Rat Brain Glyceraldehyde-3-Phosphate Dehydrogenase Interacts with the Recombinant Cytoplasmic Domain of Alzheimer's β-Amyloid Precursor Protein

Abstract: Abundant senile plaques are a histological hallmark in the brain of Alzheimer's disease patients. Such plaques consist of, among many other constituents, aggregated βA4 amyloid peptide. This peptide is derived from an amyloid precursor protein (APP) by irregular proteolytic processing and is considered to be involved in the development of Alzheimer's disease. To study possible interactions of brain proteins with 0A4 amyloid or other fragments of APP, βA4 amyloid and βA4 amyloid extended to the C-terminus of APP were recombinantly produced as fusion proteins termed "Amy" and "AmyC," respectively. Using Amy and AmyC affinity chromatography, a 35-kDa protein from rat brain was isolated that bound tightly to AmyC but not to Amy, thus indicating an interaction of the protein with the C-terminus of APP. This 35-kDa protein was identified as the glycolytic enzyme gIyceraldehyde-3-phosphate dehydrogenase (GAPDH). Binding of GAPDH to AmyC but not to Amy was confirmed by gel filtration. Although AmyC slightly reduced the V_max of GAPDH, the same reduction was observed in the presence of Amy. These findings suggest that the interaction of the cytoplasmic domain of APP with GAPDH is unlikely to influence directly the rate of glycolysis but may serve another function.     

An Alternative Secretase Cleavage Produces Soluble Alzheimer Amyloid Precursor Protein Containing a Potentially Amyloidogenic Sequence

Cell culture studies have shown that the Alzheimer amyloid precursor protein (APP) is secreted after full-length APP is cleaved by a putative secretase at the Lys16-Leu17 bond (secretase cleavage I) of the amyloid peptide sequence. Because this cleavage event is incompatible with amyloid production, it has been assumed that secreted APP cannot serve as a precursor of the amyloid depositions observed in Alzheimer's disease. Here we show that in neuronally differentiated PC12 cells and human kidney 293 cell cultures a portion of the secreted extracytoplasmic APP reacted specifically with both a monoclonal antibody recognizing amyloid protein residues Leu17-Val24 and a polyclonal antiserum directed against amyloid protein residues Ala21-Lys28. Furthermore, this APP failed to react with antisera recognizing the cytoplasmic domain of the full-length protein. These data indicate the presence of an alternative APP secretase cleavage site (secretase cleavage II), C-terminal to the predominant secretase cleavage I. Depending on the exact location of cleavage site II, potentially amyloidogenic secreted APP species may be produced.     

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.     

Polarized transport of Alzheimer amyloid precursor protein is mediated by adaptor protein complex AP1-1B

Alzheimer amyloid precursor protein (APP) is the precursor for the Abeta peptide involved in pathogenesis of Alzheimer's disease. The soluble ectodomain fragment of APP (sAPP) functions as a growth factor for epithelial cells, suggesting an important function for APP outside neuronal tissue. Previous studies have shown that in polarized epithelial cells, APP is targeted to the basolateral domain. Tyr653 within the cytoplasmic tail of APP mediates the basolateral targeting of APP, but the sorting machinery that binds to this residue has largely remained unknown. In this study, we analyzed the role of adaptor complexes in the polarized sorting of APP. We show that the medium subunit mu1B of the epithelia-specific adaptor protein (AP)-1B binds onto the cytoplasmic tail of APP in a Tyr653-dependent way. Moreover, ectopic expression of mu1B in cells lacking AP-1B resulted in correction of apical missorting of wild-type but not Tyr653Ala APP. Basolateral secretion of sAPP was found to be independent of Tyr653. We propose a model for polarized targeting of APP according to which sorting of APP to basolateral domain is dependent on binding of AP-1B on Tyr653 in basolateral endosomes. This model is in accordance with the current understanding of sorting mechanisms mediating polarized targeting of membrane proteins.     

An antibody against the alzheimer's disease amyloid precursor protein recognizes distinct conformational isoforms

The Alzheimer's disease amyloid precursor protein (APP) consists of several isoforms, which are extensively post-translationally modified and processed. A monoclonal antibody, MAbE1, was raised against a synthetic peptide from an extracellular domain that is common to all isoforms of APP. Immunoblots and immunolocalization studies on cells of neuronal and other origins demonstrated that this antibody recognized a subclass of APP isoforms when compared to a monoclonal antibody raised against a bacterial fusion protein of APP, MAb22C11. Prominent protein bands of 71 kDa and 120 kDa were only detected on immunoblots of cell lysates and no immunoreactivity was observed in protein samples obtained from cell conditioned media. Immunofluorescence labelling with MAbE1 revealed predominantly perinuclear staining of cells of neuronal and glial origin. The data suggest that this monoclonal antibody detects distinct conformational isoforms of APP present in intracellular compartments.     

Amyloid precursor-like protein 1 influences endocytosis and proteolytic processing of the amyloid precursor protein

Ectodomain shedding of the amyloid precursor protein (APP) is a key regulatory step in the generation of the Alzheimer disease amyloid beta peptide (Abeta). The molecular mechanisms underlying the control of APP shedding remain little understood but are in part dependent on the low density lipoprotein receptor-related protein (LRP), which is involved in APP endocytosis. Here, we show that the APP homolog APLP1 (amyloid precursor-like protein 1) influences APP shedding. In human embryonic kidney 293 cells expression of APLP1 strongly activated APP shedding by alpha-secretase and slightly reduced beta-secretase cleavage. As revealed by domain deletion analysis, the increase in APP shedding required the NPTY amino acid motif within the cytoplasmic domain of APLP1. This motif is conserved in APP and is essential for the endocytosis of APP and APLP1. Unrelated membrane proteins containing similar endocytic motifs did not affect APP shedding, showing that the increase in APP shedding was specific to APLP1. In LRP-deficient cells APLP1 no longer induced APP shedding, suggesting that in wild-type cells APLP1 interferes with the LRP-dependent endocytosis of APP and there by increases APP alpha-cleavage. In fact, an antibody uptake assay revealed that expression of APLP1 reduced the rate of APP endocytosis. In summary, our study provides a novel mechanism for APP shedding, in which APLP1 affects the endocytosis of APP and makes more APP available for alpha-secretase cleavage.     

Apoptotic cell death influences the signaling activity of the amyloid precursor protein through ShcA and Grb2 adaptor proteins in neuroblastoma SH-SY5Y cells

The amyloid precursor protein (APP) is an ubiquitous receptor-like molecule involved in the pathogenesis of Alzheimer's disease (AD). APP and some of its C-terminal proteolytic fragments (CTFs) have been shown to be phosphorylated and to interact with cytosolic phosphotyrosine binding (PTB) domain containing proteins involved in cell signaling and vesicular transport. Among others, the interaction between tyrosine-phosphorylated CTFs and ShcA-Grb2 adaptors is highly enhanced in AD brain. Here we have identified in SH-SY5Y neuroblastoma cells an interaction between APP holoprotein and the adaptor Grb2. Upon activation of apoptotic cell death this interaction is rapidly degraded, APP is partially cleaved and the complex APP/Grb2 is replaced by a new complex between CTFs and ShcA that still involves Grb2. The formation of these complexes is regulated by beta-site APP-cleaving enzyme 1 and influences the phosphorylation of mitogen-activated protein kinase p44/42 extracellular signal-regulated kinase as well as the level of apoptotic death of the cells. These data suggest a dual role in cell signaling for APP and its CTFs in neuroblastoma cells, in a manner similar to that previously reported for other tyrosine kinase receptor, through a tightly regulated coupling with alternative intracellular adaptors to control the signaling of the cell.     

Antisera to an amino-terminal peptide detect the amyloid protein precursor of Alzheimer's disease and recognize senile plaques

The cerebral amyloid deposited in Alzheimer's disease (AD) contains a 4.2 kDa beta amyloid polypeptide (beta AP) that is derived from a larger beta amyloid protein precursor (beta APP). Three beta APP mRNAs encoding proteins of 695, 751, and 770 amino acids have previously been identified. In each of these, there is a single membrane-spanning domain close to the carboxyl-terminus of the beta APP, and the 42 amino acid beta AP sequence extends from within the membrane-spanning domain into the large extracellular region of the beta APP. We raised rabbit antisera to a peptide corresponding to amino acids 45-62 near the amino-terminus of the beta APP. We show that these antisera detect the beta APP by demonstrating that they (i) label a set of approximately 120 kDa membrane-associated proteins in human brain previously detected by antisera to the carboxyl-terminus of beta APP and (ii) label a set of approximately 120 kDa membrane-associated proteins that are selectively overexpressed in cells transfected with a full length beta APP expression construct. The beta APP45-62 antisera specifically stain senile plaques in AD brains. This finding, along with the previous demonstration that antisera to the carboxyl-terminus of the beta APP label senile plaques, indicates that both near amino-terminal and carboxyl-terminal domains of the beta APP are present in senile plaques and suggests that proteolytic processing of the full length beta APP molecule into insoluble amyloid fibrils occurs in a highly localized fashion at the sites of amyloid deposition in AD brains.     

Dab1 binds to Fe65 and diminishes the effect of Fe65 or LRP1 on APP processing

Fe65 and Dab1 are adaptor proteins that interact with the cytoplasmic domain of amyloid precursor protein (APP) via phosphotyrosine‐binding (PTB) domain and that affect APP processing and Aβ production. Co‐expression of Dab1 with Fe65 and APP resumed nuclear translocation of Fe65 despite of its cytoplasmic anchor, APP. The decreased amount of Fe65 bound to APP was shown in co‐immunoprecipitation assay from the cells with Dab1 which also displayed the effect on APP processing. These data suggested that Fe65 and Dab1 compete for binding to APP. Surprisingly, we found that Fe65 interacts with Dab1 via C‐terminal region of Dab1 and unphosphorylated Dab1 is capable of binding Fe65. Dab1 interacts with the low‐density lipoprotein receptor‐related protein (LRP) as well as APP through its PTB domain. Dab1 significantly decreased the amount of APP bound to LRP and the level of secreted APP and APP‐CTF in LRP expressing cells, unlike Fe65. It implies that overexpression of Dab1 diminish LRP–APP complex formation, resulting in altered APP processing. The competition for overlapped binding site among adaptor proteins may be related to the regulation mechanism of APP metabolism in various conditions. J. Cell. Biochem. 111: 508–519, 2010. © 2010 Wiley‐Liss, Inc.     

Insertion of Lysosomal Targeting Sequences to the Amyloid Precursor Protein Reduces Secretion of βA4

Abstract: The processing of the amyloid precursor protein (APP) was investigated in cells stably expressing different APP hybrid proteins. The cytoplasmic domain of APP was either deleted or replaced by the corresponding domain of the membrane protein TGN38, lamp-1, or LIMPII. The cytosolic domain of TGN38 in the APP molecule did not alter the secretion of βA4 when compared with the wild-type APP; however, APP associated with the cell surface and the nonamyloidogenic processing of APP were reduced. With the APP molecules carrying the lysosomal targeting signals of lamp-1 or LIMPII, a decrease in the secretion of βA4 was observed. Cell surface association and nonamyloidogenic processing were also impaired. This suggests increased degradation of APP and thus efficient targeting to the lysosomal system. Cells expressing the Swedish APP variant generated intracellular βA4 that accumulated after treatment with chloroquine. This effect was more dramatic with APP mutants carrying lysosomal targeting signals than with full-length APP. Our data suggest the existence of an intracellular site of βA4 generation from where βA4 is degraded rather than secreted.     

Direct visualization of the gamma secretase-generated carboxyl-terminal domain of the amyloid precursor protein: association with Fe65 and translocation to the nucleus

Amyloid-beta, the peptide that deposits as senile plaques in Alzheimer's disease, is derived from the amyloid precursor protein (APP) by a gamma secretase-mediated intramembranous cleavage. In addition to amyloid-beta, this cleavage produces a carboxyl-terminal intracellular fragment which has an unknown function. The carboxyl-terminal domain of APP interacts in the cytoplasm with an adapter protein, Fe65. We demonstrate by laser scanning confocal microscopy that a gamma secretase generated APP carboxyl-terminal domain, tagged with green fluorescent protein (GFP), translocates to the nucleus in a manner dependent upon stabilization by the adapter protein Fe65; APP which has been mutated to block interactions with Fe65 cannot be detected in the nucleus. The APP-CT domain continues to interact with Fe65 in the nucleus, as determined by both colocalization and fluorescence resonance energy transfer (FRET). Visualization of the APP-CT-Fe65 complex in the nucleus may serve as a readout for processes that modify gamma secretase release of APP-CT.     

Alzheimer's disease amyloid precursor protein is present in senile plaques and cerebrospinal fluid: immunohistochemical and biochemical characterization

The amyloid fibrils deposited in cerebral vessel walls and senile plaques in Alzheimer's disease are polymeric forms of a 4 kDa fragment produced by proteolysis of a putative precursor protein (APP). Using antibodies to several fragments of the deduced precursor, we were able to demonstrate the presence of APP in senile plaques, brain extracts and cerebrospinal fluid. Membrane-associated APP is detected as a group of 105-135 kDa proteins while soluble APP is predominantly 105 kDa, does not react with an anti C-terminal antibody, and is 10 kDa shorter than the membrane-bound APP. Amino terminal sequence of the tissue 105 kDa protein indicates that APP begins at residue 18 of the cDNA sequence. These findings imply that i) two forms of APP are detected: membrane-bound and secreted, and ii) APP can be processed in situ.     

Characterization of T-DNA Insertion Mutants and RNAi Silenced Plants of Arabidopsis thaliana UV-damaged DNA Binding Protein 2 (AtUV-DDB2)

The human UV-damaged DNA binding protein (UV-DDB), a heterodimeric protein composed of 127 kDa (UV-DDB1) and 48 kDa (UV-DDB2) subunits, has been shown to be involved in DNA repair. To elucidate the in vivo function of plant UV-DDB2, we have analyzed T-DNA insertion mutants of the Arabidopsis thaliana UV-DDB2 subunit (atuv-ddb2 mutants) and AtUV-DDB2 RNAi silenced plants (atuv-ddb2 silenced plants). atuv-ddb2 mutants and atuv-ddb2 silenced plants were both viable, suggesting that AtUV-DDB2 is not essential for survival. Interestingly, both plant types showed a dwarf phenotype, implying impaired growth of the meristem. To the best of our knowledge, this is the first occasion that a dwarf phenotype has been found to be associated with a UV-DDB2 mutation in either plants or animals. The mutants also demonstrated increased sensitivity to UV irradiation, methyl methanesulfonate and hydrogen peroxide treatment, indicating that AtUV-DDB2 is also involved in DNA repair. Our results lead us to suggest that not only does AtUV-DDB2 function in DNA repair, it also has a direct or indirect influence on cell proliferation in the plant meristem. Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries.     

Identification of Heparin-Binding Domains in the Amyloid Precursor Protein of Alzheimer's Disease by Deletion Mutagenesis and Peptide Mapping

Abstract: Recent studies have shown that the binding of the amyloid protein precursor (APP) of Alzheimer's disease to heparan sulfate proteoglycans (HSPGs) can modulate a neurite outgrowth-promoting function associated with APP. We used three different approaches to identify heparin-binding domains in APP. First, as heparin-binding domains are likely to be within highly folded regions of proteins, we analyzed the secondary structure of APP using several predictive algorithms. This analysis showed that two regions of APP₆₉₅ contain a high degree of secondary structure, and clusters of basic residues, considered mandatory for heparin binding, were found principally within these regions. To determine which domains of APP bind heparin, deletion mutants of APP₆₉₅ were prepared and analyzed for binding to a heparin affinity column. The results suggested that there must be at least two distinct heparin-binding regions in APP. To identify novel heparin-binding regions, peptides homologous to candidate heparin-binding domains were analyzed for their ability to bind heparin. These experiments suggested that APP contains at least four heparin-binding domains. The presence of more than one heparin-binding domain on APP suggests the possibility that APP may interact with more than one type of glycosaminoglycan.     

The Disabled 1 Phosphotyrosine-Binding Domain Binds to the Internalization Signals of Transmembrane Glycoproteins and to Phospholipids

Disabled gene products are important for nervous system development in drosophila and mammals. In mice, the Dab1 protein is thought to function downstream of the extracellular protein Reln during neuronal positioning. The structures of Dab proteins suggest that they mediate protein-protein or protein-membrane docking functions. Here we show that the amino-terminal phosphotyrosine-binding (PTB) domain of Dab1 binds to the transmembrane glycoproteins of the amyloid precursor protein (APP) and low-density lipoprotein receptor families and the cytoplasmic signaling protein Ship. Dab1 associates with the APP cytoplasmic domain in transfected cells and is coexpressed with APP in hippocampal neurons. Screening of a set of altered peptide sequences showed that the sequence GYXNPXY present in APP family members is an optimal binding sequence, with approximately 0.5 microM affinity. Unlike other PTB domains, the Dab1 PTB does not bind to tyrosine-phosphorylated peptide ligands. The PTB domain also binds specifically to phospholipid bilayers containing phosphatidylinositol 4P (PtdIns4P) or PtdIns4,5P2 in a manner that does not interfere with protein binding. We propose that the PTB domain permits Dab1 to bind specifically to transmembrane proteins containing an NPXY internalization signal.     

The TIP1 gene of Saccharomyces cerevisiae encodes an 80 kDa cytoplasmic protein that interacts with the cytoplasmic domain of Sec20p.

The SEC20 gene of Saccharomyces cerevisiae encodes a 50 kDa type II integral membrane glycoprotein that is required for endoplasmic reticulum (ER) to Golgi transport. Here, we have used a genetic screen, based on the lethal effect of overexpressing the cytoplasmic domain of Sec20p, to identify a novel cytosolic factor that interacts with SEC20. This factor is an 80 kDa cytoplasmic protein encoded by the TIP1 (SEC twenty interacting protein) gene. Coimmunoprecipitation and immunofluorescence using Tip1p and Sec20p or its cytoplasmic domain showed that the two proteins physically interact to form a stable complex. Like SEC20, TIP1 is required for ER to Golgi transport and depletion of Tip1p results in accumulation of an extensive network of ER plus small transport vesicles. We therefore propose that Sec20p and Tip1p act together as a functional unit in the ER to Golgi transport step.     

Suppression of APP-containing vesicle trafficking and production of β-amyloid by AID/DHHC-12 protein

The metabolism of amyloid β-protein precursor (APP) is regulated by various cytoplasmic and/or membrane-associated proteins, some of which are involved in the regulation of intracellular membrane trafficking. We found that a protein containing Asp–His–His–Cys (DHHC) domain, alcadein and APP interacting DHHC protein (AID)/DHHC-12, strongly inhibited APP metabolism, including amyloid β-protein (Aβ) generation. In cells expressing AID/DHHC-12, APP was tethered in the Golgi, and APP-containing vesicles disappeared from the cytoplasm. Although DHHC domain-containing proteins are involved in protein palmitoylation, a AID/DHHC-12 mutant of which the enzyme activity was impaired by replacing the DHHC sequence with Ala–Ala–His–Ser (AAHS) made no detectable difference in the generation and trafficking of APP-containing vesicles in the cytoplasm or the metabolism of APP. Furthermore, the mutant AID/DHHC-12 significantly increased non-amyloidogenic α-cleavage of APP along with activation of a disintegrin and metalloproteinase 17, a major α-secretase, suggesting that protein palmitoylation involved in the regulation of α-secretase activity. AID/DHHC-12 can modify APP metabolism, including Aβ generation in multiple ways by regulating the generation and/or trafficking of APP-containing vesicles from the Golgi and their entry into the late secretary pathway in an enzymatic activity-independent manner, and the α-cleavage of APP in the enzymatic activity-dependent manner.     

Regulation of amyloid beta-protein precursor by phosphorylation and protein interactions

Amyloid beta-protein precursor (APP), a type I membrane protein, is cleaved by primary alpha-or beta-secretase and secondary gamma-secretase. Cleavage of APP by beta- and gamma-secretases generates amyloid beta-protein, the main constituent of the cerebrovascular amyloid that accompanies Alzheimer disease. The generation and aggregation of amyloid beta-protein in the brain are believed to be a primary cause of Alzheimer disease pathogenesis, and indeed, early onset Alzheimer disease is genetically linked to APP and also to presenilins 1 and 2, which are components of gamma-secretase. Proteolytic cleavage of APP has been investigated as a candidate target for Alzheimer disease therapy, but the mechanisms regulating APP metabolism are still unclear. APP is a type I membrane protein with a short cytoplasmic region consisting of 47 amino acids. Recent research has elucidated the significance of the cytoplasmic region in the metabolism, trafficking, and physiological function of APP. The structure and function of the APP cytoplasmic domain can be modified by phosphorylation and through interaction with cytoplasmic proteins. This minireview summarizes a large body of recent information on the regulation of APP by phosphorylation and protein interaction, along with some of the physiological functions of APP. Recent findings regarding the regulation of APP processing contribute to the development of novel drugs and/or therapies for Alzheimer disease.