetic beta-secretase inhibitors incorporating hydroxyethylamine isosteres are described. We have identified inhibitor 24 which has shown exceedingly potent activity in memapsin 2 enzyme inhibitory (K(i) 1.8 nM) and cellular (IC(50)=1 nM in Chinese hamster ovary cells) assays. Inhibitor 24 has also shown very impressive in vivo properties (up to 65% reduction of plasma A beta) in transgenic mice. The X-ray structure of protein-ligand complex of memapsin 2 revealed critical interactions in the memapsin 2 active site.     

Crystal structure of memapsin 2 (beta-secretase) in complex with an inhibitor OM00-3

The structure of the catalytic domain of human memapsin 2 bound to an inhibitor OM00-3 (Glu-Leu-Asp-LeuAla-Val-Glu-Phe, K(i) = 0.3 nM, the asterisk denotes the hydroxyethylene transition-state isostere) has been determined at 2.1 A resolution. Uniquely defined in the structure are the locations of S(3)' and S(4)' subsites, which were not identified in the previous structure of memapsin 2 in complex with the inhibitor OM99-2 (Glu-Val-Asn-LeuAla-Ala-Glu-Phe, K(i) = 1 nM). Different binding modes for the P(2) and P(4) side chains are also observed. These new structural elements are useful for the design of new inhibitors. The structural and kinetic data indicate that the replacement of the P(2)' alanine in OM99-2 with a valine in OM00-3 stabilizes the binding of P(3)' and P(4)'.     

Internalization of exogenously added memapsin 2 (beta-secretase) ectodomain by cells is mediated by amyloid precursor protein

Memapsin 2 (beta-secretase) is the protease that initiates cleavage of amyloid precursor protein (APP) leading to the production of amyloid-beta (Abeta) peptide and the onset of Alzheimer's disease. Both APP and memapsin 2 are Type I transmembrane proteins and are endocytosed into endosomes where APP is cleaved by memapsin 2. Separate endocytic signals are located in the cytosolic domains of these proteins. We demonstrate here that the addition of the ectodomain of memapsin 2 (M2(ED)) to cells transfected with native APP or APP Swedish mutant (APPsw) resulted in the internalization of M2(ED) into endosomes with increased Abeta production. These effects were reduced by treatment with glycosylphosphatidylinositol-specific phospholipase C. The nontransfected parental cells had little internalization of M2(ED). The internalization of M2(ED) was dependent on the endocytosis signal in APP, because the expression of a mutant APP that lacks its endocytosis signal failed to support M2(ED) internalization. These results suggest that exogenously added M2(ED) interacts with the ectodomain of APP on the cell surface leading to the internalization of M2(ED), supported by fluorescence resonance energy transfer experiments. The interactions between the two proteins is not due to the binding of substrate APPsw to the active site of memapsin 2, because neither a potent active site binding inhibitor of memapsin 2 nor an antibody directed to the beta-secretase site of APPsw had an effect on the uptake of M2(ED). In addition, full-length memapsin 2 and APP, immunoprecipitated together from cell lysates, suggested that the interaction of these two proteins is part of the native cellular processes.     

Flap position of free memapsin 2 (beta-secretase), a model for flap opening in aspartic protease catalysis

The three-dimensional structure of unbound human memapsin 2 (beta-secretase) protease domain determined at 2.0-A resolution has revealed a new position of the flap region, which appears to be locked in an "open" position. While the structure outside of the flap is essentially the same as the structure of memapsin 2 bound to an inhibitor, the flap positions are 4.5 A different at the tips. The open position of the flap in the current structure is stabilized by two newly formed intraflap hydrogen bonds and anchored by a new hydrogen bond involving the side chain of Tyr 71 (Tyr 75 in pepsin numbering) in a novel orientation. In molecular modeling experiments, the opening of the flap, 6.5 A at the narrowest point, permits entrance of substrates into the cleft. The narrowest point of the opening may function to discriminate among substrates based on sequence and shape. The observed flap opening may also serve as a model for the flap movement in the catalytic mechanism of eukaryotic aspartic proteases and provide insight for the side-chain selection in the design of memapsin 2 inhibitors.     

Biochemical and structural characterization of the interaction of memapsin 2 (beta-secretase) cytosolic domain with the VHS domain of GGA proteins

Memapsin 2 (beta-secretase) is a membrane-associated aspartic protease that initiates the hydrolysis of beta-amyloid precursor protein (APP) leading to the production of amyloid-beta and the onset of Alzheimer's disease (AD). Both memapsin 2 and APP are transported from the cell surface to endosomes where APP hydrolysis takes place. Thus, the intracellular transport mechanism of memapsin 2 is important for understanding the pathogenesis of AD. We have previously shown that the cytosolic domain of memapsin 2 contains an acid-cluster-dileucine (ACDL) motif that binds the VHS domain of GGA proteins (He et al. (2002) FEBS Lett. 524, 183-187). This mechanism is the presumed recognition step for the vesicular packaging of memapsin 2 for its transport to endosomes. The phosphorylation of a serine residue within the ACDL motif has been reported to regulate the recycling of memapsin 2 from early endosomes back to the cell surface. Here, we report a study on the memapsin 2/VHS domain interaction. Using isothermal titration calorimetry, the dissociation constant, K(d), values are 4.0 x 10(-4), 4.1 x 10(-4), and 3.1 x 10(-4) M for VHS domains from GGA1, GGA2, and GGA3, respectively. With the serine residue replaced by phosphoserine, the K(d) decreased about 10-, 4-, and 14-fold for the same three VHS domains. A crystal structure of the complex between memapsin 2 phosphoserine peptide and GGA1 VHS was solved at 2.6 A resolution. The side chain of the phosphoserine group does not interact with the VHS domain but forms an ionic interaction with the side chain of the C-terminal lysine of the ligand peptide. Energy calculation of the binding of native and phosphorylated peptides to VHS domains suggests that this intrapeptide ionic bond in solution may reduce the change in binding entropy and thus increase binding affinity.     

Memapsin 2 (beta-secretase) cytosolic domain binds to the VHS domains of GGA1 and GGA2: implications on the endocytosis mechanism of memapsin 2

Memapsin 2, or beta-secretase, is a membrane-anchored aspartic protease that initiates the cleavage of beta-amyloid precursor protein (APP) leading to the production of beta-amyloid peptide in the brain and the onset of Alzheimer's disease. Memapsin 2 and APP are both endocytosed into endosomes for cleavage. Here we show that the cytosolic domain of memapsin 2, but not that of memapsin 1, binds the VHS domains of GGA1 and GGA2. Gel-immobilized VHS domains of GGA1 and GGA2 also bound to full-length memapsin 2 from cell mammalian lysates. Mutagenesis studies established that Asp(496), Leu(499) and Leu(500) were essential for the binding. The spacing of these three residues in memapsin 2 is identical to those in the cytosolic domains of mannose-6-phosphate receptors, sortilin and low density lipoprotein receptor-related protein 3. These observations suggest that the endocytosis and intracellular transport of memapsin 2, mediated by its cytosolic domain, may involve the binding of GGA1 and GGA2.     

mda-7 (IL-24): signaling and functional roles

One hallmark of neoplasia is abnormal differentiation. Induction of differentiation, by chemical or biological methods, provides a possible therapeutic intervention. "Differentiation therapy" is well documented in several model systems. These include melanoma, in which treatment with interferon-beta and the protein kinase C activator mezerein induces irreversible growth arrest and terminal differentiation culminating in programmed cell death. Subtraction hybridization between terminally differentiated and untreated melanoma cells identified melanoma differentiation-associated gene-7 (mda-7), which is selectively induced during the process of melanoma terminal differentiation. Since its identification seven years ago, mda-7 has been the object of intense focus because of its unique biological properties. Firstly, mda-7 is a secreted protein having cytokine-like properties and belonging to the IL-10 cytokine family. Based on this consideration, mda-7 was renamed IL-24. Secondly if delivered by means of an adenoviral vector, mda-7 induces selective apoptosis in cancer cells of diverse origin, while sparing their normal cellular counterparts. As such, mda-7 has become a novel tool for cancer gene therapy and is currently undergoing phase II clinical trials to determine its clinical efficacy in patients. The present review examines the biological properties of mda-7 and the signaling pathways that contribute to its unique cancer-specific apoptosis-inducing properties.     

A facile synthesis of (1S,5R,6S)-5-azido-6-benzyloxycyclohex-2-en-1-ol

A facile and short synthesis of (1S,5R,6S)-5-azido-6-benzyloxycyclohex-2-en-1-ol (1) has been achieved in high yield starting from 4,5-epoxycyclohex-1-ene by using a catalytic asymmetric allylic oxidation reaction.     

Structural analysis of septin 2, 6, and 7 complexes

Mammalian septins comprise a family of 13 genes that encode GTP-binding proteins. Specific combinations of septins can hetero-oligomerize and form filaments in vivo and in vitro, by mechanisms that are not understood. Using fluorescence resonance energy transfer, size exclusion chromatography, and multi-angle light scattering techniques, we have characterized the conformation of a complex of filamentous human septins, Sept2, Sept6, and Sept7. We now show that Sept6 and Sept7 interact through a parallel coiled-coil, and that Sept2 interacts with Sept6 through their C-terminal domains. We have also been able to produce soluble, stable individual septins that behave as rod-like monomers and dimers. Taken together, these observations suggest that polymerized filaments could be comprised of laterally arranged septin core subunits.     

Synthesis of (4R,6S,7R)-7-hydroxy-4,6-dimethyl-3-nonanone and (3R,5S,6R)- 6-hydroxy-3,5-dimethyl-2-octanone,the pheromone components of the bostrychid beetle,Dinoderus bifoveolatus

(4R,6S,7R)-7-Hydroxy-4,6-dimethyl-3-nonanone and (3R,5S,6R)-6-hydroxy-3,5-dimethyl-2-octanone, the pheromone components of the bostrychid beetle, Dinoderus bifoveolatus, as well as their (4R,6S,7S)- and (3R,5S,6S)-isomers were synthesized from (2R,4S,5R)- and (2R,4S,5S)-2,4-dimethyl-5-heptanolide, respectively.     

The role of Glu(192) in the allosteric control of the S(2)' and S(3)' subsites of thrombin

Thrombin is an allosteric protease controlled through exosites flanking the catalytic groove. Binding of a peptide derived from hirudin (Hir(52-65)) and/or of heparin to these opposing exosites alters catalysis. We have investigated the contribution of subsites S(2)' and S(3)' to this allosteric transition by comparing the hydrolysis of two sets of fluorescence-quenched substrates having all natural amino acids at positions P(2)' and P(3)'. Regardless of the amino acids, Hir(52-65) decreased, and heparin increased the k(cat)/K(m) value of hydrolysis by thrombin. Several lines of evidence have suggested that Glu(192) participates in this modulation. We have examined the role of Glu(192) by comparing the catalytic activity of thrombin and its E192Q mutant. Mutation substantially diminishes the selectivity of thrombin. The substrate with the "best" P(2)' residue was cleaved with a k(cat)/K(m) value only 49 times higher than the one having the "least favorable" P(2)' residue (versus 636-fold with thrombin). Mutant E192Q also lost the strong preference of thrombin for positively charged P(3)' residues and its strong aversion for negatively charged P(3)' residues. Furthermore, both Hir(52-65) and heparin increased the k(cat)/K(m) value of substrate hydrolysis. We conclude that Glu(192) is critical for the P(2)' and P(3)' specificities of thrombin and for the allostery mediated through exosite 1.     

Structural and functional roles of ether lipids

Ether lipids, such as plasmalogens, are peroxisomederived glycerophospholipids in which the hydrocarbon chain at the sn-1 position of the glycerol backbone is attached by an ether bond, as opposed to an ester bond in the more common diacyl phospholipids. This seemingly simple biochemical change has profound structural and functional implications. Notably, the tendency of ether lipids to form non-lamellar inverted hexagonal structures in model membranes suggests that they have a role in facilitating membrane fusion processes. Ether lipids are also important for the organization and stability of lipid raft microdomains, cholesterol-rich membrane regions involved in cellular signaling. In addition to their structural roles, a subset of ether lipids are thought to function as endogenous antioxidants, and emerging studies suggest that they are involved in cell differentiation and signaling pathways. Here, we review the biology of ether lipids and their potential significance in human disorders, including neurological diseases, cancer, and metabolic disorders.     

Synthesis of 1,2-oxygenated 6-arylfurofuran lignan: stereoselective synthesis of (1S,2S,5R,6S)-1-hydroxysamin

(1S,2S,5R,6S)-6-(3,4-Methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]octan-1,2-diol ((+)-1-hydroxysamin 1) was synthesized, starting from olefin 8. Stereoselective alpha-hydroxylation was achieved after converting 8 to aldehyde 13. Resulting unstable alpha-hydroxy aldehyde 14 was then transformed to (+)-1-hydroxysamin (1). This is a new efficient synthetic route to 1,2-oxygenated 6-arylfurofuran lignans.     

The specificity of the S1 and S2 subsites of elastase

Esters of tetrapeptides of the general formula ethoxycarbonyl-prolyl-alanyl-X-Y where either X or Y was an alanine residue were synthesised and their cleavage by elastase studied. It was found that variation of the alcohol moiety between methyl, cyclohexyl and nitrophenyl residues had no effect on the catalytic rate constant for cleavage of ethoxycarbonyl-prolyl-dialanyl-alanine esters demonstrating that acylation is much faster than deacylation for this system and also that non-productive binding is not kinetically significant. The effect of changing the amino acid residue in position X was small compared with that of change in position Y. The presence of valine and serine residues in position Y produced the highest specificity constant but the highest catalytic rate constant was found for a leucine residue in this position. The results are discussed in terms of the binding of the substrate to the enzyme.