Amyloid Precursor Protein Metabolism in Primary Cell Cultures of Neurons, Astrocytes, and Microglia

Abstract: Amyloid precursor protein (APP) gives rise by proteolytic processing to the amyloid β peptide (Aβ) found abundantly in cerebral senile plaques of individuals with Alzheimer's disease. APP is highly expressed in the brain. To assess the source of cerebral Aβ, the metabolism of APP was investigated in the major cell types of the newborn rat cerebral cortex by pulse/chase labeling and immunoprecipitation of the APP and APP metabolic fragments. We describe a novel C-terminally truncated APP isoform that appears to be made only in neurons. The synthesis, degradation, and metabolism of APP were quantified by phosphorimaging in neurons, astrocytes, and microglia. The results show that although little APP is metabolized through the amyloidogenic pathways in each of the three cultures, neurons appear to generate more Aβ than astrocytes or microglia.     

Neurotoxicity of a Carboxyl-Terminal Fragment of the Alzheimer's Amyloid Precursor Protein

Abstract: We have previously shown that a recombinant carboxyl-terminal 105-amino-acid fragment (CT105) of the amyloid precursor protein (APP) induced strong non-selective inward currents in Xenopus oocytes. Here we investigated the toxic effect of CT105 peptide on the cultured mammalian cells. The CT105 peptide induced a significant lactate dehydrogenase (LDH) release from cultured rat cortical neurons and PC12 cells in a concentration (from 10 µ M )- and time (from 48 h)-dependent manner. The toxic effect of CT105 was more potent than that of any fragments of amyloid β protein (Aβ). However, CT105 peptide did not affect the viability of U251 human glioblastoma cells. In contrast to CT105, Aβ increased LDH release only slightly even at 50 µ M but significantly inhibited 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction at submicromolar concentrations. Among the various neuroprotective drugs tested, only cholesterol, which alters membrane fluidity, could attenuate the cytotoxicity of CT105 significantly. The CT105 peptide formed multiple self-aggregates on solubilization. Pretreatment with a sublethal concentration of CT105 did not significantly alter the susceptibility of cells to hydrogen peroxide and glutamate. Endogenous CT peptides were found not only in the cell lysates but also in the conditioned medium of PC12 cells. These results imply that CT peptide can directly attack the cell membrane probably by making pores or nonselective ion channels, whereas Aβ impairs the intracellular metabolic pathway first. Thus, it is thought that both CT and Aβ, which are formed during the processing of APP, may participate in the neuronal degeneration in Alzheimer's disease by different mechanisms.     

Processing of Amyloid Precursor Protein in Human Primary Neuron and Astrocyte Cultures

Abstract: Increased production of amyloid β peptide (Aβ) is highly suspected to play a major role in Alzheimer's disease (AD) pathogenesis. Because Aβ deposits in AD senile plaques appear uniquely in the brain and are fairly restricted to humans, we assessed amyloid precursor protein (APP) metabolism in primary cultures of the cell types associated with AD senile plaques: neurons, astrocytes, and microglia. We find that neurons secrete 40% of newly synthesized APP, whereas glia secrete only 10%. Neuronal and astrocytic APP processing generates five C-terminal fragments similar to those observed in human adult brain, of which the most amyloidogenic higher-molecular-weight fragments are more abundant. The level of amyloidogenic 4-kDa Aβ exceeds that of nonamyloidogenic 3-kDa Aβ in both neurons and astrocytes. In contrast, microglia make more of the smallest C-terminal fragment and no detectable Aβ. We conclude that human neurons and astrocytes generate higher levels of amyloidogenic fragments than microglia and favor amyloidogenic processing compared with previously studied culture systems. Therefore, we propose that the higher amyloidogenic processing of APP in neurons and astrocytes, combined with the extended lifespan of individuals, likely promotes AD pathology in aging humans.     

Pea,Chickpea and Lentil Protein Isolates: Physicochemical Characterization and Emulsifying Properties

This work is focused on physicochemical and emulsifying properties of pea (PP), chickpea (CP) and lentil (LP) proteins. We evaluated the molecular weight distributions, surface net charge, free sulfhydryl group (SH) and disulfide bond (SS) contents, protein solubility and thermal stability of the protein isolates. Their emulsifying properties (droplet size distribution, flocculation, coalescence and creaming) were also determined as function of pH values. The three protein isolates exhibit similar physicochemical properties, including good solubility and high thermal stability despite a high degree of denaturation. In addition, we analysed the influence of pH on stability of oil-in-water (O/W; 10 wt%/90 wt%) emulsions stabilized by the legume protein isolates. Concerning emulsifying ability and stability, the most unfavourable results for all three protein isolates relate to their isoelectric point (pI?=?4.5). A significant improvement in emulsion stability takes place as the pH value departs from the pI. Overall, this study indicates that pea, chickpea and lentil proteins have great potential as food emulsifiers.     

Regulation of Amyloid Precursor Protein Cleavage

Abstract : Multiple lines of evidence suggest that increased production and/or deposition of the β-amyloid peptide, derived from the amyloid precursor protein, contributes to Alzheimer's disease. A growing list of neuro-transmitters, 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.     

Isolation and Characterization of Biotin Carboxylase from Pea Chloroplasts

Pea (Pisum sativum L.) leaf acetyl-coenzyme A carboxylase (ACCase) exists as two structurally different forms: a major, chloroplastic, dissociable form and a minor, multifunctional enzyme form located in the leaf epidermis. The dissociable form is able to carboxylate free D-biotin as an alternate substrate in place of the natural substrate, biotin carboxyl carrier protein. Here we report the purification of the biotin carboxylase component of the chloroplastic pea leaf ACCase. The purified enzyme, free from carboxyltransferase activity, is composed of two firmly bound polypeptides, one of which (38 kD) is biotinylated. In contrast to bacterial biotin carboxylase, which retains full activity upon removal of the biotin carboxyl carrier component, attempts to dissociate the two subunits of the plant complex led to a complete loss of biotin carboxylase activity. Steady-state kinetic studies of the biotin carboxylase reaction reveal that addition of all substrates on the enzyme is sequential and that no product release is possible until all three substrates (MgATP, D-biotin, bicarbonate) are bound to the enzyme and all chemical processes at the active site are completed. In agreement with this mechanism, bicarbonate-dependent ATP hydrolysis by the enzyme is found to be strictly dependent on the presence of exogenous D-biotin in the reaction medium.     

An Etiological Role of Amyloidogenic Carboxyl-Terminal Fragments of the β-Amyloid Precursor Protein in Alzheimer's Disease

Abstract: Amyloid β protein (Aβ), 39–43 amino acids long, is the principal constituent of the extracellular amyloid deposits in brain that are characteristic of Alzheimer's disease (AD). Several lines of evidence indicate that Aβ may play an important role in the pathogenesis of AD. However, there are several discrepancies between the production of Aβ and the development of the disease. Thus, Aβ may not be the sole active fragment of β-amyloid precursor protein (βAPP) in the neurotoxicity associated with AD. Consequently, the possible effects of other cleaved products of βAPP need to be explored. The recent concentration on other potentially amyloidogenic products of βAPP has produced interesting candidates, the most promising of which are the amyloidogenic carboxyl-terminal (CT) fragments of βAPP. This review discusses a possible etiological role of CT fragments of βAPP in AD.     

C-Terminal Protein Characterization by Mass Spectrometry: Isolation of C-Terminal Fragments from Cyanogen Bromide-Cleaved Protein

A sample preparation method for protein C-terminal peptide isolation from cyanogen bromide (CNBr) digests has been developed. In this strategy, the analyte was reduced and carboxyamidomethylated, followed by CNBr cleavage in a one-pot reaction scheme. The digest was then adsorbed on ZipTip_C18 pipette tips for conjugation of the homoserine lactone-terminated peptides with 2,2′-dithiobis (ethylamine) dihydrochloride, followed by reductive release of 2-aminoethanethiol from the derivatives. The thiol-functionalized internal and N-terminal peptides were scavenged on activated thiol sepharose, leaving the C-terminal peptide in the flow-through fraction. The use of reversed-phase supports as a venue for peptide derivatization enabled facile optimization of the individual reaction steps for throughput and completeness of reaction. Reagents were replaced directly on the support, allowing the reactions to proceed at minimal sample loss. By this sequence of solid-phase reactions, the C-terminal peptide could be recognized uniquely in mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-level amounts of a whole, intact model protein. The C-terminal fragments were retrieved selectively and efficiently from the affinity support. The use of covalent chromatography for C-terminal peptide purification enabled recovery of the depleted material for further chemical and/or enzymatic manipulation. The sample preparation method provides for robustness and simplicity of operation and is anticipated to be expanded to gel-separated proteins and in a scaled-up format to high-throughput protein profiling in complex biological mixtures.     

豌豆核酮糖1,5-二磷酸羧化酶/加氧酶亚基结合蛋白的纯化及特性

用~(35)S-Met在照光下与豌豆完整叶绿体保温,显示新合成的标记的RubisCO大亚基与结合蛋白形成一复合物,经ATP处理后解离为结合蛋白亚基,同时释放出的标记的RubisCO大亚基参与了RubisCO的装配。豌豆叶片提取液经热处理,硫酸铵分部,DEAE-Sepharose fast flow和Sephacryl S-300柱层析在ND-PAGE,SDS-PAGE上显示为一条带,估计纯度达90％以上,得率比以前报道的高12倍。纯化的结合蛋白表面巯基数经测定为12±1个,总巯基数为36±1个。远紫外CD光谱具有典型的α-螺旋结构的光谱特性,α-螺旋度为39％。此外,以纯化的豌豆结合蛋白制备了多克隆抗体。琼脂糖双扩散实验显示,结合蛋白的抗体与结合蛋白产生一条沉淀线,而与豌豆的RubisCO无沉淀反应,这表明所得到的抗体是高度专一的。     

hFE65L Influences Amyloid Precursor Protein Maturation and Secretion

The amyloid precursor protein (APP) is processed in the secretory and endocytic pathways, where both the neuroprotective alpha-secretase-derived secreted APP (APPs alpha) and the Alzheimer's disease-associated beta-amyloid peptide are generated. All three members of the FE65 protein family bind the cytoplasmic domain of APP, which contains two sorting signals, YTS and YENPTY. We show here that binding of APP to the C-terminal phosphotyrosine interaction domain of hFE65L requires an intact YENPTY clathrin-coated pit internalization sequence. To study the effects of the hFE65L/APP interaction on APP trafficking and processing, we performed pulse/chase experiments and examined APP maturation and secretion in an H4 neuroglioma cell line inducible for expression of the hFE65L protein. Pulse/chase analysis of endogenous APP in these cells showed that the ratio of mature to total cellular APP increased after the induction of hFE65L. We also observed a three-fold increase in the amount of APPs alpha recovered from conditioned media of cells overexpressing hFE65L compared with uninduced controls. The effect of hFE65L on the levels of APPs alpha secreted is due neither to a simple increase in the steady-state levels of APP nor to activation of the protein kinase C-regulated APP secretion pathway. We conclude that the effect of hFE65L on APP processing is due to altered trafficking of APP as it transits through the secretory pathway.     

Human Brain βA4 Amyloid Protein Precursor of Alzheimer''s Disease: Purification and Partial Characterization

The major component of the amyloid deposition that characterizes Alzheimer's disease is the 4-kDa beta A4 protein, which is derived from a much larger amyloid protein precursor (APP). A procedure for the complete purification of APP from human brain is described. The same amino terminal sequence of APP was found in two patients with Alzheimer's disease and one control subject. Two major forms of APP were identified in human brain with apparent molecular masses of 100-110 kDa and 120-130 kDa. Soluble and membrane fractions of brain contained nearly equal amounts of APP in both humans and rats. Immunoprecipitation with carboxyl terminus-directed antibodies indicates that the soluble forms of APP are truncated. Carboxyl terminus truncation of membrane-associated forms of human brain APP was also found to occur during postmortem autolysis. The availability of purified human brain APP will facilitate the investigation of its normal function and the events that lead to its abnormal cleavage in patients with Alzheimer's disease.     

Effect of Ischemic Neuronal Insults on Amyloid Precursor Protein Processing

The nature of the association between ischemic stroke and Alzheimer’s disease (AD) at the cellular and molecular level is still unknown. We evaluated the effect of ischemic neuronal insults on the regulation of amyloid precursor protein (APP) processing. We used an in vitro model of cerebral ischemia (oxygen-glucose deprivation) to evaluate the effect of ischemic neuronal insults on the amyloidogenic and non-amyloidogenic pathways using human neuroblastoma cell line and primary cultured cells of transgenic mice which expressed human APP (Tg2576). Ischemic neuronal insults increased the production of Aβ in Tg2576 primary culture cells compared to controls. A disintegrin and metalloprotease 10 (ADAM 10) was markedly increased in early stage of ischemic insults, which was followed by decreased level of ADAM 10 expression in later stage. The protein and mRNA expression of β-site cleavage enzyme (BACE) and BACE activity was not significantly different between the group of ischemic insults and control. By contrast, the activity of γ-secretase was significantly increased after 4 h of ischemic insults, as compared to controls. The present study showed that the ischemic neuronal insults increased the production of Aβ by influencing APP metabolism, which may link the role of ischemic insults to the pathogenesis of AD.     

Iron Levels Modulate α-Secretase Cleavage of Amyloid Precursor Protein

Abstract: The amyloid precursor protein (APP) is a membrane-spanning glycoprotein that is the source of βA4 peptides, which aggregate in Alzheimer's disease to form senile plaques. APP is cleaved within the βA4 sequence to release a soluble N-terminal derivative (APP_sol), which has a wide range of trophic and protective functions. In the current study we have examined the hypothesis that iron availability may modulate expression or processing of APP, whose mRNA contains, based on sequence homology, a putative iron response element (IRE). Radiolabeled APP and its catabolites were precipitated from lysates and conditioned medium of stably transfected HEK 293 cells using antibodies selective for C-terminal, βA4, and N-terminal domains. The relative abundance of the different APP catabolites under different conditions of iron availability was determined by quantitative densitometry after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data show a specific effect on the production of APP_sol. Using standard conditions previously established for IRE studies, it was found that iron chelation reduces APP_sol production, whereas iron level elevation augments it. No changes were observed in levels of immature and mature APP holoprotein or in the C-terminal α-secretase derivative C83, βA4, and p3 peptides. The specificity for modulatory changes in APP_sol suggests that iron acts at the level of α-secretase activity. In addition to its modulatory effects, iron at very high levels was found to inhibit maturation of APP and production of its downstream catabolites without blocking formation of immature APP. The data establish a potential physiological role for iron in controlling the processing of APP. If APP_sol were to function trophically, as suggested by other studies, the current conclusion suggests that changes in iron and iron-regulating proteins in Alzheimer's disease could contribute to neuronal degeneration by decreasing the production of APP_sol.     

The Level of Expression of Thioredoxin is Linked to Fundamental Properties and Applications of Wheat Seeds

Work with cereals （barley and wheat） and a legume （Medicago truncatula） has established thioredoxin h （Trx h） as a central regulatory protein of seeds. Trx h acts by reducing disulfide （S-S） groups of diverse seed proteins （storage proteins, enzymes, and enzyme inhibitors）, thereby facilitating germination. Early in vitro protein studies were complemented with experiments in which barley seeds with Trx h overexpressed in the endosperm showed accelerated germination and early or enhanced expression of associated enzymes （α-amylase and pullulanase）. The current study extends the transgenic work to wheat. Two approaches were followed to alter the expression of Trx h genes in the endosperm： （1） a hordein promoter and its protein body targeting sequence led to overexpression of Trx hS, and （2） an antisense construct of Trx h9 resulted in cytosolic underexpression of that gene （Arabidopsis designation）. Underexpression of Trx h9 led to effects opposite to those observed for overexpression Trx h5 in barley--retardation of germination and delayed or reduced expression of associated enzymes. Similar enzyme changes were observed in developing seeds. The wheat lines with underexpressed Trx showed delayed preharvest sprouting when grown in the greenhouse or field without a decrease in final yield. Wheat with overexpressed Trx h5 showed changes commensurate with earlier in vitro work： increased solubility of disulfide proteins and lower aUergenicity of the gliadin fraction. The results are further evidence that the level of Trx h in cereal endosperm determines fundamental properties as well as potential applications of the seed.     

Intracellular Cyclic AMP Inhibits Constitutive and Phorbol Ester-Stimulated Secretory Cleavage of Amyloid Precursor Protein

Abstract: α-Secretase cleaves the full-length Alzheimer's amyloid precursor protein (APP) within the amyloid β peptide sequence, thus precluding amyloid formation. The resultant soluble truncated APP is constitutively secreted. This nonamyloidogenic processing of APP is increased on stimulation of the phospholipase C/protein kinase C pathway by phorbol esters. Here we used C6 cells transfected with APP₇₅₁ to examine whether the α-secretase cleavage is regulated by the adenylate cyclase signal transduction pathway. Forskolin, an activator of adenylate cyclase, inhibited both the constitutive and phorbol ester-stimulated secretion of nexin II (NXII), the secreted product of the α-secretase cleavage of APP₇₅₁. At 1 µ M , forskolin inhibited secretion of NXII by ∼50% without affecting either the intracellular levels of total APP or the secretion of secretory alkaline phosphatase. In contrast, 1,9-dideoxyforskolin, an inactive analogue of forskolin, did not affect secretion of NXII. These results indicated that forskolin specifically inhibited the α-secretase cleavage of APP₇₅₁. Forskolin treatment increased the intracellular concentration of cyclic AMP (cAMP), suggesting that the forskolin effects on APP cleavage may be mediated by cAMP. In support of this suggestion, both dibutyryl cAMP, a cAMP analogue, and isoproterenol, an activator of adenylate cyclase, also inhibited secretion of NXII. These data indicate that forskolin inhibition of the nonamyloidogenic cleavage of APP is mediated by the second messenger cAMP, which together with the protein kinase C signal transduction pathway modulates the secretory cleavage of APP.     

A 127-kDa Protein (UV-DDB) Binds to the Cytoplasmic Domain of the Alzheimer's Amyloid Precursor Protein

Abstract : 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 ~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 β protein secretion, may be involved in the interaction between UV-DDB/p127 and APP.     

拟南芥中一个未知功能蛋白的叶绿体亚细胞定位研究

生物信息学分析表明,模式植物拟南芥叶绿体中含有大约4000多种蛋白质,目前只分离得到1000多种,其他预测的叶绿体蛋白的实验验证对叶绿体功能研究有重要意义。本文对一个预测的叶绿体未知功能蛋白AT5G48790进行了亚细胞定位研究。我们克隆了该基因5端长178bp的DNA片段,与绿色荧光蛋白（GFP）基因构建重组载体pMON530-cTP-GFP。转基因植株通过激光共聚焦显微镜观察,GFP只在叶绿体中特异表达。实验结果表明,AT5G48790的确为叶绿体蛋白。本实验方法也可用于其他预测的蛋白质的实验验证。     

Analysis of cDNA Clones Encoding the Entire Ferredoxin I Precursor Polypeptide from Spinach

We report the isolation and characterization of a recombinant cDNA phage from a lambda gt11 expression library made from polyadenylated spinach RNA that encodes the entire precursor polypeptide of ferredoxin I. The deduced sequence predicts a molecular mass of 10.5 kDa (97 amino acid residues) for the mature protein and a transit peptide of 50 residues (5.2 kDa). In vitro synthesized ferredoxin precursor was used for import experiments with isolated unbroken spinach chloroplasts. The polypeptide was correctly directed to the organelle stroma and processed to the size of the mature protein. Northern analysis indicates a mRNA size of ca. 850 nucleotides which is close to the size of the cDNA insert (ca. 700 bp).     

Biochemical Properties of Mitochondrial Membrane from Dry Pea Seeds and Changes in the Properties during Imbibition

An attempt to isolate intact mitochondria from dry pea seeds (Pisum sativum var. Alaska) ended in failure. Cytochrome oxidase in crude mitochondrial fraction from dry seeds was separated into three fractions by sucrose density gradient centrifugation. Two of the fractions contained malate dehydrogenase, whereas the other did not. Equilibrium centrifugation of mitochondrial membrane on sucrose gradients revealed that the membrane from the fraction without malate dehydrogenase was lighter than that from the others. Differences were observed in relative content of phospholipid to protein and in polypeptide composition analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis among the membranes from three fractions and imbibed cotyledons. Membrane from the fraction without malate dehydrogenase was rich in phospholipid and lacking in polypeptides with relatively high molecular weights as compared with that from others. During imbibition, the fraction without malate dehydrogenase and one of the other two disappeared rapidly after a lag phase lasting for at least 1 hour. Concomitantly, active and stable mitochondria increased in the cotyledons. The results were interpreted to indicate that there were at least three types of mitochondria in dry seeds, the membranes of which differed in their biochemical properties, and that the mitochondria became active and stable through assembly of protein into the membranes during imbibition.     

Isolation and Biochemical Characterization of Two Novel Metagenome-Derived Esterases

The metagenomes of uncultured microbial communities are rich sources for novel biocatalysts. In this study, esterase EstA3 was derived from a drinking water metagenome, and esterase EstCE1 was derived from a soil metagenome. Both esterases are approximately 380 amino acids in size and show similarity to β-lactamases, indicating that they belong to family VIII of the lipases/esterases. EstA3 had a temperature optimum at 50°C and a pH optimum at pH 9.0. It was remarkably active and very stable in the presence of solvents and over a wide temperature and pH range. It is active in a multimeric form and displayed a high level of activity against a wide range of substrates including one secondary ester, 7-[3-octylcarboxy-(3-hydroxy-3-methyl-butyloxy)]-coumarin, which is normally unreactive. EstCE1 was active in the monomeric form and had a temperature optimum at 47°C and a pH optimum at pH 10. It exhibited the same level of stability as EstA3 over wide temperature and pH ranges and in the presence of dimethyl sulfoxide, isopropanol, and methanol. EstCE1 was highly enantioselective for (+)-menthylacetate. These enzymes display remarkable characteristics that cannot be related to the original environment from which they were derived. The high level of stability of these enzymes together with their unique substrate specificities make them highly useful for biotechnological applications.