HtrA2 regulates beta-amyloid precursor protein (APP) metabolism through endoplasmic reticulum-associated degradation

Alzheimer disease-associated beta-amyloid peptide is generated from its precursor protein APP. By using the yeast two-hybrid assay, here we identified HtrA2/Omi, a stress-responsive chaperone-protease as a protein binding to the N-terminal cysteinerich region of APP. HtrA2 coimmunoprecipitates exclusively with immature APP from cell lysates as well as mouse brain extracts and degrades APP in vitro. A subpopulation of HtrA2 localizes to the cytosolic side of the endoplasmic reticulum (ER) membrane where it contributes to ER-associated degradation of APP together with the proteasome. Inhibition of the proteasome results in accumulation of retrotranslocated forms of APP and increased association of APP with HtrA2 and Derlin-1 in microsomal membranes. In cells lacking HtrA2, APP holoprotein is stabilized and accumulates in the early secretory pathway correlating with elevated levels of APP C-terminal fragments and increased Abeta secretion. Inhibition of ER-associated degradation (either HtrA2 or proteasome) promotes binding of APP to the COPII protein Sec23 suggesting enhanced trafficking of APP out of the ER. Based on these results we suggest a novel function for HtrA2 as a regulator of APP metabolism through ER-associated degradation.     

Proteinase yscE, the yeast proteasome/multicatalytic-multifunctional proteinase: mutants unravel its function in stress induced proteolysis and uncover its necessity for cell survival.

Proteinase yscE is the yeast equivalent of the proteasome, a multicatalytic-multifunctional proteinase found in higher eukaryotic cells. We have isolated three mutants affecting the proteolytic activity of proteinase yscE. The mutants show a specific reduction in the activity of the complex against peptide substrates with hydrophobic amino acids at the cleavage site and define two complementation groups, PRE1 and PRE2. The PRE1 gene was cloned and shown to be essential. The deduced amino acid sequence encoded by the PRE1 gene reveals weak, but significant similarities to proteasome subunits of other organisms. Two-dimensional gel electrophoresis identified the yeast proteasome to be composed of 14 different subunits. Comparison of these 14 subunits with the translation product obtained from PRE1 mRNA synthesized in vitro demonstrated that PRE1 encodes the 22.6 kd subunit (numbered 11) of the yeast proteasome. Diploids homozygous for pre1-1 are defective in sporulation. Strains carrying the pre1-1 mutation show enhanced sensitivity to stresses such as incorporation of the amino acid analogue canavanine into proteins or a combination of poor growth medium and elevated temperature. Under these stress conditions pre1-1 mutant cells exhibit decreased protein degradation and accumulate ubiquitin-protein conjugates.     

Capacitive calcium entry is directly attenuated by mutant presenilin-1, independent of the expression of the amyloid precursor protein

Mutant presenilin-1 (PS1) increases amyloid peptide production, attenuates capacitative calcium entry (CCE), and augments calcium release from the endoplasmatic reticulum (ER). Here we measured the intracellular free Ca(2+) concentration in hippocampal neurons from six different combinations of transgenic and gene-ablated mice to demonstrate that mutant PS1 attenuated CCE directly, independent of the expression of the amyloid precursor protein (APP). On the other hand, increased Ca(2+) release from the ER in mutant PS1 neurons, as induced by thapsigargin, was clearly dependent on the presence of APP and its processing by PS1, i.e. on the generation of the amyloid peptides and the APP C99 fragments. This observation was corroborated by the thapsigargin-induced increase in cytosolic [Ca(2+)](i) in PS1 deficient neurons, which accumulate C99 fragments due to deficient gamma-secretase activity. Moreover, co-expression of mutant APP[V717I] in PS1-deficient neurons further increased the apparent size of the ER calcium stores in parallel with increasing levels of the APP processing products. We conclude that mutant PS1 deregulates neuronal calcium homeostasis by two different actions: (i) direct attenuation of CCE at the cell-surface independent of APP; and (ii) indirect increase of ER-calcium stores via processing of APP and generation of amyloid peptides and C99 fragments.     

Expression of human beta-secretase in the mouse brain increases the steady-state level of beta-amyloid

beta-Site APP-cleaving enzyme (BACE) initiates the processing of the amyloid precursor protein (APP) leading to the generation of beta-amyloid, the main component of Alzheimer's disease senile plaques. BACE (Asp2, memapsin 2) is a type I transmembrane aspartyl protease and is responsible for the beta-secretase cleavage of APP producing different endoproteolytic fragments referred to as the carboxy-terminal C99, C89 and the soluble ectodomain sAPPbeta. Here we describe two transgenic mouse lines expressing human BACE in the brain. Overexpression of BACE augments the amyloidogenic processing of APP as demonstrated by decreased levels of full-length APP and increased levels of C99 and C89 in vivo. In mice expressing huBACE in addition to human APP wild-type or carrying the Swedish mutation, the induction of APP processing characterized by elevated C99, C89 and sAPPbeta, results in increased brain levels of beta-amyloid peptides Abeta40 and Abeta42 at steady-state.     

Dual Roles of Proteasome in the Metabolism of Presenilin 1

Abstract : Presenilin 1 (PS1) has been identified as a causative gene for most early-onset familial Alzheimer's disease. Biochemical studies revealed that PS1 exists predominantly as two processed fragments in cells and brain tissues. We prepared stably transfected cells expressing the wild-type and familial Alzheimer's disease-associated mutants of PS1 and investigated the enzyme that participates in the metabolism of PS1. After treatment of the cells with proteasome inhibitors, the full-length PS1 was significantly accumulated. The levels of N- and C-terminal fragments were also increased. The accumulation of PS1 with a deletion of exon 10, which is unable to be processed, on treatment of the transfected cells with lactacystin indicated that proteasome can degrade full-length PS1. A synthetic peptide that includes the processing region of PS1 was cleaved by 20S proteasome at the putative processing sites after Met²⁸⁸ and Glu²⁹⁹. Metabolic labeling experiments showed that the appearance of the N-terminal fragment was attenuated by the inhibitor. Finally, 28-kDa N- and 20-kDa C-terminal fragments were generated by purified PS1 in vitro. These data indicated that the proteasome pathway is involved in PS1 processing. These results demonstrate that the proteasome pathway plays dual roles in processing and degradation of PS1.     

Jun NH2-terminal kinase (JNK) interacting protein 1 (JIP1) binds the cytoplasmic domain of the Alzheimer's beta-amyloid precursor protein (APP).

The familial Alzheimer's disease gene product amyloid beta precursor protein (APP) is sequentially processed by beta- and gamma-secretases to generate the Abeta peptide. The biochemical pathway leading to Abeta formation has been extensively studied since extracellular aggregates of Abeta peptides are considered the culprit of Alzheimer's disease. Aside from its pathological relevance, the biological role of APP processing is unknown. Cleavage of APP by gamma-secretase releases, together with Abeta, a COOH-terminal APP intracellular domain, termed AID. This peptide has recently been identified in brain tissue of normal control and patients with sporadic Alzheimer's disease. We have previously shown that AID acts as a positive regulator of apoptosis. Nevertheless, the molecular mechanism by which AID regulates this process remains unknown. Hoping to gain clues about the function of APP, we used the yeast two-hybrid system to identify interaction between the AID region of APP and JNK-interacting protein-1 (JIP1). This molecular interaction is confirmed in vitro, in vivo by fluorescence resonance energy transfer (FRET), and in mouse brain lysates. These data provide a link between APP and its processing by gamma-secretase, and stress kinase signaling pathways. These pathways are known regulators of apoptosis and may be involved in the pathogenesis of Alzheimer's disease.     

Ligand-Dependent Activation of EphA4 Signaling Regulates the Proteolysis of Amyloid Precursor Protein Through a Lyn-Mediated Pathway

Alzheimer's disease is the most common dementia afflicting the elderly in modern society. This disease arises from the neurotoxicity elicited by abnormal aggregates of amyloid-β (Aβ) protein. Such aggregates form through the cleavage of amyloid precursor protein (APP) by β-secretase and the subsequent proteolysis of the APP C-terminal fragment (APP-βCTF or C99) by γ-secretase to yield Aβ and APP intracellular domain (AICD). Recent evidence suggests that C99 and AICD may exert harmful effects on cells, suggesting that the proteolytic products of APP, including Aβ, C99, and AICD, could play a pivotal role in neuronal viability. Here, we demonstrate that ligand-activated EphA4 signaling governs the proteostasis of C99, AICD, and Aβ, without significantly affecting γ-secretase activity. EphA4 induced accumulation of C99 and AICD through a Lyn-dependent pathway; activation of this pathway triggered phosphorylation of EphA4, resulting in positive feedback of C99 and AICD proteostasis. Inhibition of EphA4 by dasatinib, a receptor tyrosine kinase inhibitor, effectively suppressed C99 and AICD accumulation. Furthermore, EphA4 signaling controlled C99 and AICD proteolysis through the ubiquitin–proteasome system. In conclusion, we have identified an EphA4–Lyn pathway that is essential for the metabolism of APP and its proteolytic derivatives, thereby providing novel pharmacological targets for the development of anti-Aβ therapeutics for AD.     

Glu11 site cleavage and N-terminally truncated A beta production upon BACE overexpression

Amyloid beta peptides (A beta) are generated by the proteolytic processing of the amyloid beta precursor protein (APP). The newly identified beta-site APP-cleaving enzyme (BACE) cleaves APP at Asp1 as well as between Tyr10 and Glu11 of A beta, producing C-terminal fragments (CTFs) C99 and C89, respectively. Subsequent cleavage by gamma-secretase gives rise to A beta 1-40/42 and A beta 11-40/42. Although both full-length and A beta peptides truncated at residue 11 have been identified in amyloid plaques in the AD brain, the relative proportion of these two cleavage products produced by BACE and secreted into the medium by cultured cells is unknown. Using cell lines stably overexpressing BACE, we found that A beta 11-40 and A beta 11-42 are major A beta cleavage products generated by BACE. We further showed that BACE utilizes both full-length APP as well as C99 as substrates for the production of C89, and that A beta 11-40/42 can be generated by sequential cleavage of single APP molecules by BACE and gamma-secretase. Taken together, the abundance of A beta 11-40/42 produced by BACE suggests that their roles in AD pathogenesis may be underestimated.     

Clathrin adaptor AP-1–mediated Golgi export of amyloid precursor protein is crucial for the production of neurotoxic amyloid fragments

One of the hallmarks of Alzheimer’s disease is the accumulation of toxic amyloid-β (Aβ) peptides in extracellular plaques. The direct precursor of Aβ is the carboxyl-terminal fragment β (or C99) of the amyloid precursor protein (APP). C99 is detected at elevated levels in Alzheimer’s disease brains, and its intracellular accumulation has been linked to early neurotoxicity independently of Aβ. Despite this, the causes of increased C99 levels are poorly understood. Here, we demonstrate that APP interacts with the clathrin vesicle adaptor AP-1 (adaptor protein 1), and we map the interaction sites on both proteins. Using quantitative kinetic trafficking assays, established cell lines and primary neurons, we also show that this interaction is required for the transport of APP from the trans-Golgi network to endosomes. In addition, disrupting AP-1-mediated transport of APP alters APP processing and degradation, ultimately leading to increased C99 production and Aβ release. Our results indicate that AP-1 regulates the subcellular distribution of APP, altering its processing into neurotoxic fragments.     

GULP1 is a novel APP-interacting protein that alters APP processing

Altered production of Aβ (amyloid-β peptide), derived from the proteolytic cleavage of APP (amyloid precursor protein), is believed to be central to the pathogenesis of AD (Alzheimer's disease). Accumulating evidence reveals that APPc (APP C-terminal domain)-interacting proteins can influence APP processing. There is also evidence to suggest that APPc-interacting proteins work co-operatively and competitively to maintain normal APP functions and processing. Hence, identification of the full complement of APPc-interacting proteins is an important step for improving our understanding of APP processing. Using the yeast two-hybrid system, in the present study we identified GULP1 (engulfment adaptor protein 1) as a novel APPc-interacting protein. We found that the GULP1-APP interaction is mediated by the NPTY motif of APP and the GULP1 PTB (phosphotyrosine-binding) domain. Confocal microscopy revealed that a proportion of APP and GULP1 co-localized in neurons. In an APP-GAL4 reporter assay, we demonstrated that GULP1 altered the processing of APP. Moreover, overexpression of GULP1 enhanced the generation of APP CTFs (C-terminal fragments) and Aβ, whereas knockdown of GULP1 suppressed APP CTFs and Aβ production. The results of the present study reveal that GULP1 is a novel APP/APPc-interacting protein that influences APP processing and Aβ production.     

Fyn modulation of Dab1 effects on amyloid precursor protein and ApoE receptor 2 processing

Dab1 is an intracellular adaptor protein that interacts with amyloid precursor protein (APP) and apoE receptor 2 (apoEr2), increases their levels on the cell surface, and increases their cleavage by alpha-secretases. To investigate the mechanism underlying these alterations in processing and trafficking of APP and apoEr2, we examined the effect of Fyn, an Src family-tyrosine kinase known to interact with and phosphorylate Dab1. Co-immunoprecipitation, co-immunostaining, and fluorescence lifetime imaging demonstrated an association between Fyn and APP. Fyn induced phosphorylation of APP at Tyr-757 of the (757)YENPTY(762) motif and increased cell surface expression of APP. Overexpression of Fyn alone did not alter levels of sAPPalpha or cytoplasmic C-terminal fragments, although it significantly decreased production of Abeta. However, in the presence of Dab1, Fyn significantly increased sAPPalpha and C-terminal fragments. Fyn-induced APP phosphorylation and cell surface levels of APP were potentiated in the presence of Dab1. Fyn also induced phosphorylation of apoEr2 and increased its cell surface levels and, in the presence of Dab1, affected processing of its C-terminal fragment. In vivo studies showed that sAPPalpha was decreased in the Fyn knock-out, supporting a role for Fyn in APP processing. These data demonstrate that Fyn, due in part to its effects on Dab1, regulates the phosphorylation, trafficking, and processing of APP and apoEr2.     

Compounds that bind APP and inhibit Abeta processing in vitro suggest a novel approach to Alzheimer disease therapeutics

Extracellular deposits of aggregated amyloid-beta (Abeta) peptides are a hallmark of Alzheimer disease; thus, inhibition of Abeta production and/or aggregation is an appealing strategy to thwart the onset and progression of this disease. The release of Abeta requires processing of the amyloid precursor protein (APP) by both beta- and gamma-secretase. Using an assay that incorporates full-length recombinant APP as a substrate for beta-secretase (BACE), we have identified a series of compounds that inhibit APP processing, but do not affect the cleavage of peptide substrates by BACE1. These molecules also inhibit the processing of APP and Abeta by BACE2 and selectively inhibit the production of Abeta(42) species by gamma-secretase in assays using CTF99. The compounds bind directly to APP, likely within the Abeta domain, and therefore, unlike previously described inhibitors of the secretase enzymes, their mechanism of action is mediated through APP. These studies demonstrate that APP binding agents can affect its processing through multiple pathways, providing proof of concept for novel strategies aimed at selectively modulating Abeta production.     

DC2 and Keratinocyte-associated Protein 2 (KCP2), Subunits of the Oligosaccharyltransferase Complex,Are Regulators of the γ-Secretase-directed Processing of Amyloid Precursor Protein (APP)

The oligosaccharyltransferase complex catalyzes the transfer of oligosaccharide from a dolichol pyrophosphate donor en bloc onto a free asparagine residue of a newly synthesized nascent chain during the translocation in the endoplasmic reticulum lumen. The role of the less known oligosaccharyltransferase (OST) subunits, DC2 and KCP2, recently identified still remains to be determined. Here, we have studied DC2 and KCP2, and we have established that DC2 and KCP2 are substrate-specific, affecting amyloid precursor protein (APP), indicating that they are not core components required for N-glycosylation and OST activity per se. We show for the first time that DC2 and KCP2 depletion affects APP processing, leading to an accumulation of C-terminal fragments, both C99 and C83, and a reduction in full-length mature APP. This reduction in mature APP levels was not due to a block in secretion because the levels of sAPPα secreted into the media were unaffected. We discover that DC2 and KCP2 depletion affects only the γ-secretase complex, resulting in a reduction of the PS1 active fragment blocking Aβ production. Conversely, we show that the overexpression of DC2 and KCP2 causes an increase in the active γ-secretase complex, particularly the N-terminal fragment of PS1 that is generated by endoproteolysis, leading to a stimulation of Aβ production upon overexpression of DC2 and KCP2. Our findings reveal that components of the OST complex for the first time can interact with the γ-secretase and affect the APP processing pathway.     

Increased generation of alternatively cleaved beta-amyloid peptides in cells expressing mutants of the amyloid precursor protein defective in endocytosis

The subcellular location of the secretases processing the beta-amyloid precursor protein (APP) is not established yet. We analyzed the generation of the beta-amyloid peptide (Abeta) in human embryonic kidney 293 cell lines stably expressing wild-type and noninternalizing mutants of human APP. APP lacking the entire cytoplasmic domain or with both tyrosine residues of the motif GYENPTY mutated to alanine showed at least fivefold reduced endocytosis. In these cell lines, the production of Abeta1-40 was substantially reduced, but accompanied by the appearance of two prominent alternative Abeta peptides differing at the amino-termini. Based on antibody reactivity and mobility in high-resolution gels in comparison with defined Abeta fragments, these peptides were identified as Abeta3-40 and Abeta5-40. Notably, these alternative Abeta peptides were not generated when the APP mutants were retained in the early secretory pathway by treatment with brefeldin A. These results indicate that the alternative processing is the result of APP accumulation at the plasma membrane and provide evidence of distinct beta-secretase activities. Cleavage amino-terminal to position 1 of Abeta occurs predominantly in endosomes, whereas the processing at positions 3 or 5 takes place at the plasma membrane.     

The link between 20S proteasome activity and post-replication DNA repair in Saccharomyces cerevisiae

We have shown previously that deletion of the Saccharomyces cerevisiae UMP1 gene encoding the 20S proteasome maturase causes sensitivity to UV radiation. In the current report, we have extended this finding to show that mutations specifically compromising chymotrypsin-like or trypsin-like activity of 20S proteasome peptidases also result in increased UV sensitivity. We have also established that mutations affecting proteasome activity, namely ump1Delta, pre2-K108R and pup1-T20A, result in spontaneous and UV-induced mutator phenotypes. To elucidate the origin of these DNA repair phenotypes of the proteasomal mutants, we performed epistasis analysis, with respect to UV sensitivity, using yeast strains with the UMP1 deletion in different DNA repair backgrounds. We show that UMP1 is not epistatic to RAD23 and RAD2, which are involved in the nucleotide excision repair (NER) pathway. Instead, our results indicate that UMP1 as well as PUP1 and PRE2 (encoding catalytic subunits of 20S proteasome) belong to an epistatic group of genes functioning in post-replication DNA repair (PRR) and are hypostatic to RAD18, which, in complex with RAD6, plays a central role in PRR. We also show that UMP1 is epistatic to REV3 and RAD30, although the relationship of UMP1 with these genes is different.     

Regulation of amyloid precursor protein processing by presenilin 1 (PS1) and PS2 in PS1 knockout cells

The presenilin 1 (PS1) and PS2 proteins are thought to play roles in processing of amyloid precursor protein (APP), but the nature of this role is not fully understood. Recent studies have shown that PS1 is necessary for cleavage of APP at the gamma-secretase site. We now show that PS1 and PS2 participate in other aspects of APP processing. Fibroblasts generated from PS1 knockout mice have increased levels of the APP cleavage products, secreted APP (APPs), and APP C-terminal fragments, but lower secretion of APPs and Abeta. We have also observed that loss of PS1 prevents protein kinase C or extracellular regulated kinase from increasing production of the APP cleavage products, APPs, and APP C-terminal fragments. Transfection of PS1 -/- cells with PS1 restores the responsiveness of APP processing to protein kinase C and extracellular regulated kinase. This suggests that the changes in APP processing in PS1 -/- cells result strictly from the absence of PS1. Transfection of PS1 -/- cells with PS2 is also able to correct the deficits in APP secretion, which suggests that the PS2 also has the ability to regulate APP processing. Finally, transfection of the truncated PS2 construct, Alg3, into cells lacking PS1 increases APP C-terminal fragments. This suggests that Alg3 can interfere with the processing of APP by PS2. These data point to roles for both PS1 and PS2 in regulating APP processing and suggest that the role of these proteins also includes coupling APP to signal transduction pathways.     

Functional characterization of an alpha-factor-like Sordaria macrospora peptide pheromone and analysis of its interaction with its cognate receptor in Saccharomyces cerevisiae

The homothallic filamentous ascomycete Sordaria macrospora possesses genes which are thought to encode two pheromone precursors and two seven-transmembrane pheromone receptors. The pheromone precursor genes are termed ppg1 and ppg2. The putative products derived from the gene sequence show structural similarity to the alpha-factor precursors and a-factor precursors of the yeast Saccharomyces cerevisiae. Likewise, sequence similarity has been found between the putative products of the pheromone receptor genes pre2 and pre1 and the S. cerevisiae Ste2p alpha-factor receptor and Ste3p a-factor receptor, respectively. To investigate whether the alpha-factor-like pheromone-receptor pair of S. macrospora is functional, a heterologous yeast assay was used. Our results show that the S. macrospora alpha-factor-like pheromone precursor PPG1 is processed into an active pheromone by yeast MATalpha cells. The S. macrospora PRE2 protein was demonstrated to be a peptide pheromone receptor. In yeast MATa cells lacking the endogenous Ste2p receptor, the S. macrospora PRE2 receptor facilitated all aspects of the pheromone response. Using a synthetic peptide, we can now predict the sequence of one active form of the S. macrospora peptide pheromone. We proved that S. macrospora wild-type strains secrete an active pheromone into the culture medium and that disruption of the ppg1 gene in S. macrospora prevents pheromone production. However, loss of the ppg1 gene does not affect vegetative growth or fertility. Finally, we established the yeast assay as an easy and useful system for analyzing pheromone production in developmental mutants of S. macrospora.     

Structural studies of the transmembrane C-terminal domain of the amyloid precursor protein (APP): does APP function as a cholesterol sensor?

The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-beta (Abeta) peptides or to non-amyloidogenic fragments. Here, we report the first structural study of C99, the 99-residue transmembrane C-terminal domain of APP liberated by beta-secretase cleavage. We also show that cholesterol, an agent that promotes the amyloidogenic pathway, specifically binds to this protein. C99 was purified into model membranes where it was observed to homodimerize. NMR data show that the transmembrane domain of C99 is an alpha-helix that is flanked on both sides by mostly disordered extramembrane domains, with two exceptions. First, there is a short extracellular surface-associated helix located just after the site of alpha-secretase cleavage that helps to organize the connecting loop to the transmembrane domain, which is known to be essential for Abeta production. Second, there is a surface-associated helix located at the cytosolic C-terminus, adjacent to the YENPTY motif that plays critical roles in APP trafficking and protein-protein interactions. Cholesterol was seen to participate in saturable interactions with C99 that are centered at the critical loop connecting the extracellular helix to the transmembrane domain. Binding of cholesterol to C99 and, most likely, to APP may be critical for the trafficking of these proteins to cholesterol-rich membrane domains, which leads to cleavage by beta- and gamma-secretase and resulting amyloid-beta production. It is proposed that APP may serve as a cellular cholesterol sensor that is linked to mechanisms for suppressing cellular cholesterol uptake.     

ADAM10 is the physiologically relevant, constitutive ��-secretase of the amyloid precursor protein in primary neurons

The amyloid precursor protein (APP) undergoes constitutive shedding by a protease activity called α‐secretase. This is considered an important mechanism preventing the generation of the Alzheimer's disease amyloid‐β peptide (Aβ). α‐Secretase appears to be a metalloprotease of the ADAM family, but its identity remains to be established. Using a novel α‐secretase‐cleavage site‐specific antibody, we found that RNAi‐mediated knockdown of ADAM10, but surprisingly not of ADAM9 or 17, completely suppressed APP α‐secretase cleavage in different cell lines and in primary murine neurons. Other proteases were not able to compensate for this loss of α‐cleavage. This finding was further confirmed by mass‐spectrometric detection of APP‐cleavage fragments. Surprisingly, in different cell lines, the reduction of α‐secretase cleavage was not paralleled by a corresponding increase in the Aβ‐generating β‐secretase cleavage, revealing that both proteases do not always compete for APP as a substrate. Instead, our data suggest a novel pathway for APP processing, in which ADAM10 can partially compete with γ‐secretase for the cleavage of a C‐terminal APP fragment generated by β‐secretase. We conclude that ADAM10 is the physiologically relevant, constitutive α‐secretase of APP.