Serine proteases and their serpin inhibitors in Alzheimer's disease (original) (raw)
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Protease nexin-2/amyloid β-protein precursor in blood is a platelet-specific protein
Biochemical and Biophysical Research Communications, 1991
The protease inhibitor, protease nexin-2 (PN-2), is the secreted form of the amyloid 13-protein precursor (APP) which contains the Kunitz protease inhibitor domain. PN-2/APP is an abundant platelet s-granule protein which is secreted upon platelet activation. PN-2/APP mRNA is present in cultured endothelial cells and the protein has been detected in plasma. In the present studies we quantitated PN-2/APP in platelets, plasma and several different cell types of the vasculature to identify the repository of the protein in the circulatory system. We report that PN-2/APP is predominantly a platelet protein in the vascular compartment. Lysates of unstimulated umbilical vein endothelial cells, granulocytes or monocytes contained little PN-2/APP based on sensitive functional protease binding and immunoblotting assays. Quantitative immunoblotting studies demonstrated that normal citrated-plasma contains < 60 pM PN-2/APP. In contrast, platelets can contribute up to 30 nM PN-2/APP, indicating that they are the major source of the protein in blood. ~ 1991 Academic P ..... Inc. The amyloid 13-protein and its parent molecule, the amyloid I]-protein precursor (APP), are constituents of senile plaques in brain and cerebrovascular deposits in individuals with Alzheimer's disease (AD) and Down's syndrome (1-5). APP can be translated from at least three altematively spliced mRNA species to yield proteins of 695, 751 and 770 amino acids (6-8). The latter two forms contain an additional domain that is homologous to Kunitz-type serine protease inhibitors (6-8). The forms of mRNA encoding for APP-751 and 770 appear to be ubiquitous. On the other hand, mRNA encoding for APP-695, the form which lacks the Kunitz protease inhibitor domain, is found primarily in brain. The previously described protease inhibitor protease nexin-2 (PN-2) *To whom correspondence should be addressed.
APP Processing and the APP-KPI Domain Involvement in the Amyloid Cascade
Neurodegenerative Diseases, 2005
Alternative APP mRNA splicing can generate isoforms of APP containing a Kunitz protease inhibitor (KPI) domain. KPI is one of the main serine protease inhibitors. Protein and mRNA KPI(+)APP levels are elevated in Alzheimer's disease (AD) brain and are associated with increased amyloid beta deposition. In the last years increasing evidence on multiple points in the amyloid cascade where KPI(+)APP is involved has been accumulated, admitting an outstanding position in the pathogenesis of AD to the KPI domain. This review focuses on the APP processing, the molecular activity of KPI and its physiological and pathological roles and the KPI involvement in the amyloid cascade through the nerve growth factor, the lipoprotein receptor-related protein, the tumor necrosis factor-alpha converting enzyme and the Notch1 protein.
Journal of Biological Chemistry, 1998
Although a number of studies have examined amyloid precursor protein (APP) mRNA levels in Alzheimer's disease (AD), no clear consensus has emerged as to whether the levels of transcripts for isoforms containing a Kunitz protease inhibitory (KPI)-encoded region are increased or decreased in AD. Here we compare AD and control brain for the relative amounts of APP protein containing KPI to APP protein lacking this domain. APP protein was purified from the soluble subcellular fraction and Triton X-100 membrane pellet extract of one hemisphere of AD (n ؍ 10), normal (n ؍ 7), and neurological control (n ؍ 5) brains. The amount of KPI-containing APP in the purified protein samples was determined using two independent assay methods. The first assay exploited the inhibitory action of KPI-containing APP on trypsin. The second assay employed reflectance analysis of Western blots. The proportion of KPI-containing forms of APP in the soluble subcellular fraction of AD brains is significantly elevated (p < 0.01) compared with controls. Species containing a KPI domain comprise 32-41 and 76 -77% of purified soluble APP from control and AD brains, respectively. For purified membraneassociated APP, 72-77 and 65-82% of control and AD samples, respectively, contain a KPI domain. Since KPIcontaining species of APP may be more amyloidogenic (Ho, L., Fukuchi, K., and Yonkin, S. G. (1996) J. Biol. Chem. 271, 30929 -30934), our findings support an imbalance of isoforms as one possible mechanism for amyloid deposition in sporadic AD.
Protease nexin-2/amyloid beta protein precursor. A tight-binding inhibitor of coagulation factor IXa
Journal of Clinical Investigation, 1993
Protease nexin-2/amyloid 6 protein precursor (PN-2/A#PP) is an abundant, secreted platelet protein which is a potent inhibitor of coagulation Factor XIa. We examined other potential anticoagulant activities of PN-2/Ai8PP. Purified Kunitz protease inhibitor domain of PN-2/A8PP and PN-2/ABPP itself were found to prolong the coagulation time of plasma and pure Factor IXa. The Kunitz protease inhibitor domain also inhibited the ability of Factor IXa to activate Factor X. PN-2/AAPP inhibited Factor IXa with a K1 of 7.9 to 3.9 X 10-1 M in the absence and presence of heparin, respectively. When the second-order rate constant of PN-2/AO8PP's inhibition of Factor IXa (2.7 X 108 M-'min-') was compared to that of antithrombin III (3.8 X 106 M-'min-'), PN-2/AfiPP was at least a 71-fold more potent inhibitor of Factor IXa than antithrombin III. PN-2/AfiPP formed a complex with Factor IXa as detected by gel filtration and ELISA. The finding that PN-2/ AfiPP is a potent inhibitor of Factor IXa could help to explain the spontaneous intracerebral hemorrhages seen in patients with hereditary cerebral hemorrhage with amyloidosis Dutchtype where there is an extensive accumulation of PN-2 /A46PP in their cerebral blood vessels.
Biochemistry, 2000
Cerebrovascular deposition of fibrillar 39-42 amino acid amyloid-protein (A), a condition known as cerebral amyloid angiopathy (CAA), is a key pathological feature of Alzheimer's disease and related disorders including hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Severe cases of CAA, particularly in HCHWA-D, lead to recurrent and often fatal hemorrhagic strokes. Although the reasons for this pathological consequence remain unclear, alterations in proteolytic hemostasis mechanisms have been implicated. For example, the A parent molecule protease nexin-2/amyloid-protein precursor (PN-2/A PP), which is elevated in HCHWA-D cerebral vessels with A deposits, is a potent inhibitor of coagulation factor XIa (FXIa). Here we show that fibrillar HCHWA-D A binds PN-2/A PP, but not its isolated Kunitz-type proteinase inhibitor (KPI) domain, in a saturable, dose-dependent manner with a K d of ≈28 nM. Neither PN-2/A PP nor its KPI domain bound to nonfibrillar HCHWA-D A. The fibrillar A binding domain on PN-2/A PP was localized to residues 18-119. PN-2/A PP that bound to fibrillar HCHWA-D A immobilized either in plastic wells or on the surface of cultured cerebrovascular smooth muscle cells was active in inhibiting FXIa. Quantitative kinetic measurements revealed that fibrillar HCHWA-D A caused a >5-fold enhancement of FXIa inhibition by PN-2/A PP. Similar stimulatory effects on FXIa inhibition by PN-2/A PP were also observed with fibrillar wild-type A. However, fibrillar A had no effect on the inhibition of trypsin by PN-2/A PP. These findings suggest that fibrillar A deposits in cerebral vessels can effectively localize and enhance the anticoagulant functions of PN-2/ A PP, thereby contributing to a microenvironment conducive to hemorrhaging.
Journal of Biological Chemistry, 1996
The insoluble amyloid deposited extracellularly in the brains of patients with Alzheimer's disease (AD) is composed of amyloid  protein, a ϳ4-kDa secreted protein that is derived from a set of large proteins collectively referred to as the amyloid  protein precursor (APP). During normal processing the APP is cleaved by  secretase, producing a large NH 2-terminal secreted derivative (sAPP) and a COOH-terminal fragment beginning at A1, which is subsequently cleaved by ␥ secretase releasing secreted A. Most secreted A is A1-40, but ϳ10% of secreted A is A1-42. Alternative APP cleavage by ␣ secretase produces a slightly longer NH 2terminal secreted derivative (sAPP␣) and a COOH-terminal fragment beginning at A17, which is subsequently cleaved by ␥ secretase releasing a ϳ3-kDa secreted form of A (P3). Several of the APP isoforms that are produced by alternative splicing contain a 56amino acid Kunitz protease inhibitor (KPI) domain known to inhibit proteases such as trypsin and chymotrypsin. To determine whether the KPI domain influences the proteolytic cleavages that generate A, we compared A production in transfected cells expressing human KPI-containing APP751 or KPI-free APP695. We focused on As ending at A42 because these forms appear to be most relevant to AD. Using specific sandwich enzyme-linked immunosorbent assays, we analyzed full-length A1-42 and total A ending at A42 (A1-42 ؉ P3(42)). In addition, we analyzed the large secreted derivatives produced by ␣ secretase (sAPP␣) and  secretase (sAPP). In mouse teratocarcinoma (P19) cells expressing APP695 or APP751, expression of the KPI-containing APP751 resulted in the secretion of a lower percentage of P3(42) and sAPP␣ and a correspondingly higher percentage of A1-42 and sAPP. Similar results were obtained in human embryonic kidney (293) cells. These results indicate that expression of the KPI domain reduces ␣ secretase cleavage so that less P3 and relatively more full-length A are produced. Thus, in human brain and in animal models of AD, the amount of KPI-containing APP produced may be an important factor influencing A deposition.
The American journal of pathology, 1993
The classical plasma protein antithrombin III (ATIII), an inhibitor of the blood coagulation cascade, is a member of the serpins that are gaining import in the nervous system. In this study, we examined the presence of ATIII in the pathological lesions of Alzheimer's disease (AD). Antibodies to ATIII consistently detected approximately 58-kd protein(s) on immunoblots of cerebral cortex and brain microvessels. Immunocytochemical studies showed ATIII reactivity within amyloid deposits, neurites associated with plaques, and neurofibrillary tangles in neocortex and hippocampus of virtually all the AD cases examined. In some cases, astrocytes were also stained, suggesting ATIII in these cells. ATIII immunoreactivity in neurofibrillary tangles was further defined by electron microscopy, which showed it to be associated with paired helical filaments. Using the polymerase chain reaction technique to amplify ATIII complementary DNA, we found low levels of messenger RNA expression, relati...
Biochemical and Biophysical Research Communications, 1992
The protease inhibitor, protease nexin-2 (PN-2), is the secreted isoform of the Alzheimer&amp;amp;amp;#39;s amyloid beta-protein precursor (A beta PP) that contains the Kunitz-type protease inhibitor (KPI) domain. Here we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the KPI domain of PN-2/A beta PP. In addition to the 57 amino acid KPI domain, the expression product contained an additional four amino acid residues at its amino terminus that correspond to amino acids 285-288 of A beta PP (Ponte et al. 1988 Nature 311:525-527). This expression system generated yields of greater than 1.0 gram of KPI domain per liter of fermentation media. The secreted 61 amino acid product was purified to homogeneity and biochemically characterized. Amino acid analysis and sequencing of the entire expressed KPI domain verified its integrity. Similar to native PN-2/A beta PP, the purified KPI domain potently inhibited trypsin, chymotrypsin, and coagulation factor XIa. Although heparin augments the inhibition of factor XIa by native PN-2/A beta PP it had no effect on the inhibition of factor XIa by expressed KPI domain suggesting that heparin binds to regions on native PN-2/A beta PP outside of the protease inhibitory domain. This KPI domain expression product should be useful in studying the physiologic and pathophysiologic functions of PN-2/A beta PP.
The multifaceted nature of amyloid precursor protein and its proteolytic fragments: friends and foes
Acta Neuropathologica, 2014
The amyloid precursor protein (APP) has occupied a central position in Alzheimer's disease (AD) pathophysiology, in large part due to the seminal role of amyloid-β peptide (Aβ), a proteolytic fragment derived from APP. Although the contribution of Aβ to AD pathogenesis is accepted by many in the research community, recent studies have unveiled a more complicated picture of APP's involvement in neurodegeneration in that other APP-derived fragments have been shown to exert pathological influences on neuronal function. However, not all APP-derived peptides are neurotoxic, and some even harbor neuroprotective effects. In this review, we will explore this complex picture by first discussing the pleiotropic effects of the major APP-derived peptides cleaved by multiple proteases, including soluble APP peptides (sAPPα, sAPPβ), various C-and Nterminal fragments, p3, and APP intracellular domain fragments. In addition, we will highlight two interesting sequences within APP that likely contribute to this duality in APP function. First, it has been found that caspase-mediated cleavage of APP in the cytosolic region may release a cytotoxic peptide, C31, which plays a role in synapse loss and neuronal death. Second, recent studies have implicated the-YENPTY-motif in the cytoplasmic region as a domain that modulates several APP activities through phosphorylation and dephosphorylation of the first tyrosine residue. Thus, this review summarizes the current understanding of various APP proteolytic products and the interplay among them to gain deeper insights into the possible mechanisms underlying neurodegeneration and AD pathophysiology.