Protein Z-dependent protease inhibitor binds to the C-terminal domain of protein Z

Protein Z (PZ) is a multidomain vitamin K-dependent plasma protein that functions as a cofactor to promote the inactivation of factor Xa (fXa) by PZ-dependent protease inhibitor (ZPI) by three orders of magnitude. To understand the mechanism by which PZ improves the reactivity of fXa with ZPI, we expressed wild-type PZ, PZ lacking the gamma-carboxyglutamic acid domain (GD-PZ), and a chimeric PZ mutant in which both Gla and EGF-like domains of the molecule were substituted with identical domains of fXa. The ZPI binding and the cofactor function of the PZ derivatives were characterized in both binding and kinetic assays. The binding assay indicated that all PZ derivatives interact with ZPI with a similar dissociation constant (K(D)) of approximately 7 nm. However, the apparent K(D) for the chimeric PZ-mediated ZPI inhibition of fXa was elevated 6-fold on PC/PS vesicles and its capacity to function as a cofactor to accelerate the ZPI inhibition of fXa was also decreased 6-fold. The cofactor activity of GD-PZ was dramatically impaired; however, the deletion mutant exhibited a normal cofactor function in solution. A chimeric activated protein C mutant containing the Gla domain of fXa was susceptible to inhibition by ZPI in the presence of PZ. These results suggest that: (i) the ZPI interactive site of PZ is located within the C-terminal domain of the cofactor and (ii) a specific interaction between the Gla domains of PZ and fXa contributes approximately 6-fold to the acceleration of the ZPI inhibition of fXa on phospholipid membranes.     

Predicted three-dimensional structure of the protease inhibitor domain of the Alzheimer's disease beta-amyloid precursor

Alzheimer's disease is characterized by the deposition of amyloid beta-protein as plaques and tangles in the brains of its victims. The amyloid precursor can be expressed with or without the inclusion of a protease inhibitor domain, the potential role of which in amyloidogenesis has prompted the generation of a model of its three-dimensional structure based on the known structure of a related inhibitor. The model structure predicts that the mutated residues are almost entirely on the surface of the inhibitor domain, while conserved residues constitute the hydrophobic core. In addition, several pairs of structurally complementary, or concerted, mutations are seen. These structural features provide strong evidence for the validity of the modeled structure, and it is suggested that the presence of complementary mutations may be used as a criterion for evaluating protein structures built by homology, in addition to the (spatial) location of the mutations. The terminal residues delimiting the domain are among those furthest from the protease binding site and are in close proximity to one another, thus suggesting the ability of the domain to function as a structural cassette within the context of a larger protein. The electrostatic potentials of the inhibitor and of the related bovine pancreatic trypsin inhibitor reveal how two inhibitors with very different net charges can bind with approximately the same binding constant to trypsin and suggest a mutation of trypsin that might selectively enhance the binding of the amyloid inhibitor domain. The model provides a structural basis for understanding the functional roles of residues in the domain and for designing simpler molecules to test as pharmacologic agents for intervention in Alzheimer's disease.     

Brain expression of presenilins in sporadic and early-onset, familial Alzheimer's disease

BACKGROUND: Mutations in the presenilin proteins cause early-onset, familial Alzheimer's disease (FAD). MATERIALS AND METHODS: We characterized the cellular localization and endoproteolysis of presenilin 2 (PS2) and presenilin 1 (PS1) in brains from 25 individuals with presenilin-mutations causing FAD, as well as neurologically normal individuals and individuals with sporadic Alzheimer's disease (AD). RESULTS: Amino-terminal antibodies to both presenilins predominantly decorated large neurons. Regional differences between the broad distributions of the two presenilins were greatest in the cerebellum, where most Purkinje cells showed high levels of only PS2 immunoreactivity. PS2 endoproteolysis in brain yielded multiple amino-terminal fragments similar in size to the PS1 amino-terminal fragments detected in brain. In addition, two different PS2 amino-terminal antibodies also detected a prominent 42 kDa band that may represent a novel PS2 form in human brain. Similar to PS1 findings, neither amino-terminal nor antiloop PS2 antibodies revealed substantial full-length PS2 in brain. Immunocytochemical examination of brains from individuals with the N141I PS2 mutation or eight different PS1 mutations, spanning the molecule from the second transmembrane domain to the large cytoplasmic loop domain, revealed immunodecoration of no senile plaques and only neurofibrillary tangles in the M139I PS1 mutation stained with PS1 antibodies. CONCLUSIONS: Overall presenilin expression and the relative abundance of full-length and amino-terminal fragments in presenilin FAD cases were similar to control cases and sporadic AD cases. Thus, accumulation of full-length protein or other gross mismetabolism of neither PS2 nor PS1 is a consequence of the FAD mutations examined.     

Interaction of human cathepsin D with the inhibitor pepstatin.

1. Because of the proposed role of cathepsin D in a variety of biological and pathological processes, the characteristics of inhibition by the potentially useful agent, pepstatin, were determined. 2. The beta and gamma forms of human cathepsin D, separated by isoelectric focusing, have identical specific extinction coefficients and specific activity in the degradation of haemoglobin. 3. Cathepsin D showed tight binding of 1 mol of pepstatin per 43000 g of protein, indicating that titration with the inhibitor represents a useful method for determination of absolute concentrations of the enzyme. 4. The titration curves were used to determine apparent dissociation constants (KD) for the binding of pepstatin and pepstatin methyl ester at pH3.5; values of approx. 5 X 10(-10)M were obtained. 5. Pepstatinyl-[3H]glycine was synthesized and shown to have a KD similar to that of pepstatin. Gel-chromatographic experiments showed that the binding of pepstatin and its derivatives is strongly pH-dependent. 6. The effect of pH on the KD for pepstatinyl-glycine was determined by equilibrium dialysis. As the pH was raised from 5.0 to 6.4, KD rose from 5 X 10(-10)M to 2 X 10(-6)M. 7. The catalytic activity of cathepsin D declines essentially to zero on going from pH5.0 to pH7.0, and we suggest that the binding site for substrate and pepstatin is abolished by a conformational change in the enzyme molecule. 8. The data indicate that, in biological experiments near neutral pH, large molar excesses of pepstatin over cathepsin D will be required for efficient inhibition.     

Inhibition of 1,4-beta-D-xylan xylanohydrolase by the specific aspartic protease inhibitor pepstatin: probing the two-step inhibition mechanism

This is the first report that describes the inhibition mechanism of xylanase from Thermomonospora sp. by pepstatin A, a specific inhibitor toward aspartic proteases. The kinetic analysis revealed competitive inhibition of xylanase by pepstatin A with an IC50 value 3.6 +/- 0.5 microm. The progress curves were time-depended, consistent with a two-step slow tight binding inhibition. The inhibition followed a rapid equilibrium step to form a reversible enzyme-inhibitor complex (EI), which isomerizes to the second enzyme-inhibitor complex (EI*), which dissociated at a very slow rate. The rate constants determined for the isomerization of EI to EI* and the dissociation of EI* were 15 +/- 1 x 10(-5) and 3.0 +/- 1 x 10(-8) s(-1), respectively. The Ki value for the formation of EI complex was 1.5 +/- 0.5 microm, whereas the overall inhibition constant Ki* was 28.0 +/- 1 nm. The conformational changes induced in Xyl I by pepstatin A were monitored by fluorescence spectroscopy, and the rate constants derived were in agreement with the kinetic data. Thus, the conformational alterations were correlated to the isomerization of EI to EI*. Pepstatin A binds to the active site of the enzyme and disturbs the native interaction between the histidine and lysine, as demonstrated by the abolished isoindole fluorescence of o-phthalaldehyde-labeled xylanase. Our results revealed that the inactivation of xylanase is due to the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis, and a model depicting the probable interaction between pepstatin A with xylanase has been proposed.     

Immunochemical identification of the serine protease inhibitor alpha 1-antichymotrypsin in the brain amyloid deposits of Alzheimer's disease

Two approaches--molecular cloning and immunochemical analysis--have identified one of the components of Alzheimer's disease amyloid deposits as the serine protease inhibitor alpha 1-antichymotrypsin. An antiserum against isolated Alzheimer amyloid deposits detected immunoreactivity in normal liver. The antiserum was then used to screen a liver cDNA expression library, yielding three related clones. DNA sequence analysis showed that these clones code for alpha 1-antichymotrypsin. Antisera against purified alpha 1-antichymotrypsin stained Alzheimer amyloid deposits, both in situ and after detergent extraction from brain. The anti-amyloid antiserum recognizes at least two distinct epitopes in alpha 1-antichymotrypsin, further supporting the presence of this protein in Alzheimer amyloid deposits. In addition to being produced in the liver and released into the serum, alpha 1-antichymotrypsin is expressed in Alzheimer brain, particularly in areas that develop amyloid lesions. Models by which alpha 1-antichymotrypsin could contribute to the development of Alzheimer amyloid deposits are discussed.     

Characterization of detergent-insoluble complexes containing the familial Alzheimer's disease-associated presenilins

Many cases of early-onset familial Alzheimer's disease have been linked to mutations within two genes encoding the proteins presenilin-1 and presenilin-2. The presenilins are 48-56-kDa proteins that can be proteolytically cleaved to generate an N-terminal fragment (approximately 25-35 kDa) and a C-terminal fragment (approximately 17-20 kDa). The N- and C-terminal fragments of presenilin-1, but not full-length presenilin-1, were readily detected in both human and mouse cerebral cortex and in neuronal and glioma cell lines. In contrast, presenilin-2 was detected almost exclusively in cerebral cortex as the full-length molecule with a molecular mass of 56 kDa. The association of the presenilins with detergent-insoluble, low-density membrane microdomains, following the isolation of these structures from cerebral cortex by solubilization in Triton X-100 and subsequent sucrose density gradient centrifugation, was also examined. A minor fraction (10%) of both the N- and C-terminal fragments of presenilin-1 was associated with the detergent-insoluble, low-density membrane microdomains, whereas a considerably larger proportion of full-length presenilin-2 was present in the same membrane microdomains. In addition, a significant proportion of full-length presenilin-2 was present in a high-density, detergent-insoluble cytoskeletal pellet enriched in beta-actin. The presence of the presenilins in detergent-insoluble, low-density membrane microdomains indicates a possible role for these specialized regions of the membrane in the lateral separation of Alzheimer's disease-associated proteins within the lipid bilayer and/or in the distinct functions of these proteins.     

The amyloid beta-protein precursor of Alzheimer's disease is degraded extracellularly by a Kunitz protease inhibitor domain-sensitive trypsin-like serine protease in cultures of chick sympathetic neurons.

The amyloid beta-protein precursor (APP) of Alzheimer's disease (AD) is cleaved either by alpha-secretase to generate an N-terminally secreted fragment, or by beta- and gamma-secretases to generate the beta-amyloid protein (Abeta). The accumulation of Abeta in the brain is an important step in the pathogenesis of AD. Alternative mRNA splicing can generate isoforms of APP which contain a Kunitz protease inhibitor (KPI) domain. However, little is known about the physiological function of this domain. In the present study, the metabolic turnover of APP was examined in cultured chick sympathetic neurons. APP was labelled by incubating neurons for 5 h with [35S]methionine and [35S]cysteine. Intracellular labelled APP decayed in a biphasic pattern suggesting that trafficking occurs through two metabolic compartments. The half-lives for APP in each compartment were 1.5 and 5.7 h, respectively. A small fraction (10%) of the total APP was secreted into the culture medium where it was degraded with a half-life of 9 h. Studies using specific protease inhibitors demonstrated that this extracellular breakdown was due to cleavage by a trypsin-like serine protease that was secreted into the culture medium. Significantly, this protease was inhibited by a recombinant isoform of APP (sAPP751), which contains a region homologous to the Kunitz protease inhibitor (KPI) domain. These results suggest that KPI forms of APP regulate extracellular cleavage of secreted APP by inhibiting the activity of a secreted APP-degrading protease.     

X-ray crystal structure of the protease inhibitor domain of Alzheimer's amyloid beta-protein precursor

Alzheimer's amyloid beta-protein precursor contains a Kunitz protease inhibitor domain (APPI) potentially involved in proteolytic events leading to cerebral amyloid deposition. To facilitate the identification of the physiological target of the inhibitor, the crystal structure of APPI has been determined and refined to 1.5-A resolution. Sequences in the inhibitor-protease interface of the correct protease target will reflect the molecular details of the APPI structure. While the overall tertiary fold of APPI is very similar to that of the Kunitz inhibitor BPTI, a significant rearrangement occurs in the backbone conformation of one of the two protease binding loops. A number of Kunitz inhibitors have similar loop sequences, indicating the structural alteration is conserved and potentially an important determinant of inhibitor specificity. In a separate region of the protease binding loops, APPI side chains Met-17 and Phe-34 create an exposed hydrophobic surface in place of Arg-17 and Val-34 in BPTI. The restriction this change places on protease target sequences is seen when the structure of APPI is superimposed on BPTI complexed to serine proteases, where the hydrophobic surface of APPI faces a complementary group of nonpolar side chains on kallikrein A versus polar side chains on trypsin.     

Apoptotic activities of wild-type and Alzheimer's disease-related mutant presenilins in Drosophila melanogaster.

Mutant human presenilins cause early-onset familial Alzheimer's disease and render cells susceptible to apoptosis in cultured cell models. We show that loss of presenilin function in Drosophila melanogaster increases levels of apoptosis in developing tissues. Moreover, overexpression of presenilin causes apoptotic and neurogenic phenotypes resembling those of Presenilin loss-of-function mutants, suggesting that presenilin exerts a dominant negative effect when expressed at high levels. In Drosophila S2 cells, Psn overexpression leads to reduced Notch receptor synthesis affecting levels of the intact approximately 300-kD precursor and its approximately 120-kD processed COOH-terminal derivatives. Presenilin-induced apoptosis is cell autonomous and can be blocked by constitutive Notch activation, suggesting that the increased cell death is due to a developmental mechanism that eliminates improperly specified cell types. We describe a genetic model in which the apoptotic activities of wild-type and mutant presenilins can be assessed, and we find that Alzheimer's disease-linked mutant presenilins are less effective at inducing apoptosis than wild-type presenilin.     

A novel cell penetrating aspartic protease inhibitor blocks processing and presentation of tetanus toxoid more efficiently than pepstatin A

Selective inhibition of enzymes involved in antigen processing such as cathepsin E and cathepsin D is a valuable tool for investigating the roles of these enzymes in the processing pathway. However, the aspartic protease inhibitors, including the highly potent pepstatin A (PepA), are inefficiently transported across the cell membrane and thus have limited access to antigen processing compartments. Previously described mannose-pepstatin conjugates were efficiently taken up by the cells via receptor mediated uptake. However, cells without mannose receptors are unable to take up these conjugates efficiently. The aim of the present study was to synthesize new cell-permeable aspartic protease inhibitors by conjugating pepstatin A with well-known cell penetrating peptides (CPPs). To achieve this, the most commonly used CPPs namely pAntp_(43-58) (penetratin), Tat_(49-60), and 9-mer of l-arginine (R9), were synthesized and coupled to pepstatin. The enzyme inhibitory properties of these bioconjugates and their cellular uptake into MCF7 (human breast cancer cell line), Boleths (EBV-transformed B-cell line) and dendritic cells (DC) were the focus of our study. We found that the bioconjugate PepA-penetratin (PepA-P) was the most efficient cell-permeable aspartic protease inhibitor tested, and was more efficient than unconjugated PepA. Additionally, we found that PepA-P efficiently inhibited the tetanus toxoid C-fragment processing in peripheral blood mononuclear cells (PBMC), primary DC and in primary B cells. Therefore, PepA-P can be used in studying the role of intracellular aspartic proteases in the MHC class II antigen processing pathway. Moreover, inhibition of tetanus toxoid C-fragment processing by PepA-P clearly implicates the role of aspartic proteinases in antigen processing.     

The Alzheimer's disease-associated presenilins are differentially phosphorylated proteins located predominantly within the endoplasmic reticulum.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the deposition of extracellular senile plaques composed of amyloid beta-peptide (A beta). Whereas most cases of AD occur sporadically, about 10% of AD cases are inherited as a fully penetrant autosomal dominant trait. Mutations in the recently cloned Presenilin genes (PS-1 and PS-2) are by far the most common cause of early onset familial AD. MATERIALS AND METHODS: Cellular expression of endogenous and overexpressed PS proteins was analyzed by immunocytochemistry and metabolic labeling followed by immunoprecipitation. In vivo phosphorylation sites of PS proteins were analyzed by extensive mutagenesis. RESULTS: PS-1 as well as PS-2 proteins were localized predominantly within the endoplasmic reticulum (ER). However, small amounts of the PS proteins were detected within the Golgi compartment, where they colocalize with the beta-amyloid precursor protein (beta APP). The PS-2 protein was found to be highly phosphorylated, whereas very little phosphorylation was observed for PS-1. The selective phosphorylation of PS-2 occurs exclusively on serine residues. In vivo phosphorylation of PS-2 was mapped to serine residues 7, 9, and 19 within an acidic stretch at the N terminus, which is absent in PS-1. casein kinase (CK)-1 and CK-2 were shown to phosphorylate the N terminus of PS-2 in vitro. CONCLUSIONS: The majority of PS proteins were detected in the ER where little if any proteolytic processing of beta APP was reported. ER retention of PS proteins might occur by intramolecular aggregation. Small amounts of PS proteins were also detected in the Golgi where they colocalized with beta APP. This might suggest that potential interactions between PS proteins and beta APP could occur within the Golgi. Selective phosphorylation of PS-2 proteins within the acidic domain missing in PS-1 indicates differences in the biological functions and regulation of the two highly homologous proteins.     

Antiviral effects of a thiol protease inhibitor on foot-and-mouth disease virus.

The thiol protease inhibitor E-64 specifically blocks autocatalytic activity of the leader protease of foot-and-mouth disease virus (FMDV) and interferes with cleavage of the structural protein precursor in an in vitro translation assay programmed with virion RNA. Experiments with FMDV-infected cells and E-64 or a membrane-permeable analog, E-64d, have confirmed these results and demonstrated interference in virus assembly, causing a reduction in virus yield. In addition, there is a lag in the appearance of virus-induced cellular morphologic alterations, a delay in cleavage of host cell protein p220 and in shutoff of host protein synthesis, and a decrease in viral protein and RNA synthesis. The implications of using E-64-based compounds as potential antiviral agents for FMDV are discussed.     

Linear patterns of Alzheimer's disease mutations along alpha-helices of presenilins as a tool for PS-1 model construction

We performed an analysis of mutation patterns in all 10 hydrophobic regions (HRs) of presenilin-1 (PS-1) and PS-2 using a recent database of Alzheimer's disease (AD) mutations. The linear patterns were confirmed and extended to areas spanning as many as three faces of a given HR. The complementary areas of residues free of AD mutations were identified based on the location of non-pathogenic polymorphisms and PS-1 versus PS-2 amino acid discordances. Taking into account the location of areas of AD mutations and mutation-free areas/regions, we proposed a preliminary model of PS-1 structure using a general stick-out-mutation rule. To build a molecular structure of PS-1 and preserve features of the preliminary model, we used bacteriorhodopsin template in homology/comparative modelling. Two molecular models were built differing in the location of C-terminal fragment helices. The models properly distinguish residues belonging to AD-affected sites and non-pathogenic areas, and may be used for classification purposes. They also comply with experimental results, such as differences in accessibility of the catalytic residues in uncleaved PS-1, and binding of PEN-2 by the PS-1 NF motif.     

Biochemical characterization of an aspartic protease from Vigna radiata: Kinetic interactions with the classical inhibitor pepstatin implicating a tight binding mechanism

Aspartic proteases are the focus of recent research interest in understanding the physiological importance of this class of enzymes in plants. This is the first report of an aspartic protease from the seeds of Vigna radiata. The aspartic protease was purified to homogeneity by fractional ammonium sulfate precipitation and pepstatin-A agarose affinity column. It was found to have a molecular weight of 67,406 Da by gel filtration chromatography. SDS-PAGE analysis revealed the presence of a heterodimer with subunits of molecular weights of 44,024 and 23,349 Da respectively. The enzyme was pH stable with the amino acid analysis confirming the molecular weight of the protein. The substrate cleavage site as analyzed by using the synthetic substrate was found to be the Phe-Tyr bond. The kinetic interactions of the enzyme were studied with the universal inhibitor, pepstatin A. This is the first report on the interactions of a plant aspartic protease with pepstatin-A, an inhibitor from a microbial source. A competitive one-step mechanism of binding is observed. The progress curves are time-dependent and consistent with tight binding inhibition. The K(i) value of the reversible complex of pepstatin with the enzyme was 0.87 microM whereas the overall inhibition constant K(i)* was 0.727 microM.     

Molecular characterization of the minimal protease resistant tau unit of the Alzheimer's disease paired helical filament.

The Alzheimer's disease paired helical filament (PHF), after digestion with Pronase, retains its characteristic morphological features. We term this the protease resistant core PHF. A 12 kDa tau fragment can be released from the core as an essentially pure preparation. Sequence analysis of this fragment revealed six distinct N-termini beginning in the repeat region of tau. The precise C-terminus is unknown, but the fragment is approximately 100 residues long. A monoclonal antibody, mAb 423, which recognizes the core PHF and the 12 kDa tau fragment, does not recognize normal full-length tau. We describe cDNA synthesis and expression of candidate 12 kDa tau analogues which permit the mapping of the mAb 423 epitope. mAb 423 recognizes all and only those analogues which terminate at Glu391, which lies beyond the homology repeat region. Addition or removal of a single residue at the C-terminus abolishes immunoreactivity. Therefore, mAb 423, together with knowledge of the N-terminus, can be used to measure the precise extent of 12 kDa PHF core tau fragment which we term the minimal protease resistant tau unit of the core PHF. This unit is 93-95 residues long, which is equivalent to three repeats, but is 14-16 residues out of phase with respect to the maximum homology organization of the repeat region. mAb 423 labels isolated PHFs prior to Pronase digestion and intracellular granular and neurofibrillary degeneration in Alzheimer's disease tissues. The constraints which determine endogenous truncation at Glu391 appear to be characteristic of an assembled configuration of tau, either within the PHF or its precursor.