硫化氢对淀粉样前体蛋白代谢途径的影响

目的:观察硫化氢(H2S)对PC12细胞淀粉样前体蛋白(APP)代谢途径的影响,探讨可能的细胞信号机制.方法:用不同浓度硫氢化钠(NaHS,50、100和200 μmol·L-1)作用PC12细胞1和18 h,Western blotting法检测APP、C99、C83、磷脂酰肌醇-3激酶/丝氨酸苏氨酸蛋白激酶(PI3-K/Akt)及丝裂原活化蛋白激酶/细胞外信号调节激酶1/2(MAPK/ERK1/2)信号通路相关蛋白的水平;ELISA检测细胞培养液中Aβ42的水平.50 μmol·L-1 NaHS作用于PC12细胞前30 min加入PI3-K抑制剂LY294002(25和50 μmol·L-1)或 MAPK激酶(MEK)抑制剂PD98059(25和50 μmol·L-1),重复上述检测.各种检测实验均设正常对照组.结果:与正常对照组比较,50 μmol·L-1 NaHS组C99及Aβ42 水平降低(P<0.05),C83的表达提高(P<0.05),Akt及ERK1/2的磷酸化增加(P<0.05),而LY294002可抑制NaHS的上述作用,但PD98059不具有与LY294002类似的作用.结论:H2S能使APP的代谢向非淀粉样途径转变,其机制可能涉及PI3-K/Akt信号通路.     

Regulation of amyloid precursor protein cleavage

Multiple lines of evidence suggest that increased production and/or deposition of the beta-amyloid peptide, derived from the amyloid precursor protein, contributes to Alzheimer's disease. A growing list of neurotransmitters, growth factors, cytokines, and hormones have been shown to regulate amyloid precursor protein processing. Although traditionally thought to be mediated by activation of protein kinase C, recent data have implicated other signaling mechanisms in the regulation of this process. Moreover, novel mechanisms of regulation involving cholesterol-, apolipoprotein E-, and stress-activated pathways have been identified. As the phenotypic changes associated with Alzheimer's disease encompass many of these signaling systems, it is relevant to determine how altered cell signaling may be contributing to increasing brain amyloid burden. We review the myriad ways in which first messengers regulate amyloid precursor protein catabolism as well as the signal transduction cascades that give rise to these effects.     

Interaction of a neuron-specific protein containing PDZ domains with Alzheimer's amyloid precursor protein

A novel protein, human X11-like (human X11L), contains a phosphotyrosine interaction (PI) domain and two PDZ domains and displays 55.2% amino acid homology with the human X11 (human X11). The PI domain of human X11L interacts with a sequence containing the NPXY motif found in the cytoplasmic domain of Alzheimer's amyloid precursor protein. A construct lacking the carboxyl-terminal domain, which comprises two PDZ domains (N + PI), enhances PI binding to APP, whereas another construct lacking an amino-terminal domain relative to PI domain (PI + C) suppresses PI binding to APP. Overexpression of full-length human X11L (N + PI + C) in cells that express APP695 stably decreased the secretion of Abeta40 but not that of Abeta42. However, overexpression of the PI domain alone and the N + PI construct in cells did not affect the secretion of Abeta despite their ability to bind to the cytoplasmic domain of Alzheimer's amyloid precursor protein. These observations suggest that the amino-terminal domain regulates PI binding to APP and that the carboxyl-terminal domain containing PDZ motifs is essential to modulate APP processing. Because expression of the human X11L gene is specific to brain, the present observations should contribute to shedding light on the molecular mechanism of APP processing in Alzheimer's disease.     

Physical interaction of ApoE with amyloid precursor protein independent of the amyloid Abeta region in vitro

Variation at the APOE gene locus has been shown to affect the risk for Alzheimer's disease. To gain deeper insight into the postulated apoE-mediated amyloid formation, we have characterized the three common apoE isoforms (apoE2, apoE3, and apoE4) regarding their binding to amyloid precursor protein (APP). We employed the yeast two-hybrid system and co-immunoprecipitation experiments in cell culture supernatants of COS-1 cells, ectopically expressing apoE isoforms and APP751 holoprotein or a COOH-terminal Abeta deletion mutant protein, designated APPtrunc. We found that all three apoE isoforms were able to bind APP751 holoprotein in an Abeta-independent fashion. The interacting domains could be mapped to the NH2 termini of APP (amino acids 1-207) and apoE (amino acids 1-191). As a functional consequence of this novel APP751 ectodomain-mediated apoE binding, the secretion of soluble APP751 is differentially affected by distinct apoE isoforms in vitro, suggesting a new "chaperon-like" mechanism by which apoE isoforms may modulate APP metabolism and consequently the risk for Alzheimer's disease.     

PAT1, a microtubule-interacting protein, recognizes the basolateral sorting signal of amyloid precursor protein

In epithelial cells, sorting of membrane proteins to the basolateral surface depends on the presence of a basolateral sorting signal (BaSS) in their cytoplasmic domain. Amyloid precursor protein (APP), a basolateral protein implicated in the pathogenesis of Alzheimer's disease, contains a tyrosine-based BaSS, and mutation of the tyrosine residue results in nonpolarized transport of APP. Here we report identification of a protein, termed PAT1 (protein interacting with APP tail 1), that interacts with the APP-BaSS but binds poorly when the critical tyrosine is mutated and does not bind the tyrosine-based endocytic signal of APP. PAT1 shows homology to kinesin light chain, which is a component of the plus-end directed microtubule-based motor involved in transporting membrane proteins to the basolateral surface. PAT1, a cytoplasmic protein, associates with membranes, cofractionates with APP-containing vesicles, and binds microtubules in a nucleotide-sensitive manner. Cotransfection of PAT1 with a reporter protein shows that PAT1 is functionally linked with intracellular transport of APP. We propose that PAT1 is involved in the translocation of APP along microtubules toward the cell surface.     

Inhibition of platelet activation by the Alzheimer's disease amyloid precursor protein

The amyloid precursor protein (APP) of Alzheimer's disease is abundantly expressed in the platelet alpha-granule where its role remains unclear. This study describes a novel function for APP in regulating human platelet activation. Preincubation of platelet-rich plasma with recombinant secreted APP (sAPP) isoforms dose-dependently inhibited platelet aggregation and secretion induced by ADP or adrenaline. Similarly, sAPP potently inhibited low-dose thrombin-induced activation in washed platelet suspensions, indicating that the activity does not require plasma cofactors. There were no functional differences between sAPP forms with or without the Kunitz protease inhibitor domain or derived from either alpha- or beta-secretase cleavage. In fact, the N-terminal cysteine-rich region of APP (residues 18-194) was as effective as the entire sAPP region in the inhibition of platelet activation. The inhibitory activity of sAPP correlated with a significant reduction in the agonist-induced production of the arachidonic acid (AA) metabolites thromboxane B2 and prostaglandin E2. However, sAPP did not affect AA-induced platelet aggregation or secretion, indicating the enzymatic conversion of AA was not inhibited. The addition of a threshold dose of AA reversed the sAPP-inhibition of agonist-induced platelet activation. This suggests that sAPP decreases the availability of free AA, although the mechanism is not yet known. These data provide evidence that the release of sAPP upon platelet degranulation may result in negative feedback regulation during platelet activation.     

Novel domain-specific actions of amyloid precursor protein on developing synapses

The effect of the secretory form of amyloid precursor protein (sAPP) on synaptic transmission was examined by using developing neuromuscular synapses in Xenopus cell cultures. The frequency of spontaneous postsynaptic currents (SSCs) was reduced by the addition of sAPP, whereas the amplitude of impulse-evoked postsynaptic currents (ESCs) was increased by sAPP. These opposing effects on spontaneous versus evoked release were separated by using the specific domain of APP. The C-terminal fragment of sAPP (CAPP) only reduced SSC frequency and did not affect ESCs. By contrast, the N-terminal fragment of sAPP (NAPP) did not affect SSC frequency but did increase ESC amplitude. The reduction of SSC frequency by sAPP appears to be mediated by activation of potassium channels through a cGMP-dependent pathway, whereas the increase of ESC amplitude is mediated by a different pathway involving activation of protein kinase(s). These results suggest the potential role of sAPP as a modulator of synaptic activity by two specific domains.     

Increased production of amyloid precursor protein provides a substrate for caspase-3 in dying motoneurons

Biochemical and molecular mechanisms of neuronal cell death are currently an area of intense research. It is well documented that the lumbar spinal motoneurons of the chick embryo undergo a period of naturally occurring programmed cell death (PCD) requiring new gene expression and activation of caspases. To identify genes that exhibit changed expression levels in dying motoneurons, we used a PCR-based subtractive hybridization protocol to identify messages uniquely expressed in motoneurons deprived of trophic support as compared with their healthy counterparts. We report that one upregulated message in developing motoneurons undergoing cell death is the mRNA for amyloid precursor protein (APP). Increased levels of APP and beta-amyloid protein are also detected within dying motoneurons. The predicted peptide sequence of APP indicates two potential cleavage sites for caspase-3 (CPP-32), a caspase activated in dying motoneurons. When peptide inhibitors of caspase-3 are administered to motoneurons destined to undergo PCD, decreased levels of APP protein and greatly reduced beta-amyloid production are observed. Furthermore, we show that APP is cleaved by caspase-3. Our results suggest that differential gene expression results in increased levels of APP, providing a potential substrate for one of the cell death-activated caspases that may ultimately cause the demise of the cell. These results, combined with information on the toxic role of APP and its proteolytic by-product beta-amyloid, in the neurodegenerative disease Alzheimer's, suggest that events of developmental PCD may be reactivated in early stages of pathological neurodegeneration.     

Endothelial cell dysfunction in response to intracellular overexpression of amyloid precursor protein

Previous reports have shown that exposure of vascular endothelial and smooth muscle cells to exogenous amyloid beta (Abeta) peptide results in cell damage and toxicity via oxidative injury. In this study we demonstrate that overexpression of the amyloid precursor protein (APP) is toxic to bovine aortic endothelial cells but not to bovine aortic smooth muscle cells. Intracellular coexpression of the free radical scavenger proteins metallothionein or MnSOD abolished the toxic effect of APP overexpression in endothelial cells. Our results demonstrate that endothelial cells are specifically susceptible to intracellular overexpression of APP and free radical generation is the likely mechanism of cell damage due to APP overexpression.     

Altered processing of Alzheimer amyloid precursor protein in response to neuronal degeneration

In the brains of individuals with Alzheimer disease, senile plaques containing aggregates of beta-amyloid peptide, derived from the beta-amyloid precursor protein (APP), are seen in association with degenerating nerve terminals. It is not known whether the degenerating nerve terminals cause the formation of these aggregates or whether beta-amyloid peptide in the aggregates causes nerve-terminal degeneration. In the present study of rat brain, degeneration either of local neurons or of nerve terminals caused decreased levels of a neuron-enriched isoform of APP, increased levels of a glia-enriched isoform of APP, and increased levels of potentially amyloidogenic, as well as nonamyloidogenic, COOH-terminal fragments of APP. Our results demonstrate that neuronal degeneration affects APP processing and suggest that it may contribute to amyloid formation in mammalian brain.     

Proteasome inhibitors induce the association of Alzheimer's amyloid precursor protein with Hsc73

Amyloid precursor protein (APP) is a secretory membrane-bound protein that undergoes restrictive proteolysis and degradation with a short life span in the constitutive secretory pathway or in the endosomal/lysosomal compartment. The degradation machinery, including cellular trafficking and the restrictive cleavage of APP, is poorly understood. To gain further insight into the intracellular degradation mechanism of APP, we searched for effector proteins that interact with APP. We found that a cytosolic molecular chaperon, Hsc73, effectively interacts with the cytoplasmic domain of APP in the presence of proteasome inhibitors. Hsc73 binds to the cytoplasmic domain near the post-transmembrane region of APP and not to the KFERQ-related sequence, KFFEQ, at the C-terminal tail that is assumed to be the selective targeting signal for lysosomal proteolysis. The amounts of Hsc73 that bind to several APP species such as those found in pathological Familial Alzheimer's disease (FAD), Swedish, or Dutch type mutation, are almost identical, suggesting that an abnormal conformation around the secretory cleavage site or a pathological imbalance in APP processing are not irrelevant to the efficiency of Hsc73 binding.     

Overexpression of amyloid precursor protein of Alzheimer's type in brains of intoxicated children

The coincidence of neuronal stress induced by intoxication and an overexpression of amyloid precursor protein (APP) in the brains of children was examined. Brains of ten children accidentally intoxicated by poisonous mushroom were studied by means of immunohistochemical methods using monoclonal antibodies generated against different domains of APP and glial cell markers. Overexpression of APP was found in the brain neurons of all intoxicated children. Neurons were immunopositive with the antibodies generated against the middle (amyloid beta protein) domain of APP. No extracellular deposits were found in the tissue. Our results provided, for the first time, the evidence that overexpression of APP concomitant with the neuronal stress is age-independent phenomenon appearing not only in the brain of adults but in very young individuals as well.     

Increased susceptibility to ischemic brain damage in transgenic mice overexpressing the amyloid precursor protein

We studied the role of the amyloid precursor protein (APP) in ischemic brain damage using transgenic mice overexpressing APP. The middle cerebral artery (MCA) was occluded in FVB/N mice expressing APP695.SWE (Swedish mutation) and in nontransgenic littermates. Infarct volume (cubic millimeters) was assessed 24 hr later in thionin-stained brain sections. The infarct produced by MCA occlusion was enlarged in the transgenics (+32 +/- 6%; n = 12; p < 0. 05; t test). Measurement of APP by ELISA revealed that, although relatively high levels of Abeta were present in the brain of the transgenics (Abeta1-40 = 80 +/- 19 pmol/g; n = 6), there were no differences between ischemic and nonischemic hemispheres (p > 0.05). The reduction in cerebral blood flow produced by MCA occlusion at the periphery of the ischemic territory was more pronounced in APP transgenics (-42 +/- 8%; n = 9) than in controls (-20 +/- 8%; n = 9). Furthermore, the vasodilatation produced by neocortical application of the endothelium-dependent vasodilator acetylcholine (10 microM) was reduced by 82 +/- 5% (n = 8; p < 0.05) in APP transgenics. The data demonstrate that APP overexpression increases the susceptibility of the brain to ischemic injury. The effect is likely to involve the Abeta-induced disturbance in endothelium-dependent vascular reactivity that leads to more severe ischemia in regions at risk for infarction. The cerebral vascular actions of peptides deriving from APP metabolism may play a role in the pathogenic effects of APP.     

Molecular cloning of the cDNA for a human amyloid precursor protein homolog: evidence for a multigene family

Alzheimer's disease is a degenerative neurological disorder characterized by neural loss and brain lesions associated with plaques containing large amounts of the beta/A4 amyloid peptide. Molecular cloning of the cDNA for this peptide from human brain has shown it to be derived by proteolysis from a much larger precursor called the amyloid precursor protein (APP). The biological role of the precursor is unknown, but it has been shown to be transcribed in many human tissues in addition to brain. In the present report, we describe the molecular cloning from a human placental library of a full-length cDNA for a molecule closely related to APP. This novel molecule, which we have called amyloid precursor protein homolog (APPH), shares overall domain organization with APP. It is 763 amino acids in length and appears to encode a signal peptide, a large apparent extracellular domain including a Kunitz inhibitor domain, a transmembrane region, and a short cytoplasmic domain. Northern analysis indicates that it occurs in at least two molecular forms and is transcribed in human brain, heart, lung, liver, and kidney, in addition to placenta. On the basis of its extensive sequence similarity and conservation of domain structure, APPH is the nearest relative of APP yet identified in an emerging multigene family.     

The alternatively spliced Kunitz protease inhibitor domain alters amyloid beta protein precursor processing and amyloid beta protein production in cultured cells

The insoluble amyloid deposited extracellularly in the brains of patients with Alzheimer's disease (AD) is composed of amyloid beta protein, a approximately 4-kDa secreted protein that is derived from a set of large proteins collectively referred to as the amyloid beta protein precursor (betaAPP). During normal processing the betaAPP is cleaved by beta secretase, producing a large NH2-terminal secreted derivative (sAPPbeta) and a COOH-terminal fragment beginning at Abeta1, which is subsequently cleaved by gamma secretase releasing secreted Abeta. Most secreted Abeta is Abeta1-40, but approximately 10% of secreted Abeta is Abeta1-42. Alternative betaAPP cleavage by alpha secretase produces a slightly longer NH2-terminal secreted derivative (sAPPalpha) and a COOH-terminal fragment beginning at Abeta17, which is subsequently cleaved by gamma secretase releasing a approximately 3-kDa secreted form of Abeta (P3). Several of the betaAPP isoforms that are produced by alternative splicing contain a 56-amino 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 Abeta, we compared Abeta production in transfected cells expressing human KPI-containing betaAPP751 or KPI-free betaAPP695. We focused on Abetas ending at Abeta42 because these forms appear to be most relevant to AD. Using specific sandwich enzyme-linked immunosorbent assays, we analyzed full-length Abeta1-42 and total Abeta ending at Abeta42 (Abeta1-42 + P3(42)). In addition, we analyzed the large secreted derivatives produced by alpha secretase (sAPPalpha) and beta secretase (sAPPbeta). In mouse teratocarcinoma (P19) cells expressing betaAPP695 or betaAPP751, expression of the KPI-containing betaAPP751 resulted in the secretion of a lower percentage of P3(42) and sAPPalpha and a correspondingly higher percentage of Abeta1-42 and sAPPbeta. Similar results were obtained in human embryonic kidney (293) cells. These results indicate that expression of the KPI domain reduces alpha secretase cleavage so that less P3 and relatively more full-length Abeta are produced. Thus, in human brain and in animal models of AD, the amount of KPI-containing betaAPP produced may be an important factor influencing Abeta deposition.     

Modulation of secreted beta-amyloid precursor protein and amyloid beta-peptide in brain by cholesterol

The effects of dietary cholesterol on brain amyloid precursor protein (APP) processing were examined using an APP gene-targeted mouse, genetically humanized in the amyloid beta-peptide (Abeta) domain and expressing the Swedish familial Alzheimer's disease mutations. These mice express endogenous levels of APP holoprotein and abundant human Abeta. Increased dietary cholesterol led to significant reductions in brain levels of secreted APP derivatives, including sAPPalpha, sAPPbeta, Abeta1-40, and Abeta1-42, while having little to no effect on cell-associated species, including full-length APP and the COOH-terminal APP processing derivatives. The changes in levels of sAPP and Abeta in brain all were negatively correlated with serum cholesterol levels and levels of serum and brain apoE. These results demonstrate that secreted APP processing derivatives and Abeta can be modulated in the brain of an animal by diet and provide evidence that cholesterol plays a role in the modulation of APP processing in vivo. APP gene-targeted mice lacking apoE, also have high serum cholesterol levels but do not show alterations in APP processing, suggesting that effects of cholesterol on APP processing require the presence of apoE.     

The precursor molecule of a V lambda II-immunoglobulin light chain-derived amyloid fibril protein circulates precleaved

The putative precursor molecule of a human AL type amyloid fibril protein was isolated from an ultrafiltrate after hemofiltration. Subsequent separation of this protein was achieved by high performance liquid chromatography (HPLC) after reduction and carboxymethylation of the disulfide bonds. The protein was separated into several fractions which were further analyzed by automatic amino acid sequence determination. It was deduced from the sequence data that the precursor molecule is an immunoglobulin L-chain of the lambda-type. The V-region of this protein is most closely related to the proteins of subgroup II. Internal splits occurred in the molecule after lysine residues in positions 110, 129 and 179. The predominant fragment commences with either serine or alanine in position 9 and extends to a serine in position 65 of the V-region. Tryptic peptides generated from the fragments cover nearly the entire V- and C-region of the L-chain, with the exception of positions 1-8, from which no peptide has been isolated.     

A 127-kDa protein (UV-DDB) binds to the cytoplasmic domain of the Alzheimer's amyloid precursor protein

Alzheimer amyloid precursor protein (APP) is an integral membrane protein with a short cytoplasmic domain of 47 amino acids. It is hoped that identification of proteins that interact with the cytoplasmic domain will provide new insights into the physiological function of APP and, in turn, into the pathogenesis of Alzheimer's disease. To identify proteins that interact with the cytoplasmic domain of APP, we employed affinity chromatography using an immobilized synthetic peptide corresponding to residues 645-694 of APP695 and identified a protein of approximately 130 kDa in rat brain cytosol. Amino acid sequencing of the protein revealed the protein to be a rat homologue of monkey UV-DDB (UV-damaged DNA-binding protein, calculated molecular mass of 127 kDa). UV-DDB/p127 co-immunoprecipitated with APP using an anti-APP antibody from PC12 cell lysates. APP also co-immunoprecipitated with UV-DDB/p127 using an anti-UV-DDB/p127 antibody. These results indicate that UV-DDB/p127, which is present in the cytosolic fraction, forms a complex with APP through its cytoplasmic domain. In vitro binding experiments using a glutathione S-transferase-APP cytoplasmic domain fusion protein and several mutants indicated that the YENPTY motif within the APP cytoplasmic domain, which is important in the internalization of APP and amyloid beta protein secretion, may be involved in the interaction between UV-DDB/p127 and APP.     

Alzheimer amyloid protein precursor in the rat hippocampus: transport and processing through the perforant path

Amyloid deposition is a neuropathological hallmark of Alzheimer's disease. The principal component of amyloid deposits is beta amyloid peptide (Abeta), a peptide derived by proteolytic processing of the amyloid precursor protein (APP). APP is axonally transported by the fast anterograde component. Several studies have indicated that Abeta deposits occur in proximity to neuritic and synaptic profiles. Taken together, these latter observations have suggested that APP, axonally transported to nerve terminals, may be processed to Abeta at those sites. To examine the fate of APP in the CNS, we injected [35S]methionine into the rat entorhinal cortex and examined the trafficking and processing of de novo synthesized APP in the perforant pathway and at presynaptic sites in the hippocampal formation. We report that both full-length and processed APP accumulate at presynaptic terminals of entorhinal neurons. Finally, we demonstrate that at these synaptic sites, C-terminal fragments of APP containing the entire Abeta domain accumulate, suggesting that these species may represent the penultimate precursors of synaptic Abeta.