Kunitz-type Serine Protease Inhibitor Domain Research Articles

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.

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.

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.

Amyloid precursor protein mRNA levels in Alzheimer's disease brain

Insoluble β-amyloid deposits in Alzheimer's disease (AD) brain are proteolytically derived from the membrane bound amyloid precursor protein (APP). The APP gene is differentially spliced to produce isoforms that can be classified into those containing a Kunitz-type serine protease inhibitor domain (K +, APP 751, APP 770, APRP 365 and APRP 563), and those without (K −, APP 695 and APP 714,). Given the hypothesis that Aβ is a result of aberrant catabolism of APP, differential expression of mRNA isoforms containing protease inhibitors might play an active role in the pathology of AD. We took 513 cerebral cortex samples from 90 AD and 81 control brains and quantified the mRNA isoforms of APP with TaqMan™ real-time RT-PCR. After adjustment for age at death, brain pH and gender we found a change in the ratio of KPI+ to KPI− mRNA isoforms of APP. Three separate probes, designed to recognise only KPI+ mRNA species, gave increases of between 28% and 50% in AD brains relative to controls ( p=0.002). There was no change in the mRNA levels of KPI-(APP 695) ( p=0.898). Therefore, whilst KPI-mRNA levels remained stable the KPI+species increased specifically in the AD brains.

Hypoglycemia enhances the expression of mRNA encoding β-amyloid precursor protein in rat primary cortical astroglial cells

Deposition of β-amyloid (A β) is a characteristic feature of the pathology of Alzheimer's disease (AD). Since glucose metabolism and the consequential ATP production are depressed in the temporal and parietal regions of the cortex in patients with AD, we designed the present study to investigate the possible role of hypometabolism in the pathogenesis of AD. We incubated rat primary cortical astroglial cells for 2 h to 4 days in a media deprived of 95% of its glucose and assessed the expression and alternative splicing of the mRNA that encoding β-amyloid precursor protein (APP) using RT-PCR. Hypoglycemia caused a time-dependent increase in APP mRNA expression, which reaches a peak level of 173.2% of control expression ( P<0.05) at 24 h of hypoglycemia. Noteworthy, hypoglycemia favors the alternative splicing that includes the exon 7 segment, which encodes a Kunitz-type serine protease inhibitor domain. This study demonstrates that hypoglycemia increases APP mRNA expression in astroglial cells and processing of APP mRNA to a form that may encourage A β deposits in AD. These data suggest that the observed hypometabolism in AD may contribute to its deposition of A β in affected brain regions.

Expression of beta-amyloid precursor protein mRNAs following transient focal ischaemia.

beta-Amyloid precursor protein (beta APP) can be alternatively processed to result in release of either neurotoxic amyloid beta-peptide, or secreted forms of beta APP, which might have a role in neuronal plasticity and survival. Four different forms of beta APP mRNAs have been described in rodents: APP695, APP714, APP751 and APP770. The two larger forms contain a Kunitz-type serine protease inhibitor domain (KPI). Since previous studies have shown increased APP immunoreactivity following brain ischaemia, we used in situ hybridization histochemistry to determine whether induction of any APP mRNA transcripts take place following focal brain ischaemia. While the hybridization signal of all APP isoforms in the infarct was lost 4-24 h following the insult, APP770 mRNA was slightly upregulated in the ipsilateral cortex and striatum 3 days after 90 min ischaemia. At 7 days post-ischaemia APP770 and APP751 mRNAs were induced in the infarct core and in a thin perifocal zone. KPI-containing APP forms are differentially induced following focal brain ischaemia, possibly as a neuroprotective response to neuronal injury.

Alzheimer Amyloid Protein Precursor Enhances Proliferation of Neural Stem Cells from Fetal Rat Brain

We have investigated the effect of the Alzheimer amyloid protein precursor (APP) on the proliferation of neural stem cells. Two secretory forms of APP (sAPP770 and sAPP695, with and without the Kunitz-type serine protease inhibitor domain, respectively) were purified from conditioned media of COS-7 cells transfected with genetically modified APP cDNAs. Both secretory APPs promoted the growth of neural stem cells, and the effect of sAPP770 was greater than that of sAPP695. sAPP770 and known growth factors in combination exerted a cooperative stimulation of the stem cell proliferation. These results suggest that APP, especially APP possessing the protease inhibitor domain, regulates the growth of neuronal precursor cells during development of the nervous system.

Amyloid β-protein precursor in cultured leptomeningeal smooth muscle cells

Smooth muscle cell cultures were established from human leptomeningeal blood vessels obtained at autopsy from a patient with normal cortical blood vessels and a patient with extensive cerebral amyloid angiopathy (CAA). The normal leptomeningeal smooth muscle cells and CAA/ leptomeningeal smooth muscle cells were compared to normal human aorta smooth muscle cells with respect to amyloid β-protein precursor (AβPP) expression. All the cultured smooth muscle cells secreted a prominent =110 kDaform of AβPP that contained the Kunitz-type serine protease inhibitor domain analogous to protease nexin-2 (PN-2). Quantitative measurements revealed that aorta smooth muscle cells secrete -1.5-fold more PN-2/ABPP than normal leptomeningeal smooth muscle cells and CAA/ leptomeningeal smooth muscle cells. In contrast, the normal leptomeningeal smooth muscle cells and CAA/ leptomeningeal smooth muscle cells retain =2.4- and =3.3-fold, respectively, more cell-associated AβPP than aorta smooth muscle cells. These preliminary...

The Rat Central Nervous System Expresses Alzheimer's Amyloid Precursor Protein APP695, but Not APP677 (L‐APP Form)

A novel splicing form of beta A4 amyloid precursor protein (APP) lacking exon 15, corresponding to 18 residues, was first reported in leukocytes and then in ubiquitous organs. To determine which APP molecules (APP695, APP751, or APP770) either with (N-APP) or without (L-APP; leukocyte-derived APP) exon 15 were expressed in various organs, we investigated the alternative splicing at exon 15 in the rat brain, kidney, heart, and testis by a PCR analysis of reverse-transcribed RNA and Southern blot analysis. Regarding APP695 without exons 7 and 8, L-APP was either seldom or never expressed in the brain, whereas both N- and L-APP were expressed in other organs. On the other hand, regarding APP751/770 containing exon 7, which codes for the Kunitz-type serine protease inhibitor domain, both N- and L-APP were expressed in all the organs examined, including the brain. These results suggest that a particular alternative regulation system related to exon 15 might be present in only APP695 of the brain and influence the proteolytic processing of APP.

Sequential NMR resonance assignment and structure determination of the Kunitz-type inhibitor domain of the Alzheimer's .beta.-amyloid precursor protein

Certain precursor proteins (APP751 and APP770) of the amyloid beta-protein (AP) present in Alzheimer's disease contain a Kunitz-type serine protease inhibitor domain (APPI). In this study, the domain is obtained as a functional inhibitor through both recombinant (APPIr) and synthetic (APPIs) methodologies, and the solution structure of APPI is determined by 1H 2D NMR techniques. Complete sequence-specific resonance assignments (except for P13 and G37 NH) for both APPIr and APPIs are achieved using standard procedures. Ambiguities arising from degeneracies in the NMR resonances are resolved by varying sample conditions. Qualitative interpretation of short- and long-range NOEs reveals secondary structural features similar to those extensively documented by NMR for bovine pancreatic trypsin inhibitor (BPTI). A more rigorous interpretation of the NOESY spectra yields NOE-derived interresidue distance restraints which are used in conjunction with dynamic simulated annealing to generate a family of APPI structures. Within this family, the beta-sheet and helical regions are in good agreement with the crystal structure of BPTI, whereas portions of the protease-binding loops deviate from those in BPTI. These deviations are consistent with those recently described in the crystal structure of APPI (Hynes et al., 1990). Also supported in the NMR study is the hydrophobic patch in the protease-binding domain created by side chain-side chain NOE contacts between M17 and F34. In addition, the NMR spectra indicate that the rotation of the W21 ring in APPI is hindered, unlike Y21 in BPTI, showing a greater than 90% preference for one orientation in the hydrophobic groove.

Platelet Coagulation Factor XI a -Inhibitor, a Form of Alzheimer Amyloid Precursor Protein

An inhibitor of coagulation factor XIa was purified from serum-free conditioned medium of HepG2 liver cells. Platelets stimulated with thrombin or calcium ionophore (A23187) secrete a protein functionally and immunologically identical to the inhibitor, implying a role for this inhibitor in hemostasis. Analysis of the amino-terminal amino acid sequence and immunologic reactivity showed the inhibitor to be a truncated form of the Alzheimer's amyloid precursor protein that contains a Kunitz-type serine protease inhibitor domain and at least a portion of the amyloid beta protein. It inhibits factor XIa and trypsin with a Ki of 450 +/- 50 pM and 20 +/- 10 pM, respectively. Heparin (1 unit/ml) did not significantly effect inhibition of trypsin, but inhibition of XIa was 15 times greater (Ki = 25 +/- 15 pM) in the presence of heparin.