Kunitz-type Protease Inhibitor Domain Research Articles

Characterization of the role of Kunitz-type protease inhibitor domain in dimerization of amyloid precursor protein.

A major difference between amyloid precursor protein (APP) isoforms (APP695 and APP751) is the existence of a Kunitz type protease inhibitor (KPI) domain which has a significant impact on the homo- and hetero-dimerization of APP isoforms. However, the exact molecular mechanisms of dimer formation remain elusive. To characterize the role of the KPI domain in APP dimerization, we performed a single molecule pull down (SiMPull) assay where homo-dimerization between tethered APP molecules and soluble APP molecules was highly preferred regardless of the type of APP isoforms, while hetero-dimerization between tethered APP751 molecules and soluble APP695 molecules was limited. We further investigated the domain level APP-APP interactions using coarse-grained models with the Martini force field. Though the model initial ternary complexes (KPI-E1, KPI-KPI, KPI-E2, E1-E1, E2-E2, and E1-E2) generated using HADDOCK (HD) and AlphaFold2 (AF2), the binding free energy profiles and the binding affinities of the domain combinations were investigated via the umbrella sampling with Martini force field. Additionally, membrane-bound microenvironments at the domain level were modeled. As a result, it was revealed that the KPI domain has a stronger attractive interaction with itself than the E1 and E2 domains, as reported elsewhere. Thus, the KPI domain of APP751 may form additional attractive interactions with E1, E2 and the KPI domain itself, whereas it is absent in APP695. In conclusion, we found that the APP751 homo-dimer formation is predominant than the homodimerization in APP695, which is facilitated by the presence of the KPI domain.

BFPI: A Minimal Prothrombinase Inhibitor from the Saliva of Simulium Vittatum

Background: The black fly, Simulium Vittatum, has an anticoagulant protein in its saliva that allows it to feed on mammalian blood (black fly protease inhibitor; BFPI). Remarkably, BFPI is similar to the human anticoagulant tissue factor pathway inhibitor alpha (TFPIα). TFPIα contains three Kunitz-type protease inhibitor domains (K1, K2, K3), which inhibit factor VIIa (FVIIa) and factor Xa (FXa) and bind the co-factor protein S (PS), respectively; BFPI contains a single Kunitz domain that inhibits FXa. In addition, TFPIα and BFPI contain homologous basic regions (BRs) near their C-termini (252LIKTKRKRKK261 in human TFPIα, LIKTRKRKPKK in BFPI). The TFPIα BR binds a regulatory acidic region (AR) in factor Va (FVa). The AR is present in forms of FVa released by collagen-activated platelets and generated through limited proteolysis by FXa (FVaXa), and is removed by thrombin (FVaIIa). We hypothesized that BFPI, through its Kunitz domain and basic C-terminus, inhibits early forms of the prothrombinase complex, but does not possess the other inhibitory functions of TFPIα: (1) K1-dependent inhibition of the tissue factor (TF)-FVIIa complex; and (2) PS/K3-dependent FXa inhibition.Results: Recombinant BFPI inhibited FXa in an amidolytic activity assay, and PS did not promote this inhibition. BFPI did not inhibit TF-FVIIa-mediated FX activation. As described with TFPIα, FV promoted FXa inhibition by BFPI but FVaIIa did not, suggesting that the BFPI BR is capable of binding the FVa AR. In a purified protein assay, BFPI inhibited prothrombinase assembled with FVaXa (IC50=4.9nM), but not FVaIIa. Similarly, 5nM BFPI increased the lag time for FXa-initiated plasma thrombin generation by 10.4±1.5%.We next used BFPI as a backbone to evaluate a reported human mutation in the TFPIα BR, K254E. Every mammalian, avian, or reptilian TFPIα sequence available contains either a Lys or Arg residue at this position, suggesting that this residue is functionally important. In purified protein assays, BFPI-K254E inhibited FXa amidolytic activity identically to BFPI, but FV did not promote this inhibition, suggesting that BFPI-K254E has a specific defect in its interaction with FV. Consistent with this, BFPI-K254E was a weaker inhibitor of prothrombinase assembled with FVaXa (IC50 = 15.8nM) and FXa-initiated plasma thrombin generation.The results obtained with BFPI-K254E were confirmed using peptides and full-length TFPIα proteins. First, a peptide mimicking the wild type TFPIα BR (LIKTKRKRKK) inhibited prothrombinase assembled with FVaXa (IC50 = 1.0 µM), while the substituted peptide (LIETKRKRKK) was substantially weaker (20% inhibition observed with 340 µM peptide). Second, full-length TFPIα-K254E was a weaker inhibitor of prothrombinase containing FXa-activated FVa (IC50 = 14.8 nM, vs. 1.8 nM for TFPIα) and had greatly reduced anticoagulant activity in plasma-based thrombin generation assays.Conclusions: In summary, the anticoagulant effect of BFPI is mediated through inhibition of early forms of prothrombinase, independent of TF-FVIIa inhibition or PS-dependent FXa inhibition. The natural mutation TFPIα K254E disrupts prothrombinase inhibition, despite the presence of six other conserved basic residues, and is thus procoagulant in human plasma. The absolute conservation of the TFPIα BR, and its usurpation to allow feeding by black flies, point to formation of the initial prothrombinase complex as a key regulatory step in blood coagulation. DisclosuresMast:Novo Nordisk: Research Funding.

Factor Xa and VIIa inhibition by tissue factor pathway inhibitor is prevented by a monoclonal antibody to its Kunitz‐1 domain

Background Initiation of coagulation is induced by binding of activated factor VII (FVIIa) to tissue factor (TF) and activation of factor X (FX) in a process regulated by tissue factor pathway inhibitor (TFPI). TFPI contains three Kunitz-type protease inhibitor domains (K1-K3), of which K1 and K2 block the active sites of FVIIa and FXa, respectively. Objective To produce a monoclonal antibody (mAb) directed towards K1, to characterize the binding epitope, and to study its effect on TFPI inhibition. Methods A monoclonal antibody, mAb2F22, was raised against the N-terminal TFPI(1-79) fragment. Binding data were obtained by surface plasmon resonance analysis. The Fab-fragment of mAb2F22, Fab2F22, was expressed and the structure of its complex with TFPI(1-79) determined by X-ray crystallography. Effects of mAb2F22 on TFPI inhibition were measured in buffer- and plasma-based systems. Results mAb2F22 bound exclusively to K1 of TFPI (KD ~1 nm) and not to K2. The crystal structure of Fab2F22/TFPI (1-79) mapped an epitope on K1 including seven residues upstream of the domain. TFPI inhibition of TF/FVIIa amidolytic activity was neutralized by mAb2F22, although the binding epitope on K1 did not include the P1 residue. Binding of mAb2F22 to K1 blocked TFPI inhibition of the FXa amidolytic activity and normalized hemostasis in hemophilia human A-like plasma and whole blood. Conclusion mAb2F22 blocked TFPI inhibition of both FVIIa and FXa activities and mapped a FXa exosite for binding to K1. It reversed TFPI feedback inhibition of TF/FVIIa-induced coagulation and restored clot formation in FVIII-neutralized human plasma and blood.

Human Kunitz-type protease inhibitor engineered for enhanced matrix retention extends longevity of fibrin biomaterials.

Aprotinin is a broad-spectrum serine protease inhibitor used in the clinic as an anti-fibrinolytic agent in fibrin-based tissue sealants. However, upon re-exposure, some patients suffer from hypersensitivity immune reactions likely related to the bovine origin of aprotinin. Here, we aimed to develop a human-derived substitute to aprotinin. Based on sequence homology analyses, we identified the Kunitz-type protease inhibitor (KPI) domain of human amyloid-β A4 precursor protein as being a potential candidate. While KPI has a lower intrinsic anti-fibrinolytic activity than aprotinin, we reasoned that its efficacy is additionally limited by its fast release from fibrin material, just as aprotinin's is. Thus, we engineered KPI variants for controlled retention in fibrin biomaterials, using either covalent binding through incorporation of a substrate for the coagulation transglutaminase Factor XIIIa or through engineering of extracellular matrix protein super-affinity domains for sequestration into fibrin. We showed that both engineered KPI variants significantly slowed plasmin-mediated fibrinolysis invitro, outperforming aprotinin. Invivo, our best engineered KPI variant (incorporating the transglutaminase substrate) extended fibrin matrix longevity by 50%, at a dose at which aprotinin did not show efficacy, thus qualifying it as a competitive substitute of aprotinin in fibrin sealants.

The intact Kunitz domain protects the amyloid precursor protein from being processed by matriptase-2.

Proteolytic processing of the amyloid precursor protein (APP) leads to amyloid-β (Aβ) peptides. So far, the mechanism of APP processing is insufficiently characterized at the molecular level. Whereas the knowledge of Aβ generation by several proteases has been expanded, the contribution of the Kunitz-type protease inhibitor domain (KPI) present in two major APP isoforms to the complex proteolytic processing of APP is poorly understood. In this study, we have identified KPI-containing APP as a very potent, slow-binding inhibitor for the membrane-bound proteolytic regulator of iron homeostasis matriptase-2 by forming stable complexes with its target protease in HEK cells. Inhibition and complex formation depend on the intact KPI domain. By inhibiting matriptase-2, KPI-containing APP is protected from matriptase-2-mediated proteolysis within the Aβ region, thus preventing the generation of N-terminally truncated Aβ.

Water-soluble chlorophyll protein is involved in herbivore resistance activation during greening of Arabidopsis thaliana

Water-soluble chlorophyll proteins (WSCPs) constitute a small family of unusual chlorophyll (Chl)-binding proteins that possess a Kunitz-type protease inhibitor domain. In Arabidopsis thaliana, a WSCP has been identified, named AtWSCP, that forms complexes with Chl and the Chl precursor chlorophyllide (Chlide) in vitro. AtWSCP exhibits a quite unexpected expression pattern for a Chl binding protein and accumulated to high levels in the apical hook of etiolated plants. AtWSCP expression was negatively light-regulated. Transgenic expression of AtWSCP fused to green fluorescent protein (GFP) revealed that AtWSCP is localized to cell walls/apoplastic spaces. Biochemical assays identified AtWSCP as interacting with RD21 (responsive to desiccation 21), a granulin domain-containing cysteine protease implicated in stress responses and defense. Reconstitution experiments showed tight interactions between RD21 and WSCP that were relieved upon Chlide binding. Laboratory feeding experiments with two herbivorous isopod crustaceans, Porcellio scaber (woodlouse) and Armadillidium vulgare (pillbug), identified the apical hook as Achilles' heel of etiolated plants and that this was protected by RD21 during greening. Because Chlide is formed in the apical hook during seedling emergence from the soil, our data suggest an unprecedented mechanism of herbivore resistance activation that is triggered by light and involves AtWSCP.

Amyloid precursor protein expression is enhanced in human platelets from subjects with Alzheimer's disease and frontotemporal lobar degeneration: A Real-time PCR study

Frontotemporal lobar degeneration (FTLD) and Alzheimer's disease (AD) represent the most frequent causes of early-onset and late-onset degenerative dementia, respectively. A correct diagnosis entails the choice of appropriate therapies. In this view the present study aimed to identify biomarkers that could improve the differential diagnosis. We recently found an overexpression of platelet amyloid precursor protein (APP) in AD; furthermore, recent studies have suggested the presence of changes in APP processing in FTLD. In this context, we analyzed the mRNA expression level of Total APP (TOT) and APP containing a Kunitz-type serine protease inhibitor domain (KPI) in platelets obtained from AD patients, subjects with FTLD, and healthy subjects. In addition, we evaluated the correlation between platelet APP mRNA expression levels and cognitive impairment.Differential gene expression measurements revealed a significant up-regulation of APP TOT and APP KPI in both AD and FTLD patients compared to the controls (being AD/Controls: 1.67 for APP TOT and 1.47 for APP KPI; FTLD/Controls: 1.62 for APP TOT and 1.51 for APP KPI; p<0.05), although it is interesting to note that in FTLD patients this expression did not correlate with the severity of cognitive impairment.This could be related to a reduced beta-amyloid (Aβ) formation, caused by an alteration of secretase enzymatic activity, even though a post-transcriptional regulation of APP mRNAs in FTLD cannot be excluded.

Hemostatic effect of a monoclonal antibody mAb 2021 blocking the interaction between FXa and TFPI in a rabbit hemophilia model

AbstractHemophilia is treated by IV replacement therapy with Factor VIII (FVIII) or Factor IX (FIX), either on demand to resolve bleeding, or as prophylaxis. Improved treatment may be provided by drugs designed for subcutaneous and less frequent administration with a reduced risk of inhibitor formation. Tissue factor pathway inhibitor (TFPI) down-regulates the initiation of coagulation by inhibition of Factor VIIa (FVIIa)/tissue factor/Factor Xa (FVIIa/TF/FXa). Blockage of TFPI inhibition may facilitate thrombin generation in a hemophilic setting. A high-affinity (KD = 25pM) mAb, mAb 2021, against TFPI was investigated. Binding of mAb 2021 to TFPI effectively prevented inhibition of FVIIa/TF/FXa and improved clot formation in hemophilia blood and plasma. The binding epitope on the Kunitz-type protease inhibitor domain 2 of TFPI was mapped by crystallography, and showed an extensive overlap with the FXa contact region highlighting a structural basis for its mechanism of action. In a rabbit hemophilia model, an intravenous or subcutaneous dose significantly reduced cuticle bleeding. mAb 2021 showed an effect comparable with that of rFVIIa. Cuticle bleeding in the model was reduced for at least 7 days by a single intravenous dose of mAb 2021. This study suggests that neutralization of TFPI by mAb 2021 may constitute a novel treatment option in hemophilia.

Contribution of Kunitz Protease Inhibitor and Transmembrane Domains to Amyloid Precursor Protein Homodimerization

Background: The two major isoforms of the human amyloid precursor protein (APP) are APP695 and APP751. They differ by the insertion of a Kunitz-type protease inhibitor (KPI) sequence in the extracellular domain of APP751. APP-KPI isoforms are increased in Alzheimer’s disease brains, and they could be associated with disease progression. Recent studies have shown that APP processing to Aβ is regulated by homodimerization, which involves both extracellular and juxtamembrane/transmembrane (JM/TM) regions. Objective: Our aim is to understand the mechanisms controlling APP dimerization and the contribution of the ectodomain and JM/TM regions to this process. Methods: We used bimolecular fluorescence complementation approaches coupled to fluorescence-activated cell sorting analysis to measure the dimerization level of different APP isoforms and APP C-terminal fragments (C99) mutated in their JM/TM region. Results: APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain of APP or C99 did not significantly affect fluorescence complementation. Conclusion: These findings indicate that the KPI domain plays a major role in APP dimerization. They set the basis for further investigation of the relation between dimerization, metabolism and function of APP.

TFPIβ is the GPI-anchored TFPI isoform on human endothelial cells and placental microsomes

AbstractTissue factor pathway inhibitor (TFPI) produces factor Xa-dependent feedback inhibition of factor VIIa/tissue factor-induced coagulation. Messages for 2 isoforms of TFPI have been identified. TFPIα mRNA encodes a protein with an acidic N-terminus, 3 Kunitz-type protease inhibitor domains and a basic C-terminus that has been purified from plasma and culture media. TFPIβ mRNA encodes a form in which the Kunitz-3 and C-terminal domains of TFPIα are replaced with an alternative C-terminus that directs the attachment of a glycosylphosphatidylinositol (GPI) anchor, but whether TFPIβ protein is actually expressed is not clear. Moreover, previous studies have suggested that the predominant form of TFPI released from cells by phosphatidylinositol-specific phospholipase C (PIPLC) treatment is TFPIα, implying it is bound at cell surfaces to a separate GPI-anchored coreceptor. Our studies show that the form of TFPI released by PIPLC treatment of cultured endothelial cells and placental microsomes is actually TFPIβ based on (1) migration on SDS-PAGE before and after deglycosylation, (2) the lack of a Kunitz-3 domain, and (3) it contains a GPI anchor. Immunoassays demonstrate that, although endothelial cells secrete TFPIα, greater than 95% of the TFPI released by PIPLC treatment from the surface of endothelial cells and from placental microsomes is TFPIβ.

The many domains of TFPI

If tissue factor pathway inhibitor (TFPI) were a product like a smart phone or computer operating system, no doubt we would all be complaining that it came in too many versions. This relatively small (32 kDa) protein, thought to be the major plasma inhibitor of the initiation of clotting (reviewed by Broze and Girard (1)), has five domains: an acidic N-terminal domain, three Kunitz-type protease inhibitor domains, and a basic C-terminal domain. TFPI exists in multiple versions in plasma, including the “full-length” TFPIalpha molecule as well as various C-terminally truncated forms, some of which are in mixed disulfides with LDL. Although the latter have little or no anticoagulant activity, they are the predominant TFPI species in plasma. An alternatively spliced variant, TFPIbeta, is on the surface of endothelial cells and other cell types; it lacks the Kunitz 3 and basic C-terminal domains and instead is GPI-anchored via an alternate C-terminal sequence (2). Other alternatively spliced TFPI mRNAs have also been detected, although it is not clear if they are expressed into protein. The first two Kunitz domains of TFPI function as protease inhibitors, with Kunitz 1 inactivating factor VIIa and Kunitz 2 inactivating factor Xa. The Kunitz 3 domain, N-terminal acidic domain and C-terminal basic domain are all thought to contribute to TFPI function, as removing them diminishes TFPI anticoagulant activity (1). In the present issue of Thrombosis and Haemostasis, Peraramelli et al. employ chemical synthesis of peptides (including the use of peptide ligation to assemble them into larger proteins) to create versions of TFPI that contain just the isolated Kunitz 2 domain of TFPI, as well as combinations of Kunitz domains 1 and 2, or of Kunitz domains 2 and 3. They then utilize these precisely defined truncations of TFPI to explore the contributions of Kunitz domains 1 and 3, as well as the basic C-terminal domain, to the inactivation of factor Xa—which appears to be the major target of this protease inhibitor. This clever approach confirms previous studies of the functional contributions of the various TFPI domains (1), and extends them to identify, among other things, which portions of TFPI outside Kunitz domain 2 contribute to the initial encounter complex, and which contribute to the isomerization of TFPI bound to factor Xa to bring about the formation of the tightly-bound (and fully inhibited) TFPI/factor Xa complex.

Characterization of a novel variant of the second domain of bikunin with increased leukocyte elastase inhibitory activity

The light chain of inter-α inhibitor, also known as bikunin or urinary trypsin inhibitor, is composed of two tandemly arranged Kunitz-type protease inhibitor domains. The second domain of bikunin has factor Xa inhibitory activity which previously was enhanced by mutating two amino acids, glutamine 19 and tyrosine 46 to lysine and aspartate, respectively. In this study, we tried to potentiate its inhibitory activity against leukocyte elastase. A molecular docking model of the second domain of bikunin with leukocyte elastase revealed that P5 arginine 11 was a candidate residue for a third substitution. We generated six triple point mutants using site-directed mutagenesis, compared their leukocyte elastase-inhibitory activities, and selected the most potent variant with arginine 11 substituted to serine. The IC50 values for factor XIa, factor Xa, and leukocyte elastase were 182, 302, and 273 nM, respectively. Moreover, this triple point mutant prolonged the activated partial thromboplastin time and moderately reduced leukocyte elastase-induced endothelial injury. Additionally, favorable conformations created by these mutations were speculated using the structure of the Kunitz protease inhibitor domain of protease nexin 2 complexed with factor XIa as a reference. We discovered a novel triple point mutant of the second domain of bikunin that has potent inhibitory activities against factor XIa, factor Xa, and leukocyte elastase. This variant exhibited anticoagulant activity in plasma and suppressed endothelial cell injury.

APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms

The amyloid precursor protein (APP) is part of a larger gene family, which has been found to form homo- or heterotypic complexes with its homologues, whereby the exact molecular mechanism and origin of dimer formation remains elusive. In order to assess the cellular location of dimerization, we have generated a cell culture model system in CHO-K1 cells, stably expressing human APP, harboring dilysine-based organelle sorting motifs [KKAA-endoplasmic reticulum (ER); KKFF-Golgi], accomplishing retention within early secretory compartments. We show that APP exists as disulfide-bonded dimers upon ER retention after it was isolated from cells, and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. In contrast, strong denaturing and reducing conditions, or deletion of the E1 domain, resulted in the disappearance of those dimers. Thus we provide first evidence that a fraction of APP can associate via intermolecular disulfide bonds, likely generated between cysteines located in the extracellular E1 domain. We particularly visualize APP dimerization itself and identified the ER as subcellular compartment of its origin using biochemical or split GFP approaches. Interestingly, we also found that minor amounts of SDS-resistant APP dimers were located to the cell surface, revealing that once generated in the oxidative environment of the ER, dimers remained stably associated during transport. In addition, we show that APP isoforms encompassing the Kunitz-type protease inhibitor (KPI) domain exhibit a strongly reduced ability to form cis-directed dimers in the ER, whereas trans-mediated cell aggregation of Drosophila Schneider S2-cells was isoform independent. Thus, suggesting that steric properties of KPI-APP might be the cause for weaker cis-interaction in the ER, compared to APP695. Finally, we provide evidence that APP/APLP1 heterointeractions are likewise initiated in the ER.

Structural features of the KPI domain control APP dimerization, trafficking, and processing

The two major isoforms of human APP, APP695 and APP751, differ by the presence of a Kunitz-type protease inhibitor (KPI) domain in the extracellular region. APP processing and function is thought to be regulated by homodimerization. We used bimolecular fluorescence complementation (BiFC) to study dimerization of different APP isoforms and mutants. APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain did not affect fluorescence complementation, but native folding of KPI is critical for APP751 homodimerization. APP751 and APP695 dimers were mostly localized at steady state in the Golgi region, suggesting that most of the APP751 and 695 dimers are in the secretory pathway. Mutation of the KPI led to the retention of the APP homodimers in the endoplasmic reticulum. We finally showed that APP751 is more efficiently processed through the nonamyloidogenic pathway than APP695. These findings provide new insight on the particular role of KPI domain in APP dimerization. The correlation observed between dimerization, subcellular localization, and processing suggests that dimerization acts as an efficient regulator of APP trafficking in the secretory compartments that has major consequences on its processing.

Genes encoding WFDC- and Kunitz-type protease inhibitor domains: are they related?

We have previously demonstrated that the genes of SCPs (semen coagulum proteins) and the WFDC (whey acidic protein four-disulfide core)-type protease inhibitor elafin are homologous in spite of lacking similarity between their protein products. This led to the discovery of a locus on human chromosome 20, encompassing genes of the SCPs, SEMG1 (semenogelin I) and SEMG2, and 14 genes containing the sequence motif that is characteristic of WFDC-type protease inhibitors. We have now identified additional genes at the locus that are similarly organized, but which give rise to proteins containing the motif of Kunitz-type protease inhibitors. Here, we discuss the evolution of genes encoding SCPs and describe mechanisms by which they and genes with Kunitz motifs might have evolved from genes with WFDC motifs. We can also demonstrate an expansion of the WFDC locus with 0.6 Mb in the cow. The region, which seems to be specific to ruminants, contains several genes and pseudogenes with Kunitz motifs, one of which is the much-studied BPTI (bovine pancreatic trypsin inhibitor).

Biological functions of the WAP domain-containing multidomain proteins WFIKKN1 and WFIKKN2

WFIKKN1 and WFIKKN2 are two closely related multidomain proteins consisting of a WAP (whey acidic protein)-, a follistatin-, an immunoglobulin-, two Kunitz-type protease inhibitor-domains and an NTR domain (netrin domain). Recent experiments have shown that both WFIKKN1 and WFIKKN2 bind myostatin and GDF11 (growth and differentiation factor 11) with high affinity and are potent antagonists of these growth factors. Structure-function studies on WFIKKN proteins have revealed that their interactions with GDF8 and GDF11 are mediated primarily by the follistatin and NTR domains.

Platelet Amyloid Precursor Protein Isoform Expression in Alzheimer's Disease: Evidence for Peripheral Marker

Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized by a progressive cognitive and memory decline. Among peripheral markers of AD, great interest has been focused on the amyloid precursor protein (APP). In this regard, platelets represent an important peripheral source of APP since it has been demonstrated that the three major isoforms, that are constituted of 770, 751 and 695 aa residues, are inserted in the membrane of resting platelets. APP 751 and APP 770 contain a Kunitz-type serine protease inhibitor domain (APP KPI) and APP 695 lacks this domain. To address this issue, we first examined the platelet APP isoform mRNAs prospectively as biomarker for the diagnosis of AD by means of real-time quantitative PCR, and then evaluated the correlation between APP mRNA expression levels and cognitive impairment of enrolled subjects. Differential gene expression measurements in the AD patient group (n=18) revealed a significant up-regulation of APP TOT (1.52-fold), APP KPI (1.32-fold), APP 770 (1.33-fold) and APP 751 (1.26-fold) compared to controls (n=22). Moreover, a statistically significant positive correlation was found between APP mRNA levels (TOT, KPI, 770 and 751) and cognitive impairment. Since AD definitive diagnosis still relies on pathological evaluation at autopsy, the present results are consistent with the hypothesis that platelet APP could be considered a potential reliable peripheral marker for studying AD and could contribute to define a signature for the presence of AD pathology.

Structural and Mutational Analysis of Canonical Loop Residues In Protease Nexin 2 Involved In Factor XIa and Trypsin Inhibition.

AbstractAbstract 1147Factor XIa (FXIa) activates FIX and is regulated by platelet-secreted protease nexin 2 (PN2) that contains a Kunitz-type protease inhibitor (KPI) domain. Trypsin is regulated by basic pancreatic trypsin inhibitor (BPTI). The primary and tertiary structures of trypsin and the catalytic domain of FXIa are highly homologous, and KPI and BPTI are nearly identical structurally. We have previously identified two loop structures (loops 1 and 2) in the KPI domain of PN2 that interact with residues in the FXIa catalytic domain. Based on the structure of the FXIa/KPI complex crystal structure, residues within loops 1 and 2 were mutated for experiments examining the inhibition of FXIa and trypsin.Results show that the loop-1-region P1 site residue Arg15 of PN2KPI plays a major role in FXIa inhibition by protruding into the S1 specificity pocket of FXIa. Ala mutation at this site renders PN2KPI non-inhibitory for both FXIa and trypsin. BPTI has Lys15 at the P1 site. BPTI inhibits both FXIa and trypsin significantly less effectively than PN2KPI. PN2KPI-R15K lost FXIa inhibitory activity, whereas BPTI-K15R substantially gained affinity for FXIa. Like FXIa, trypsin preferred BPTI-K15R showing a significant enhancement in affinity. Thus, a major determinant of the inhibitory activity of PN2KPI and BPTI against FXIa and trypsin is the P1 residue, with Arg being preferred over Lys for both inhibitors and both proteases. In addition, loop 1 residues Pro13 and Arg20 make important contributions to both FXIa and trypsin inhibition as demonstrated by significantly elevated Ki values for Ala mutations (P13A, and R20A) at these sites. In contrast, Ser19 makes no significant contribution to inhibition of either FXIa or trypsin whereas Met17 makes a significant contribution to the inhibition of trypsin, but not FXIa. In loop 2, only Phe34 is identified as a residue making significant contributions to the inhibition of both FXIa and trypsin, since the PN2KPI-F34A mutant displayed reduced inhibitory activity for both FXIa (6-fold) and for trypsin (3-fold). To rationalize these findings, we examined the crystal structures of the FXIa(catalytic domain)/PN2KPI complex, and the trypsin/BPTI complex. Structurally, the PN2KPI loop-1, P1-site residue Arg15 makes a complex primary interaction with Asp189 of both FXIa and trypsin. Disruption of this site by R15A mutation renders PN2KPI non-inhibitory because it preempts salt bridge interactions from two nitrogen atoms of the guanidinium group of Arg15 with Asp189 and Gly218 in FXIa. In addition, the Arg15 carbonyl oxygen forms hydrogen bonds with main-chain nitrogen atoms of one of the catalytic triad residues, Ser195, and with Gly193. The other important interaction in FXIa/PN2KPI or trypsin/BPTI is hydrophobic, between PN2KPI-Phe34 and FXIa-Tyr143 and between BPTI-Val34 and trypsin-Tyr151. This intermolecular interaction is further strengthened by an intramolecular interaction in which the side chain of Phe34 packs closely with the side chain of Met17 within PN2KPI, altogether forming a strong hydrophobic patch in FXIa-PN2KPI and trypsin-PN2KPI. PN2KPI-F34A disrupts both inter- and intramolecular hydrophobic interactions, leading to discernable reductions in affinity for both FXIa and trypsin. Despite occupying extreme positions in the autolysis loops, (143YRKLRDKI151 in FXIa and 143NTKSSGTSY151 in trypsin), Tyr143/151 residues still orient themselves in close proximity to Phe34. Thus, loop-1 residues of PN2KPI establish complex ionic interactions that play a major role, which is supplemented by the loop-2 residue, Phe34 (in PN2KPI) or Val34 (in BPTI), which establish hydrophobic interactions with residues in FXIa and trypsin leading to very high-affinity enzyme-inhibitor complexes. Disclosures:No relevant conflicts of interest to declare.

WFIKKN1 and WFIKKN2 bind growth factors TGFβ1, BMP2 and BMP4 but do not inhibit their signalling activity

WFIKKN1 and WFIKKN2 are large extracellular multidomain proteins consisting of a WAP domain, a follistatin domain, an immunoglobulin domain, two Kunitz-type protease inhibitor domains and an NTR domain. Recent experiments have shown that both proteins have high affinity for growth and differentiation factor (GDF)8 and GDF11. Here we study the interaction of WFIKKN proteins with several additional representatives of the transforming growth factor (TGF)β family using SPR measurements. Analyses of SPR sensorgrams suggested that, in addition to GDF8 and GDF11, both WFIKKN proteins bind TGFβ1, bone morphogenetic protein (BMP)2 and BMP4 with relatively high affinity (K(d) ∼ 10(-6) m). To assess the biological significance of these interactions we studied the effect of WFIKKN proteins on the activity of GDF8, GDF11, TGFβ1, BMP2 and BMP4 using reporter assays. These studies revealed that WFIKKN1 and WFIKKN2 inhibited the biological activity of GDF8 and GDF11 in the nanomolar range, whereas they did not inhibit the activities of TGFβ1, BMP2 and BMP4 even in the micromolar range. Our data indicate that WFIKKN proteins are antagonists of GDF8 and GDF11, but in the case of TGFβ1, BMP2 and BMP4 they function as growth factor binding proteins. It is suggested that the physical association of WFIKKN proteins with these growth factors may localize their action and thus help to establish growth factor gradients in the extracellular space.

Effect of a conserved peptide derived from Kunitz domain of hepatitis B virus x protein on the cell cycle and apoptosis of HepG2 cells via the proteasome pathway

Background Hepatitis B virus (HBV) x protein (HBx) in HepG2 cells causes a moderate decrease in proteolysis activity of the proteasome. A highly conserved Kunitz-type serine protease inhibitor domain within 154 amino acid residues of HBx has been identified. In this study, a peptide chain derived from the Kunitz domain (PKD) was used to study its effect on the cell cycle and apoptosis of HepG2 cells, and investigated the function of PKD on the activities of proteasomes and AAA-ATPase p97, which involves in the ubiquitin-proteasome protein degradation pathway. Methods The PKD peptide (Phe-Val-Leu-Gly-Gly-Cys-Arg-His-Lys) was chemically synthesized. MTT assays were used to determine the effects of PKD on HepG2 cell growth. Mouse anti-p97 antibody was developed for Western blotting to detect the expression of p97. ATPase activity of proteasomes was measured using a colorimetric assay. Peptidase activities of proteasomes were analyzed with various peptidase-specific fluorogenic peptide substrates. Flow cytometry was used to determinate cell cycle phase and apoptosis. Results Viability of HepG2 cells decreased in a PKD-dose-dependent manner. Cells exhibited significant cytotoxicity in the presence of 15 mmol/L of PKD. Western blotting analysis showed that expression of p97 was suppressed in HepG2 cells treated with PKD compared to untreated cells. The ATPase activity of proteasomes from immunoprecipitates of HepG2 cells pretreated with PKD was apparently decreased. Chymotryptic activity of proteasomes in HepG2 cells was significantly inhibited by 10mmol/L PKD; tryptic activity and peptidylglutamyl peptide hydrolase activity of proteasomes were less inhibited by PKD than chymotryptic activity. The cell cycle phase of HepG2 cells treated with PKD for 36 hours was blocked largely at the G0-G1 phase, while untreated control cells were mainly in S phase. PKD also significantly induced apoptosis. Conclusions The peptide derived from Kunitz domain of HBx protein induces HepG2 cell growth arrest and apoptosis, which may result from down-regulation of p97 expression, and decrease of both the ATPase and chymotryptic activities of proteasomes.