Intracellular Trafficking of Presenilin 1 Is Regulated by ��-Amyloid Precursor Protein and Phospholipase D1

Excessive accumulation of β-amyloid peptides in the brain is a major cause for the pathogenesis of Alzheimer disease. β-Amyloid is derived from β-amyloid precursor protein (APP) through sequential cleavages by β- and γ-secretases, whose enzymatic activities are tightly controlled by subcellular localization. Delineation of how intracellular trafficking of these secretases and APP is regulated is important for understanding Alzheimer disease pathogenesis. Although APP trafficking is regulated by multiple factors including presenilin 1 (PS1), a major component of the γ-secretase complex, and phospholipase D1 (PLD1), a phospholipid-modifying enzyme, regulation of intracellular trafficking of PS1/γ-secretase and β-secretase is less clear. Here we demonstrate that APP can reciprocally regulate PS1 trafficking; APP deficiency results in faster transport of PS1 from the trans-Golgi network to the cell surface and increased steady state levels of PS1 at the cell surface, which can be reversed by restoring APP levels. Restoration of APP in APP-deficient cells also reduces steady state levels of other γ-secretase components (nicastrin, APH-1, and PEN-2) and the cleavage of Notch by PS1/γ-secretase that is more highly correlated with cell surface levels of PS1 than with APP overexpression levels, supporting the notion that Notch is mainly cleaved at the cell surface. In contrast, intracellular trafficking of β-secretase (BACE1) is not regulated by APP. Moreover, we find that PLD1 also regulates PS1 trafficking and that PLD1 overexpression promotes cell surface accumulation of PS1 in an APP-independent manner. Our results clearly elucidate a physiological function of APP in regulating protein trafficking and suggest that intracellular trafficking of PS1/γ-secretase is regulated by multiple factors, including APP and PLD1.An important pathological hallmark of Alzheimer disease (AD)⁴ is the formation of senile plaques in the brains of patients. The major components of those plaques are β-amyloid peptides (Aβ), whose accumulation triggers a cascade of neurodegenerative steps ending in formation of senile plaques and intraneuronal fibrillary tangles with subsequent neuronal loss in susceptible brain regions (1, 2). Aβ is proteolytically derived from the β-amyloid precursor protein (APP) through sequential cleavages by β-secretase (BACE1), a novel membrane-bound aspartyl protease (3, 4), and by γ-secretase, a high molecular weight complex consisting of at least four components: presenilin (PS), nicastrin (NCT), anterior pharynx-defective-1 (APH-1), and presenilin enhancer-2 (PEN-2) (5, 6). APP is a type I transmembrane protein belonging to a protein family that includes APP-like protein 1 (APLP1) and 2 (APLP2) in mammals (7, 8). Full-length APP is synthesized in the endoplasmic reticulum (ER) and transported through the Golgi apparatus. Most secreted Aβ peptides are generated within the trans-Golgi network (TGN), also the major site of steady state APP in neurons (9–11). APP can be transported to the cell surface in TGN-derived secretory vesicles if not proteolyzed to Aβ or an intermediate metabolite. At the cell surface APP is either cleaved by α-secretase to produce soluble sAPPα (12) or reinternalized for endosomal/lysosomal degradation (13, 14). Aβ may also be generated in endosomal/lysosomal compartments (15, 16). In contrast to neurotoxic Aβ peptides, sAPPα possesses neuroprotective potential (17, 18). Thus, the subcellular distribution of APP and proteases that process it directly affect the ratio of sAPPα to Aβ, making delineation of the mechanisms responsible for regulating trafficking of all of these proteins relevant to AD pathogenesis.Presenilin (PS) is a critical component of the γ-secretase. Of the two mammalian PS gene homologues, PS1 and PS2, PS1 encodes the major form (PS1) in active γ-secretase (19, 20). Nascent PSs undergo endoproteolytic cleavage to generate an amino-terminal fragment (NTF) and a carboxyl-terminal fragment (CTF) to form a functional PS heterodimer (21). Based on observations that PSs possess two highly conserved aspartate residues indispensable for γ-secretase activity and that specific transition state analogue γ-secretase inhibitors bind to PS1 NTF/CTF heterodimers (5, 22), PSs are believed to be the catalytic component of the γ-secretase complex. PS assembles with three other components, NCT, APH-1, and PEN-2, to form the functional γ-secretase (5, 6). Strong evidence suggests that PS1/γ-secretase resides principally in the ER, early Golgi, TGN, endocytic and intermediate compartments, most of which (except the TGN) are not major subcellular sites for APP (23, 24). In addition to generating Aβ and cleaving APP to release the APP intracellular domain, PS1/γ-secretase cleaves other substrates such as Notch (25), cadherin (26), ErbB4 (27), and CD44 (28), releasing their respective intracellular domains. Interestingly, PS1/γ-secretase cleavage of different substrates seems to occur at different subcellular compartments; APP is mainly cleaved at the TGN and early endosome domains, whereas Notch is predominantly cleaved at the cell surface (9, 11, 29). Thus, perturbing intracellular trafficking of PS1/γ-secretase may alter interactions between PS1/γ-secretase and APP, contributing to either abnormal Aβ generation and AD pathogenesis or decreased access of PS1/γ-secretase to APP such that Aβ production is reduced. However, mechanisms regulating PS1/γ-secretase trafficking warrant further investigation.In addition to participating in γ-secretase activity, PS1 regulates intracellular trafficking of several membrane proteins, including other γ-secretase components (nicastrin, APH-1, and PEN-2) and the substrate APP (reviewed in Ref. 30). Intracellular APP trafficking is highly regulated and requires other factors such as mint family members and SorLA (2). Moreover, we recently found that phospholipase D1 (PLD1), a phospholipid-modifying enzyme that regulates membrane trafficking events, can interact with PS1, and can regulate budding of APP-containing vesicles from the TGN and delivery of APP to the cell surface (31, 32). Interestingly, Kamal et al. (33) identified an axonal membrane compartment that contains APP, BACE1, and PS1 and showed that fast anterograde axonal transport of this compartment is mediated by APP and kinesin-I, implying a traffic-regulating role for APP. Increased APP expression is also shown to decrease retrograde axonal transport of nerve growth factor (34). However, whether APP indeed regulates intracellular trafficking of proteins including BACE1 and PS1/γ-secretase requires further validation. In the present study we demonstrate that intracellular trafficking of PS1, as well as that of other γ-secretase components, but not BACE1, is regulated by APP. APP deficiency promotes cell surface delivery of PS1/γ-secretase complex and facilitates PS1/γ-secretase-mediated Notch cleavage. In addition, we find that PLD1 also regulates intracellular trafficking of PS1 through a different mechanism and more potently than APP.     

Novel Role of RanBP9 in BACE1 Processing of Amyloid Precursor Protein and Amyloid �� Peptide Generation

Accumulation of the amyloid β (Aβ) peptide derived from the proteolytic processing of amyloid precursor protein (APP) is the defining pathological hallmark of Alzheimer disease. We previously demonstrated that the C-terminal 37 amino acids of lipoprotein receptor-related protein (LRP) robustly promoted Aβ generation independent of FE65 and specifically interacted with Ran-binding protein 9 (RanBP9). In this study we found that RanBP9 strongly increased BACE1 cleavage of APP and Aβ generation. This pro-amyloidogenic activity of RanBP9 did not depend on the KPI domain or the Swedish APP mutation. In cells expressing wild type APP, RanBP9 reduced cell surface APP and accelerated APP internalization, consistent with enhanced β-secretase processing in the endocytic pathway. The N-terminal half of RanBP9 containing SPRY-LisH domains not only interacted with LRP but also with APP and BACE1. Overexpression of RanBP9 resulted in the enhancement of APP interactions with LRP and BACE1 and increased lipid raft association of APP. Importantly, knockdown of endogenous RanBP9 significantly reduced Aβ generation in Chinese hamster ovary cells and in primary neurons, demonstrating its physiological role in BACE1 cleavage of APP. These findings not only implicate RanBP9 as a novel and potent regulator of APP processing but also as a potential therapeutic target for Alzheimer disease.The major defining pathological hallmark of Alzheimer disease (AD)² is the accumulation of amyloid β protein (Aβ), a neurotoxic peptide derived from β- and γ-secretase cleavages of the amyloid precursor protein (APP). The vast majority of APP is constitutively cleaved in the middle of the Aβ sequence by α-secretase (ADAM10/TACE/ADAM17) in the non-amyloidogenic pathway, thereby abrogating the generation of an intact Aβ peptide. Alternatively, a small proportion of APP is cleaved in the amyloidogenic pathway, leading to the secretion of Aβ peptides (37–42 amino acids) via two proteolytic enzymes, β- and γ-secretase, known as BACE1 and presenilin, respectively (1).The proteolytic processing of APP to generate Aβ requires the trafficking of APP such that APP and BACE1 are brought together in close proximity for β-secretase cleavage to occur. We and others have shown that the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytosis receptor (2), binds to APP and alters its trafficking to promote Aβ generation. The loss of LRP substantially reduces Aβ release, a phenotype that is reversed when full-length (LRP-FL) or truncated LRP is transfected in LRP-deficient cells (3, 4). Specifically, LRP-CT lacking the extracellular ligand binding regions but containing the transmembrane domain and the cytoplasmic tail is capable of rescuing amyloidogenic processing of APP and Aβ release in LRP deficient cells (3). Moreover, the LRP soluble tail (LRP-ST) lacking the transmembrane domain and only containing the cytoplasmic tail of LRP is sufficient to enhance Aβ secretion (5). This activity of LRP-ST is achieved by promoting APP/BACE1 interaction (6), although the precise mechanism is unknown. Although we had hypothesized that one or more NPXY domains in LRP-ST might underlie the pro-amyloidogenic processing of APP, we recently found that the 37 C-terminal residues of LRP (LRP-C37) lacking the NPXY motif was sufficient to robustly promote Aβ production independent of FE65 (7). Because LRP-C37 likely acts by recruiting other proteins, we used the LRP-C37 region as bait in a yeast two-hybrid screen, resulting in the identification of 4 new LRP-binding proteins (7). Among these, we focused on Ran-binding protein 9 (RanBP9) in this study, which we found to play a critical role in the trafficking and processing of APP. RanBP9, also known as RanBPM, acts as a multi-modular scaffolding protein, bridging interactions between the cytoplasmic domains of a variety of membrane receptors and intracellular signaling targets. These include Axl and Sky (8), MET receptor protein-tyrosine kinase (9), and β2-integrin LFA-1 (10). Similarly, RanBP9 interacts with Plexin-A receptors to strongly inhibit axonal outgrowth (11) and functions to regulate cell morphology and adhesion (12, 13). Here we show that RanBP9 robustly promotes BACE1 processing of APP and Aβ generation.     

Alzheimer Disease A�� Production in the Absence of S-Palmitoylation-dependent Targeting of BACE1 to Lipid Rafts

Alzheimer disease β-amyloid (Aβ) peptides are generated via sequential proteolysis of amyloid precursor protein (APP) by BACE1 and γ-secretase. A subset of BACE1 localizes to cholesterol-rich membrane microdomains, termed lipid rafts. BACE1 processing in raft microdomains of cultured cells and neurons was characterized in previous studies by disrupting the integrity of lipid rafts by cholesterol depletion. These studies found either inhibition or elevation of Aβ production depending on the extent of cholesterol depletion, generating controversy. The intricate interplay between cholesterol levels, APP trafficking, and BACE1 processing is not clearly understood because cholesterol depletion has pleiotropic effects on Golgi morphology, vesicular trafficking, and membrane bulk fluidity. In this study, we used an alternate strategy to explore the function of BACE1 in membrane microdomains without altering the cellular cholesterol level. We demonstrate that BACE1 undergoes S-palmitoylation at four Cys residues at the junction of transmembrane and cytosolic domains, and Ala substitution at these four residues is sufficient to displace BACE1 from lipid rafts. Analysis of wild type and mutant BACE1 expressed in BACE1 null fibroblasts and neuroblastoma cells revealed that S-palmitoylation neither contributes to protein stability nor subcellular localization of BACE1. Surprisingly, non-raft localization of palmitoylation-deficient BACE1 did not have discernible influence on BACE1 processing of APP or secretion of Aβ. These results indicate that post-translational S-palmitoylation of BACE1 is not required for APP processing, and that BACE1 can efficiently cleave APP in both raft and non-raft microdomains.Alzheimer disease-associated β-amyloid (Aβ)³ peptides are derived from the sequential proteolysis of β-amyloid precursor protein (APP) by β- and γ-secretases. The major β-secretase is an aspartyl protease, termed BACE1 (β-site APP-cleaving enzyme 1) (1–4). BACE1 cleaves APP within the extracellular domain of APP, generating the N terminus of Aβ. In addition, BACE1 also cleaves to a lesser extent within the Aβ domain between Tyr¹⁰ and Glu¹¹ (β′-cleavage site). Processing of APP at these sites results in the shedding/secretion of the large ectodomain (sAPPβ) and generating membrane-tethered C-terminal fragments +1 and +11 (β-CTF) (5). The multimeric γ-secretase cleaves at multiple sites within the transmembrane domain of β-CTF, generating C-terminal heterogeneous Aβ peptides (ranging in length between 38 and 43 residues) that are secreted, as well as cytosolic APP intracellular domains (6). In addition to BACE1, APP can be cleaved by α-secretase within the Aβ domain between Lys¹⁶ and Leu¹⁷, releasing sAPPα and generating α-CTF. γ-Secretase cleavage of α-CTF generates N-terminal truncated Aβ, termed p3.Genetic ablation of BACE1 completely abolishes Aβ production, establishing BACE1 as the major neuronal enzyme responsible for initiating amyloidogenic processing of APP (4, 7). Interestingly, both the expression and activity of BACE1 is specifically elevated in neurons adjacent to senile plaques in brains of individuals with Alzheimer disease (8). In the past few years additional substrates of BACE1 have been identified that include APP homologues APLP1 and APLP2 (9), P-selectin glycoprotein ligand-1 (10), β-galactoside α2,6-sialyltransferase (11), low-density lipoprotein receptor-related protein (12), β-subunits of voltage-gated sodium channels (13), and neuregulin-1 (14, 15), thus extending the physiological function of BACE1 beyond Alzheimer disease pathogenesis.BACE1 is a type I transmembrane protein with a long extracellular domain harboring a catalytic domain and a short cytoplasmic tail. BACE1 is synthesized as a proenzyme, which undergoes post-translational modifications that include removal of a pro-domain by a furin-like protease, N-glycosylation, phosphorylation, S-palmitoylation, and acetylation, during the transit in the secretory pathway (16–20). In non-neuronal cells the majority of BACE1 localizes to late Golgi/TGN and endosomes at steady-state and a fraction of BACE1 also cycles between the cell surface and endosomes (21). The steady-state localization of BACE1 is consistent with the acidic pH optimum of BACE1 in vitro, and BACE1 cleavage of APP is observed in the Golgi apparatus, TGN, and endosomes (22–25). BACE1 endocytosis and recycling are mediated by the GGA family of adaptors binding to a dileucine motif (⁴⁹⁶DISLL) in its cytoplasmic tail (21, 26–31). Phosphorylation at Ser⁴⁹⁸ within this motif modulates GGA-dependent retrograde transport of BACE1 from endosomes to TGN (21, 26–31).Over the years, a functional relationship between cellular cholesterol level and Aβ production has been uncovered, raising the intriguing possibility that cholesterol levels may determine the balance between amyloidogenic and non-amyloidogenic processing of APP (32–34). Furthermore, several lines of evidence from in vitro and in vivo studies indicate that cholesterol- and sphingolipid-rich membrane microdomains, termed lipid rafts, might be the critical link between cholesterol levels and amyloidogenic processing of APP. Lipid rafts function in the trafficking of proteins in the secretory and endocytic pathways in epithelial cells and neurons, and participate in a number of important biological functions (35). BACE1 undergoes S-palmitoylation (19), a reversible post-translational modification responsible for targeting a variety of peripheral and integral membrane proteins to lipid rafts (36). Indeed, a significant fraction of BACE1 is localized in lipid raft microdomains in a cholesterol-dependent manner, and addition of glycosylphosphatidylinositol (GPI) anchor to target BACE1 exclusively to lipid rafts increases APP processing at the β-cleavage site (37, 38). Antibody-mediated co-patching of cell surface APP and BACE1 has provided further evidence for BACE1 processing of APP in raft microdomains (33, 39). Components of the γ-secretase complex also associate with detergent-resistant membrane (DRM) fractions enriched in raft markers such as caveolin, flotillin, PrP, and ganglioside GM1 (40). The above findings suggest a model whereby APP is sequentially processed by BACE1 and γ-secretase in lipid rafts.Despite the accumulating evidence, cleavage of APP by BACE1 in non-raft membrane regions cannot be unambiguously ruled out because of the paucity of full-length APP (APP FL) and BACE1 in DRM isolated from adult brain and cultured cells (41). Moreover, it was recently reported that moderate reduction of cholesterol (<25%) displaces BACE1 from raft domains, and increases BACE1 processing by promoting the membrane proximity of BACE1 and APP in non-raft domains (34). Nevertheless, this study also found that BACE1 processing of APP is inhibited with further loss of cholesterol (>35%), consistent with earlier studies (32, 33). Nevertheless, given the pleiotropic effects of cholesterol depletion on membrane properties and vesicular trafficking of secretory and endocytic proteins (42–47), unequivocal conclusions regarding BACE1 processing of APP in lipid rafts cannot be reached based on cholesterol depletion studies.In this study, we explored the function of BACE1 in lipid raft microdomains without manipulating cellular cholesterol levels. In addition to the previously reported S-palmitoylation sites (Cys⁴⁷⁸/Cys⁴⁸²/Cys⁴⁸⁵) within the cytosolic tail of BACE1 (19), we have identified a fourth site (Cys⁴⁷⁴) within the transmembrane domain of BACE1 that undergoes S-palmitoylation. A BACE1 mutant with Ala substitution of all four Cys residues (BACE1-4C/A) fails to associate with DRM in cultured cells, but is not otherwise different from wtBACE1 in terms of protein stability, maturation, or subcellular localization. Surprisingly, APP processing and Aβ generation were unaffected in cells stably expressing the BACE1-4C/A mutant. Finally, we observed an increase in the levels of APP CTFs in detergent-soluble fractions of BACE1-4C/A as compared with wtBACE1 cells. Thus, our data collectively indicate a non-obligatory role of S-palmitoylation and lipid raft localization of BACE1 in amyloidogenic processing of APP.     

Neuroprotective Secreted Amyloid Precursor Protein Acts by Disrupting Amyloid Precursor Protein Dimers

The amyloid precursor protein (APP) is implied both in cell growth and differentiation and in neurodegenerative processes in Alzheimer disease. Regulated proteolysis of APP generates biologically active fragments such as the neuroprotective secreted ectodomain sAPPα and the neurotoxic β-amyloid peptide. Furthermore, it has been suggested that the intact transmembrane APP plays a signaling role, which might be important for both normal synaptic plasticity and neuronal dysfunction in dementia. To understand APP signaling, we tracked single molecules of APP using quantum dots and quantitated APP homodimerization using fluorescence lifetime imaging microscopy for the detection of Förster resonance energy transfer in living neuroblastoma cells. Using selective labeling with synthetic fluorophores, we show that the dimerization of APP is considerably higher at the plasma membrane than in intracellular membranes. Heparan sulfate significantly contributes to the almost complete dimerization of APP at the plasma membrane. Importantly, this technique for the first time structurally defines the initiation of APP signaling by binding of a relevant physiological extracellular ligand; our results indicate APP as receptor for neuroprotective sAPPα, as sAPPα binding disrupts APP dimers, and this disruption of APP dimers by sAPPα is necessary for the protection of neuroblastoma cells against starvation-induced cell death. Only cells expressing reversibly dimerized wild-type, but not covalently dimerized mutant APP are protected by sAPPα. These findings suggest a potentially beneficial effect of increasing sAPPα production or disrupting APP dimers for neuronal survival.The amyloid precursor protein (APP)⁴ is known both for its important role in the development and plasticity of the nervous system (1–6) and for its involvement in Alzheimer disease (AD) (7, 8). Despite intensive research efforts, the initial events that lead to the prevalent sporadic, i.e. non-familial, forms of AD are still unclear. Furthermore, although a higher gene dose of APP (9) or the presence of pathological APP mutations is sufficient to induce familial AD (for review, see Ref. 10), the exact pathological mechanism that is triggered by APP is still under debate.Some fragments of APP, such as the β-amyloid peptide (Aβ), are thought to contribute to synaptic dysfunction and neurotoxicity (11, 12). On the other hand, the α-secretase-derived extracellular fragment of APP (sAPPα), which is present at lower levels in AD patients than in controls (13), has been shown to be beneficial for memory function, to possess neuroprotective properties, and to counteract the effects of Aβ (14–18).Signaling by transmembrane APP may directly contribute to neurodegeneration in AD (19–24); however, the signal transduction pathway for transmembrane APP remains unknown, although several potential regulatory proteins, glycosaminoglycans, and metal ions are known to bind with high affinity to APP and sAPPα (25, 26). The most common form of signal transduction for single-pass transmembrane proteins is the ligand-induced perturbation of a monomer/dimer equilibrium. Indeed, the dimerization of transmembrane APP has been implied several times in the past. Several studies have investigated the effects of presumed dimer-breaking perturbations on biological read-outs, such as the production of Aβ (27, 28), but without directly measuring the APP aggregation state, or have investigated the aggregation state of APP subdomains, often reconstituted in cell-free systems (27–32). Dimerization interfaces in both the extracellular and the transmembrane domain have been suggested.In the studies investigating the aggregation state of full-length APP, most of the employed methods, such as chemical cross-linking and co-immunoprecipitation, do not lend themselves readily to a rigorous quantitative analysis of the abundance of potentially instable dimers (31, 33), whereas in other cases the use of chimeras may have influenced the dimerization potential or precluded the search for a natural stimulus (23, 34). The only previously reported direct observation of APP dimerization by Förster resonance energy transfer (FRET) microscopy uses an assay in which the FRET efficiency varies with the level of overexpression (35). Therefore, a concentration-dependent FRET component due to nonspecific stochastic encounters cannot be excluded in this study.Most importantly, as none of the published procedures permitted the selective detection of APP dimers on the surface of live cells, where they would encounter ligands, they could not differentiate between subpopulations of APP. This may be one reason why no natural ligand of APP has ever been shown to signal via modulation of its monomer/dimer equilibrium.Another elusive goal is the identity of the receptor for neuroprotective sAPPα (36–39). The ligand-dependent dimerization of sAPPα in solution (40) and its origination from transmembrane APP suggest that APP might serve as receptor for sAPPα, but this binding has never been experimentally shown.     

S-Palmitoylation of ��-Secretase Subunits Nicastrin and APH-1

Proteolytic processing of amyloid precursor protein (APP) by β- and γ-secretases generates β-amyloid (Aβ) peptides, which accumulate in the brains of individuals affected by Alzheimer disease. Detergent-resistant membrane microdomains (DRM) rich in cholesterol and sphingolipid, termed lipid rafts, have been implicated in Aβ production. Previously, we and others reported that the four integral subunits of the γ-secretase associate with DRM. In this study we investigated the mechanisms underlying DRM association of γ-secretase subunits. We report that in cultured cells and in brain the γ-secretase subunits nicastrin and APH-1 undergo S-palmitoylation, the post-translational covalent attachment of the long chain fatty acid palmitate common in lipid raft-associated proteins. By mutagenesis we show that nicastrin is S-palmitoylated at Cys⁶⁸⁹, and APH-1 is S-palmitoylated at Cys¹⁸² and Cys²⁴⁵. S-Palmitoylation-defective nicastrin and APH-1 form stable γ-secretase complexes when expressed in knock-out fibroblasts lacking wild type subunits, suggesting that S-palmitoylation is not essential for γ-secretase assembly. Nevertheless, fractionation studies show that S-palmitoylation contributes to DRM association of nicastrin and APH-1. Moreover, pulse-chase analyses reveal that S-palmitoylation is important for nascent polypeptide stability of both proteins. Co-expression of S-palmitoylation-deficient nicastrin and APH-1 in cultured cells neither affects Aβ40, Aβ42, and AICD production, nor intramembrane processing of Notch and N-cadherin. Our findings suggest that S-palmitoylation plays a role in stability and raft localization of nicastrin and APH-1, but does not directly modulate γ-secretase processing of APP and other substrates.Alzheimer disease is the most common among neurodegenerative diseases that cause dementia. This debilitating disorder is pathologically characterized by the cerebral deposition of 39–42 amino acid peptides termed Aβ, which are generated by proteolytic processing of amyloid precursor protein (APP)² by β- and γ-secretases (1, 2). The β-site APP cleavage enzyme 1 cleaves full-length APP within its luminal domain to generate a secreted ectodomain leaving behind a C-terminal fragment (β-CTF). γ-Secretase cleaves β-CTF within the transmembrane domain to release Aβ and APP intracellular C-terminal domain (AICD). γ-Secretase is a multiprotein complex, comprising at least four subunits: presenilins (PS1 and PS2), nicastrin, APH-1, and PEN-2 for its activity (3). PS1 is synthesized as a 42–43-kDa polypeptide and undergoes highly regulated endoproteolytic processing within the large cytoplasmic loop domain connecting putative transmembrane segments 6 and 7 to generate stable N-terminal (NTF) and C-terminal fragments (CTF) by an uncharacterized proteolytic activity (4). This endoproteolytic event has been identified as the activation step in the process of PS1 maturation as it assembles with other γ-secretase subunits (3). Nicastrin is a heavily glycosylated type I membrane protein with a large ectodomain that has been proposed to function in substrate recognition and binding (5), but this putative function has not been confirmed by others (6). APH-1 is a seven-transmembrane protein encoded by two human or three rodent genes that are alternatively spliced (7). Although PS1 (or PS2), nicastrin, APH-1, and PEN-2 are sufficient for γ-secretase processing of APP, a type I membrane protein, termed p23 (also referred toTMP21), was recently identified as a γ-secretase component that modulates γ-secretase activity and regulates secretory trafficking of APP (8, 9).A growing number of type I integral membrane proteins has been identified as γ-secretase substrates within the last few years, including Notch1 homologues, Notch ligands, Delta and Jagged, cell adhesion receptors N- and E-cadherins, low density lipoprotein receptor-related protein, ErbB-4, netrin receptor DCC, and others (10). Mounting evidence suggests that APP processing occurs within cholesterol- and sphingolipid-enriched lipid rafts, which are biochemically defined as detergentresistant membrane microdomains (DRM) (11, 12). Previously we reported that each of the γ-secretase subunits localizes in lipid rafts in post-Golgi and endosome membranes enriched in syntaxin 6 (13). Moreover, loss of γ-secretase activity by gene deletion or exposure to γ-secretase inhibitors results in the accumulation of APP CTFs in lipid rafts indicating that cleavage of APP CTFs likely occurs in raft microdomains (14). In contrast, CTFs derived from Notch1, Jagged2, N-cadherin, and DCC are processed by γ-secretase in non-raft membranes (14). The mechanisms underlying association of γ-secretase subunits with lipid rafts need further clarification to elucidate spatial segregation of amyloidogenic processing of APP in membrane microdomains.Post-translational S-palmitoylation is increasingly recognized as a potential mechanism for regulating raft association, stability, intracellular trafficking, and function of several cytosolic and transmembrane proteins (15–17). S-palmitoylation refers to the addition of 16-carbon palmitoyl moiety to certain cysteine residues through thioester linkage. Cysteines close to transmembrane domains or membrane-associated domains in non-integral membrane proteins are preferred S-palmitoylation sites, although no conserved motif has been identified (18). Palmitoylation modifies numerous neuronal proteins, including postsynaptic density protein PSD-95 (19), a-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid receptors (20), nicotinic α7 receptors (21), neuronal t-SNAREs SNAP-25, synaptobrevin 2 and synaptogagmin (22, 23), neuronal growth-associated protein GAP-43 (24), protein kinase CLICK-III (CL3)/CaMKIγ (25), β-secretase (26), and Huntingtin (27). Although palmitoylation can occur in vitro without the involvement of an enzyme, a family of palmitoyltransferases that specifically catalyze S-palmitoylation has been identified (28, 29).In this study, we have identified S-palmitoylation of γ-secretase subunits nicastrin and APH-1, and characterized its role on DRM association, protein stability, and γ-secretase enzyme activities. We show that nicastrin is S-palmitoylated at Cys⁶⁸⁹, and APH-1 at Cys¹⁸² and Cys²⁴⁵. Mutagenesis of palmitoylation sites results in increased degradation of nascent nicastrin and APH-1 polypeptides and reduced association with DRM. Nevertheless, in cultured cells overexpression of S-palmitoylation-deficient nicastrin and APH-1 does not modulate γ-secretase processing of APP or other substrates.     

Pen2 and Presenilin-1 Modulate the Dynamic Equilibrium of Presenilin-1 and Presenilin-2 ��-Secretase Complexes

γ-Secretase is known to play a pivotal role in the pathogenesis of Alzheimer disease through production of amyloidogenic Aβ42 peptides. Early onset familial Alzheimer disease mutations in presenilin (PS), the catalytic core of γ-secretase, invariably increase the Aβ42:Aβ40 ratio. However, the mechanism by which these mutations affect γ-secretase complex formation and cleavage specificity is poorly understood. We show that our in vitro assay system recapitulates the effect of PS1 mutations on the Aβ42:Aβ40 ratio observed in cell and animal models. We have developed a series of small molecule affinity probes that allow us to characterize active γ-secretase complexes. Furthermore we reveal that the equilibrium of PS1- and PS2-containing active complexes is dynamic and altered by overexpression of Pen2 or PS1 mutants and that formation of PS2 complexes is positively correlated with increased Aβ42:Aβ40 ratios. These data suggest that perturbations to γ-secretase complex equilibrium can have a profound effect on enzyme activity and that increased PS2 complexes along with mutated PS1 complexes contribute to an increased Aβ42:Aβ40 ratio.β-Amyloid (Aβ)⁵ peptides are believed to play a causative role in Alzheimer disease (AD). Aβ peptides are generated from the processing of the amyloid precursor protein (APP) by two proteases, β-secretase and γ-secretase. Although γ-secretase generates heterogenous Aβ peptides ranging from 37 to 46 amino acids in length, significant work has focused mainly on the Aβ40 and Aβ42 peptides that are the major constituents of amyloid plaques. γ-Secretase is a multisubunit membrane aspartyl protease comprised of at least four known subunits: presenilin (PS), nicastrin (Nct), anterior pharynx-defective (Aph), and presenilin enhancer 2 (Pen2). Presenilin is thought to contain the catalytic core of the complex (1–4), whereas Aph and Nct play critical roles in the assembly, trafficking, and stability of γ-secretase as well as substrate recognition (5, 6). Lastly Pen2 facilitates the endoproteolysis of PS into its N-terminal (NTF) and C-terminal (CTF) fragments thereby yielding a catalytically competent enzyme (5, 7–10). All four proteins (PS, Nct, Aph1, and Pen2) are obligatory for γ-secretase activity in cell and animal models (11, 12). There are two homologs of PS, PS1 and PS2, and three isoforms of Aph1, Aph1aS, Aph1aL, and Aph1b. At least six active γ-secretase complexes have been reported (two presenilins × three Aph1s) (13, 14). The sum of apparent molecular masses of the four proteins (PS1-NTF/CTF ≈ 53 kDa, Nct ≈ 120 kDa, Aph1 ≈ 30 kDa, and Pen2 ≈ 10kDa) is ∼200 kDa. However, active γ-secretase complexes of varying sizes, ranging from 250 to 2000 kDa, have been reported (15–19). Recently a study suggested that the γ-secretase complex contains only one of each subunit (20). Collectively these studies suggest that a four-protein complex around 200–250 kDa may be the minimal functional γ-secretase unit with additional cofactors and/or varying stoichiometry of subunits existing in the high molecular weight γ-secretase complexes. CD147 and TMP21 have been found to be associated with the γ-secretase complex (21, 22); however, their role in the regulation of γ-secretase has been controversial (23, 24).Mutations of PS1 or PS2 are associated with familial early onset AD (FAD), although it is debatable whether these familial PS mutations act as “gain or loss of function” alterations in regard to γ-secretase activity (25–27). Regardless the overall outcome of these mutations is an increased ratio of Aβ42:Aβ40. Clearly these mutations differentially affect γ-secretase activity for the production of Aβ40 and Aβ42. Despite intensive studies of Aβ peptides and γ-secretase, the molecular mechanism controlling the specificity of γ-secretase activity for Aβ40 and Aβ42 production has not been resolved. It has been found that PS1 mutations affect the formation of γ-secretase complexes (28). However, the precise mechanism by which individual subunits alter the dynamics of γ-secretase complex formation and activity is largely unresolved. A better mechanistic understanding of γ-secretase activity associated with FAD mutations has been hindered by the lack of suitable assays and probes that are necessary to recapitulate the effect of these mutations seen in cell models and to characterize the active γ-secretase complex.In our present studies, we have determined the overall effect of Pen2 and PS1 expression on the dynamics of PS1- and PS2-containing complexes and their association with γ-secretase activity. Using newly developed biotinylated small molecular probes and activity assays, we revealed that expression of Pen2 or PS1 FAD mutants markedly shifts the equilibrium of PS1-containing active complexes to that of PS2-containing complexes and results in an overall increase in the Aβ42:Aβ40 ratio in both stable cell lines and animal models. Our studies indicate that perturbations to the equilibrium of active γ-secretase complexes by an individual subunit can greatly affect the activity of the enzyme. Moreover they serve as further evidence that there are multiple and distinct γ-secretase complexes that can exist within the same cells and that their equilibrium is dynamic. Additionally the affinity probes developed here will facilitate further study of the expression and composition of endogenous active γ-secretase from a variety of model systems.     

The Cytoskeletal Protein ��-Actinin Regulates Acid-sensing Ion Channel 1a through a C-terminal Interaction

The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and peripheral neurons where it generates transient cation currents when extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic dendritic spines and has critical effects in neurological diseases associated with a reduced pH. However, knowledge of the proteins that interact with ASIC1a and influence its function is limited. Here, we show that α-actinin, which links membrane proteins to the actin cytoskeleton, associates with ASIC1a in brain and in cultured cells. The interaction depended on an α-actinin-binding site in the ASIC1a C terminus that was specific for ASIC1a versus other ASICs and for α-actinin-1 and -4. Co-expressing α-actinin-4 altered ASIC1a current density, pH sensitivity, desensitization rate, and recovery from desensitization. Moreover, reducing α-actinin expression altered acid-activated currents in hippocampal neurons. These findings suggest that α-actinins may link ASIC1a to a macromolecular complex in the postsynaptic membrane where it regulates ASIC1a activity.Acid-sensing ion channels (ASICs)² are H⁺-gated members of the DEG/ENaC family (1–3). Members of this family contain cytosolic N and C termini, two transmembrane domains, and a large cysteine-rich extracellular domain. ASIC subunits combine as homo- or heterotrimers to form cation channels that are widely expressed in the central and peripheral nervous systems (1–4). In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have two splice forms, a and b. Central nervous system neurons express ASIC1a, ASIC2a, and ASIC2b (5–7). Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2, and half-maximal activation occurs at pH 6.5–6.8 (8–10). These channels desensitize in the continued presence of a low extracellular pH, and they can conduct Ca²⁺ (9, 11–13). ASIC1a is required for acid-evoked currents in central nervous system neurons; disrupting the gene encoding ASIC1a eliminates H⁺-gated currents unless extracellular pH is reduced below pH 5.0 (5, 7).Previous studies found ASIC1a enriched in synaptosomal membrane fractions and present in dendritic spines, the site of excitatory synapses (5, 14, 15). Consistent with this localization, ASIC1a null mice manifested deficits in hippocampal long term potentiation, learning, and memory, which suggested that ASIC1a is required for normal synaptic plasticity (5, 16). ASICs might be activated during neurotransmission when synaptic vesicles empty their acidic contents into the synaptic cleft or when neuronal activity lowers extracellular pH (17–19). Ion channels, including those at the synapse often interact with multiple proteins in a macromolecular complex that incorporates regulators of their function (20, 21). For ASIC1a, only a few interacting proteins have been identified. Earlier work indicated that ASIC1a interacts with another postsynaptic scaffolding protein, PICK1 (15, 22, 23). ASIC1a also has been reported to interact with annexin II light chain p11 through its cytosolic N terminus to increase cell surface expression (24) and with Ca²⁺/calmodulin-dependent protein kinase II to phosphorylate the channel (25). However, whether ASIC1a interacts with additional proteins and with the cytoskeleton remain unknown. Moreover, it is not known whether such interactions alter ASIC1a function.In analyzing the ASIC1a amino acid sequence, we identified cytosolic residues that might bind α-actinins. α-Actinins cluster membrane proteins and signaling molecules into macromolecular complexes and link membrane proteins to the actincytoskeleton (for review, Ref. 26). Four genes encode α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an N-terminal head domain that binds F-actin, a C-terminal region containing two EF-hand motifs, and a central rod domain containing four spectrin-like motifs (26–28). The C-terminal portion of the rod segment appears to be crucial for binding to membrane proteins. The α-actinins assemble into antiparallel homodimers through interactions in their rod domain. α-Actinins-1, -2, and -4 are enriched in dendritic spines, concentrating at the postsynaptic membrane (29–35). In the postsynaptic membrane of excitatory synapses, α-actinin connects the NMDA receptor to the actin cytoskeleton, and this interaction is key for Ca²⁺-dependent inhibition of NMDA receptors (36–38). α-Actinins can also regulate the membrane trafficking and function of several cation channels, including L-type Ca²⁺ channels, K⁺ channels, and TRP channels (39–41).To better understand the function of ASIC1a channels in macromolecular complexes, we asked if ASIC1a associates with α-actinins. We were interested in the α-actinins because they and ASIC1a, both, are present in dendritic spines, ASIC1a contains a potential α-actinin binding sequence, and the related epithelial Na⁺ channel (ENaC) interacts with the cytoskeleton (42, 43). Therefore, we hypothesized that α-actinin interacts structurally and functionally with ASIC1a.     

Induced Dimerization of the Amyloid Precursor Protein Leads to Decreased Amyloid-�� Protein Production

The amyloid precursor protein (APP) plays a central role in Alzheimer disease (AD) pathogenesis because sequential cleavages by β- and γ-secretase lead to the generation of the amyloid-β (Aβ) peptide, a key constituent in the amyloid plaques present in brains of AD individuals. In several studies APP has recently been shown to form homodimers, and this event appears to influence Aβ generation. However, these studies have relied on APP mutations within the Aβ sequence itself that may affect APP processing by interfering with secretase cleavages independent of dimerization. Therefore, the impact of APP dimerization on Aβ production remains unclear. To address this question, we compared the approach of constitutive cysteine-induced APP dimerization with a regulatable dimerization system that does not require the introduction of mutations within the Aβ sequence. To this end we generated an APP chimeric molecule by fusing a domain of the FK506-binding protein (FKBP) to the C terminus of APP. The addition of the synthetic membrane-permeant drug AP20187 induces rapid dimerization of the APP-FKBP chimera. Using this system we were able to induce up to 70% APP dimers. Our results showed that controlled homodimerization of APP-FKBP leads to a 50% reduction in total Aβ levels in transfected N2a cells. Similar results were obtained with the direct precursor of β-secretase cleavage, C99/SPA4CT-FKBP. Furthermore, there was no modulation of different Aβ peptide species after APP dimerization in this system. Taken together, our results suggest that APP dimerization can directly affect γ-secretase processing and that dimerization is not required for Aβ production.The mechanism of β-amyloid protein (Aβ)² generation from the amyloid precursor protein is of major interest in Alzheimer disease research because Aβ is the major constituent of senile plaques, one of the neuropathological hallmarks of Alzheimer disease (1, 2). In the amyloidogenic pathway Aβ is released from the amyloid precursor protein (APP) (3) after sequential cleavages by β-secretase BACE1 (4–6) and by the γ-secretase complex (7, 8). BACE1 cleavage releases the large ectodomain of APP while generating the membrane-anchored C-terminal APP fragment (CTF) of 99 amino acids (C99). Cleavage of β-CTF by γ-secretase leads to the secretion of Aβ peptides of various lengths and the release of the APP intracellular domain (AICD) into the cytosol (9–11). The γ-secretase complex consists of at least four proteins: presenilin, nicastrin, Aph-I, and Pen-2 (12). Presenilin is thought to be the catalytic subunit of the enzyme complex (13), but how the intramembrane scission is carried out remains to be elucidated. Alternatively, APP can first be cleaved in the non-amyloidogenic pathway by α-secretase within the Aβ domain between Lys-16 and Leu-17 (14, 15). This cleavage releases the APP ectodomain (APPsα) while generating the membrane-bound C-terminal fragment (α-CTF) of 83 amino acids (C83). The latter can be further processed by the γ-secretase complex, resulting in the secretion of the small 3-kDa fragment p3 and the release of AICD.APP, a type I transmembrane protein (16) of unclear function, may act as a cell surface receptor (3). APP and its two homologues, APLP1 and APLP2, can dimerize in a homotypic or heterotypic manner and, in so doing, promote intercellular adhesion (17). In vivo interaction of APP, APLP1, and APLP2 was demonstrated by cross-linking studies from brain homogenates (18). To date at least four domains have been reported to promote APP dimerization; that is, the E1 domain containing the N-terminal growth factor-like domain and copper binding domain (17), the E2 domain containing the carbohydrate domain in the APP ectodomain (19), the APP juxtamembrane region (20), and the transmembrane domain (21, 22). In the latter domain the dimerization appears to be mediated by the GXXXG motif near the luminal face of the transmembrane region (21, 23). In addition to promoting cell adhesion, APP dimerization has been proposed to increase susceptibility to cell death (20, 24).Interestingly, by introducing cysteine mutations into the APP juxtamembrane region, it was shown that stable dimers through formation of these disulfide linkages result in significantly enhanced Aβ production (25). This finding is consistent with the observation that stable Aβ dimers are found intracellularly in neurons and in vivo in brain (26). Taken together, these results have led to the idea that APP dimerization can positively regulate Aβ production. However, other laboratories have not been able to confirm some of these observations using slightly different approaches (23, 27).To further address the question of how dimerization of APP affects cleavage by α-, β-, and γ-secretase, we chose to test this with a controlled dimerization system. Accordingly, we engineered a chimeric APP molecule by fusing a portion of the FK506-binding protein (FKBP) to the C terminus of APP such that the addition of the synthetic membrane-permeant bifunctional compound, AP20187, will induce dimerization of the APP-FKBP chimera in a controlled manner by binding to the FKBP domains. Using this system, efficient dimerization of APP up to 70% can be achieved in a time and concentration-dependent fashion. Our studies showed that controlled homodimerization of APP-FKBP leads to decreased total Aβ levels in transfected N2a cells. Homodimerization of the β-CTF/C99 fragment, the direct precursor of γ-secretase cleavage, showed comparable results. In addition, induced dimerization of APP did not lead to a modulation of different Aβ peptides as it was reported for GXXXG mutants within the transmembrane domain of APP (21).     

Paradoxical Condensation of Copper with Elevated ��-Amyloid in Lipid Rafts under Cellular Copper Deficiency Conditions: IMPLICATIONS FOR ALZHEIMER DISEASE*

Redox-active copper is implicated in the pathogenesis of Alzheimer disease (AD), β-amyloid peptide (Aβ) aggregation, and amyloid formation. Aβ·copper complexes have been identified in AD and catalytically oxidize cholesterol and lipid to generate H₂O₂ and lipid peroxides. The site and mechanism of this abnormality is not known. Growing evidence suggests that amyloidogenic processing of the β-amyloid precursor protein (APP) occurs in lipid rafts, membrane microdomains enriched in cholesterol. β- and γ-secretases, and Aβ have been identified in lipid rafts in cultured cells, human and rodent brains, but the role of copper in lipid raft amyloidogenic processing is presently unknown. In this study, we found that copper modulates flotillin-2 association with cholesterol-rich lipid raft domains, and consequently Aβ synthesis is attenuated via copper-mediated inhibition of APP endocytosis. We also found that total cellular copper is associated inversely with lipid raft copper levels, so that under intracellular copper deficiency conditions, Aβ·copper complexes are more likely to form. This explains the paradoxical hypermetallation of Aβ with copper under tissue copper deficiency conditions in AD.Imbalance of metal ions has been recognized as one of the key factors in the pathogenesis of Alzheimer disease (AD).² Aberrant interactions between copper or zinc with the β-amyloid peptide (Aβ) released into the glutamatergic synaptic cleft vicinity could result in the formation of toxic Aβ oligomers and aggregation into plaques characteristic of AD brains (reviewed in Ref. 1). Copper, iron, and zinc are highly concentrated in extracellular plaques (2, 3), and yet brain tissues from AD (4–6) and human β-amyloid precursor protein (APP) transgenic mice (7–10) are paradoxically copper deficient compared with age-matched controls. Elevation of intracellular copper levels by genetic, dietary, and pharmacological manipulations in both AD transgenic animal and cell culture models is able to attenuate Aβ production (7, 9, 11–15). However, the underlying mechanism is at present unclear.Abnormal cholesterol metabolism is also a contributing factor in the pathogenesis of AD. Hypercholesterolemia increases the risk of developing AD-like pathology in a transgenic mouse model (16). Epidemiological and animal model studies show that a hypercholesterolemic diet is associated with Aβ accumulation and accelerated cognitive decline, both of which are further aggravated by high dietary copper (17, 18). In contrast, biochemical depletion of cholesterol using statins, inhibitors of 3-hydroxy-3-methyglutaryl coenzyme A reductase, and methyl-β-cyclodextrin, a cholesterol sequestering agent, inhibit Aβ production in animal and cell culture models (19–25).Cholesterol is enriched in lipid rafts, membrane microdomains implicated in Aβ generation from APP cleavage by β- and γ-secretases. Recruitment of BACE1 (β-secretase) into lipid rafts increases the production of sAPP_β and Aβ (23, 26). The β-secretase-cleaved APP C-terminal fragment (β-CTF), and γ-secretase, a multiprotein complex composed of presenilin (PS1 or PS2), nicastrin (Nct), PEN-2 and APH-1, colocalize to lipid rafts (27). The accumulation of Aβ in lipid rafts isolated from AD and APP transgenic mice brains (28) provided further evidence that cholesterol plays a role in APP processing and Aβ generation.Currently, copper and cholesterol have been reported to modulate APP processing independently. However, evidence indicates that, despite tissue copper deficiency, Aβ·Cu²⁺ complexes form in AD that catalytically oxidize cholesterol and lipid to generate H₂O₂ and lipid peroxides (e.g. hydroxynonenal and malondialdehyde), which contribute to oxidative damage observed in AD (29–35). The underlying mechanism leading to the formation of pathological Aβ·Cu²⁺ complexes is unknown. In this study, we show that copper alters the structure of lipid rafts, and attenuates Aβ synthesis in lipid rafts by inhibition of APP endocytosis. We also identify a paradoxical inverse relationship between total cellular copper levels and copper distribution to lipid rafts, which appear to possess a privileged pool of copper where Aβ is more likely to interact with Cu²⁺ under copper-deficiency conditions to form Aβ·Cu²⁺ complexes. These data provide a novel mechanism by which cellular copper deficiency in AD could foster an environment for potentially adverse interactions between Aβ, copper, and cholesterol in lipid rafts.     

Sequential protein kinase C (PKC)-dependent and PKC-independent protein kinase D catalytic activation via Gq-coupled receptors: differential regulation of activation loop Ser(744) and Ser(748) phosphorylation

Protein kinase D (PKD) is a serine/threonine protein kinase rapidly activated by G protein-coupled receptor (GPCR) agonists via a protein kinase C (PKC)-dependent pathway. Recently, PKD has been implicated in the regulation of long term cellular activities, but little is known about the mechanism(s) of sustained PKD activation. Here, we show that cell treatment with the preferential PKC inhibitors GF 109203X or Gö 6983 blocked rapid (1–5-min) PKD activation induced by bombesin stimulation, but this inhibition was greatly diminished at later times of bombesin stimulation (e.g. 45 min). These results imply that GPCR-induced PKD activation is mediated by early PKC-dependent and late PKC-independent mechanisms. Western blot analysis with site-specific antibodies that detect the phosphorylated state of the activation loop residues Ser⁷⁴⁴ and Ser⁷⁴⁸ revealed striking PKC-independent phosphorylation of Ser⁷⁴⁸ as well as Ser⁷⁴⁴ phosphorylation that remained predominantly but not completely PKC-dependent at later times of bombesin or vasopressin stimulation (20–90 min). To determine the mechanisms involved, we examined activation loop phosphorylation in a set of PKD mutants, including kinase-deficient, constitutively activated, and PKD forms in which the activation loop residues were substituted for alanine. Our results show that PKC-dependent phosphorylation of the activation loop Ser⁷⁴⁴ and Ser⁷⁴⁸ is the primary mechanism involved in early phase PKD activation, whereas PKD autophosphorylation on Ser⁷⁴⁸ is a major mechanism contributing to the late phase of PKD activation occurring in cells stimulated by GPCR agonists. The present studies identify a novel mechanism induced by GPCR activation that leads to late, PKC-independent PKD activation.A rapid increase in the synthesis of lipid-derived second messengers with subsequent activation of protein phosphorylation cascades has emerged as a fundamental signal transduction mechanism triggered by multiple extracellular stimuli, including hormones, neurotransmitters, chemokines, and growth factors (1). Many of these agonists bind to G protein-coupled receptors (GPCRs),⁴ activate heterotrimeric G proteins and stimulate isoforms of the phospholipase C family, including β, γ, δ, and ε (reviewed in Refs. 1 and 2). Activated phospholipase Cs catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG). Inositol 1,4,5-trisphosphate mobilizes Ca²⁺ from intracellular stores (3, 4) whereas DAG directly activates the classic (α, β, and γ) and novel (δ, ε, η, and θ) isoforms of PKC (5–7). Although it is increasingly recognized that each PKC isozyme has specific functions in vivo (5–8), the mechanisms by which PKC-mediated signals are propagated to critical downstream targets remain incompletely defined.PKD, also known initially as PKCμ (9, 10), and two recently identified serine protein kinases termed PKD2 (11) and PKCν/PKD3 (12, 13), which are similar in overall structure and primary amino acid sequence to PKD (14), constitute a new protein kinase family within the Ca²⁺/calmodulin-dependent protein kinase group (15) and separate from the previously identified PKCs (14). Salient features of PKD structure include an N-terminal regulatory region containing a tandem repeat of cysteine-rich zinc finger-like motifs (termed the cysteine-rich domain) that confers high affinity binding to phorbol esters and DAG (9, 16, 17), followed by a pleckstrin homology (PH) domain that negatively regulates catalytic activity (18, 19). The C-terminal region of the PKDs contains its catalytic domain, which is distantly related to Ca²⁺-regulated kinases.In unstimulated cells, PKD is in a state of low kinase catalytic activity maintained by the N-terminal domain, which represses the catalytic activity of the enzyme by autoinhibition. Consistent with this model, deletions or single amino acid substitutions in the PH domain result in constitutive kinase activity (18–20). Physiological activation of PKD within cells occurs via a phosphorylation-dependent mechanism first identified in our laboratory (21). In response to cellular stimuli, PKD is converted from a low activity form into a persistently active form that is retained during isolation from cells, as shown by in vitro kinase assays performed in the absence of lipid co-activators (21, 22). PKD activation has been demonstrated in response to engagement of specific GPCRs either by regulatory peptides (23–30) or lysophosphatidic acid (27, 31, 32); signaling through G_q, G₁₂, G_i, and Rho (27, 31–34); activation of receptor tyrosine kinases, such as the platelet-derived growth factor receptor (23, 35, 36); cross-linking of B-cell receptor and T-cell receptor in B and T lymphocytes, respectively (37–40); and oxidative stress (41–44).Throughout these studies, multiple lines of evidence indicated that PKC activity is necessary for rapid PKD activation within intact cells. For example, rapid PKD activation was selectively and potently blocked by cell treatment with preferential PKC inhibitors (e.g. GF 109203X or Gö 6983) that do not directly inhibit PKD catalytic activity (21, 22), implying that PKD activation in intact cells is mediated, directly or indirectly, through PKCs. In line with this conclusion, cotransfection of PKD with active mutant forms of “novel” PKCs (PKCs δ, ε, η, and θ) resulted in robust PKD activation in the absence of cell stimulation (21, 44–46). Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade in response to multiple GPCR agonists in a broad range of cell types, including normal and cancer cells (reviewed in Ref. 14). Our previous studies identified Ser⁷⁴⁴ and Ser⁷⁴⁸ in the PKD activation loop (also referred as the activation segment or T-loop) as phosphorylation sites critical for PKC-mediated PKD activation (reviewed in Ref. 14). Collectively, these findings demonstrated the existence of rapidly activated PKC-PKD protein kinase cascade(s) and raised the possibility that some PKC-dependent biological responses involve PKD acting as a downstream effector.PKD has been reported recently to mediate several important cellular activities and processes, including signal transduction (30, 47–49), chromatin modification (50), Golgi organization and function (51, 52), c-Jun function (47, 53, 54), NFκB-mediated gene expression (43, 55, 56), and cell survival, migration, and differentiation and DNA synthesis and proliferation (reviewed in Ref. 14). Thus, mounting evidence indicates that PKD has a remarkable diversity of both its signal generation and distribution and its potential for complex regulatory interactions with multiple downstream pathways, leading to multiple responses, including long term cellular events. Despite increasing recognition of its importance, very little is known about the mechanism(s) of sustained PKD activation as opposed to the well documented rapid, PKC-dependent PKD activation.The results presented here demonstrate that prolonged GPCR-induced PKD activation is mediated by sequential PKC-dependent and PKC-independent phases of regulation. We report here, for the first time, that PKD autophosphorylation on Ser⁷⁴⁸ is a major mechanism contributing to the late phase of PKD activation occurring in cells stimulated by GPCR agonists. The present studies expand previous models of PKD regulation by identifying a novel mechanism induced by GPCR activation that leads to late, PKC-independent PKD activation.     

Dynamic ��-Helix Structure of Micelle-bound Human Amylin

Amylin is an endocrine hormone that regulates metabolism. In patients afflicted with type 2 diabetes, amylin is found in fibrillar deposits in the pancreas. Membranes are thought to facilitate the aggregation of amylin, and membrane-bound oligomers may be responsible for the islet β-cell toxicity that develops during type 2 diabetes. To better understand the structural basis for the interactions between amylin and membranes, we determined the NMR structure of human amylin bound to SDS micelles. The first four residues in the structure are constrained to form a hairpin loop by the single disulfide bond in amylin. The last nine residues near the C terminus are unfolded. The core of the structure is an α-helix that runs from about residues 5–28. A distortion or kink near residues 18–22 introduces pliancy in the angle between the N- and C-terminal segments of the α-helix. Mobility, as determined by ¹⁵N relaxation experiments, increases from the N to the C terminus and is strongly correlated with the accessibility of the polypeptide to spin probes in the solution phase. The spin probe data suggest that the segment between residues 5 and 17 is positioned within the hydrophobic lipid environment, whereas the amyloidogenic segment between residues 20 and 29 is at the interface between the lipid and solvent. This orientation may direct the aggregation of amylin on membranes, whereas coupling between the two segments may mediate the transition to a toxic structure.Type 2 diabetes affects over 100 million people worldwide (1) and is thought to cost upward of $130 billion dollars a year to treat in the United States alone (2). The endocrine hormone amylin (also known as islet amyloid polypeptide) appears to have key roles in diabetes pathology (3–5). The normal functions of amylin include the inhibition of glucagon secretion, slowing down the emptying of the stomach, and inducing a feeling of satiety through the actions of the hormone on neurons of the hypothalamus in the brain (5). The effects of amylin are exerted in concert with those of insulin and reduce the level of glucose in the blood (3, 5). Circulating amylin levels increase in a number of pathological conditions, including obesity, syndrome X, pancreatic cancer, and renal failure (3). Amylin levels together with insulin are raised initially in type 2 diabetes but fall as the disease progresses to a stage where the pancreatic islets of Langerhans β-cells that synthesize amylin no longer function (3).One of the hallmarks of type 2 diabetes, found in 90% of patients, is the formation of extracellular amyloid aggregates composed of amylin (3–5). The amyloid deposits accumulate in the interstitial fluid between islet cells and are usually juxtaposed with the β-cell membranes (3). Aggregates of amylin are toxic when added to cultures of β-cells, so that the amyloid found in situ may be responsible for β-cell death as type 2 diabetes progresses (6, 7). Genetic evidence that amylin is directly involved in pathology includes a familial S20G mutation that leads to early onset of the disease (8) and produces an amylin variant that aggregates more readily (9).As with all amyloids it is unclear whether fibrillar structures or soluble oligomers are responsible for pathology. A recurrent theme for amyloidogenic proteins is that toxicity appears to be exerted through membrane-bound oligomers that form pores and disrupt ion balance across membranes (4, 10–13). Experimental evidence for such oligomers has been found for the amyloid-β (Aβ)² peptides (14), which cause Alzheimer disease, and for α-synuclein (αS), the protein involved in Parkinson disease (15), a particular interest of our laboratory. The similar toxic effects exerted by these amyloidogenic molecules may have a common structural and physical basis. Detailed structural models are available for Aβ (16) and αS (17) bound to SDS micelle mimetics of membranes. For amylin there are models of peptide fragments 1–19 (18), 20–29 (19), and 17–29 (20) bound to micelles but as of yet no model of the complete hormone. This turns out to be particularly important as the interplay between structure and dynamics in amylin only comes to light when considering the whole molecule.Here we report the solution structure of human amylin bound to SDS micelles. We complement the structure with information on dynamics and on the immersion of amylin into micelles.     

Engineered Protein Connectivity to Actin Mimics PDZ-dependent Recycling of G Protein-coupled Receptors but Not Its Regulation by Hrs

Many G protein-coupled receptors (GPCRs) recycle after agonist-induced endocytosis by a sequence-dependent mechanism, which is distinct from default membrane flow and remains poorly understood. Efficient recycling of the β2-adrenergic receptor (β2AR) requires a C-terminal PDZ (PSD-95/Discs Large/ZO-1) protein-binding determinant (PDZbd), an intact actin cytoskeleton, and is regulated by the endosomal protein Hrs (hepatocyte growth factor-regulated substrate). The PDZbd is thought to link receptors to actin through a series of protein interaction modules present in NHERF/EBP50 (Na⁺/H+ exchanger 3 regulatory factor/ezrin-binding phosphoprotein of 50 kDa) family and ERM (ezrin/radixin/moesin) family proteins. It is not known, however, if such actin connectivity is sufficient to recapitulate the natural features of sequence-dependent recycling. We addressed this question using a receptor fusion approach based on the sufficiency of the PDZbd to promote recycling when fused to a distinct GPCR, the δ-opioid receptor, which normally recycles inefficiently in HEK293 cells. Modular domains mediating actin connectivity promoted receptor recycling with similarly high efficiency as the PDZbd itself, and recycling promoted by all of the domains was actin-dependent. Regulation of receptor recycling by Hrs, however, was conferred only by the PDZbd and not by downstream interaction modules. These results suggest that actin connectivity is sufficient to mimic the core recycling activity of a GPCR-linked PDZbd but not its cellular regulation.G protein-coupled receptors (GPCRs)² comprise the largest family of transmembrane signaling receptors expressed in animals and transduce a wide variety of physiological and pharmacological information. While these receptors share a common 7-transmembrane-spanning topology, structural differences between individual GPCR family members confer diverse functional and regulatory properties (1-4). A fundamental mechanism of GPCR regulation involves agonist-induced endocytosis of receptors via clathrin-coated pits (4). Regulated endocytosis can have multiple functional consequences, which are determined in part by the specificity with which internalized receptors traffic via divergent downstream membrane pathways (5-7).Trafficking of internalized GPCRs to lysosomes, a major pathway traversed by the δ-opioid receptor (δOR), contributes to proteolytic down-regulation of receptor number and produces a prolonged attenuation of subsequent cellular responsiveness to agonist (8, 9). Trafficking of internalized GPCRs via a rapid recycling pathway, a major route traversed by the β2-adrenergic receptor (β2AR), restores the complement of functional receptors present on the cell surface and promotes rapid recovery of cellular signaling responsiveness (6, 10, 11). When co-expressed in the same cells, the δOR and β2AR are efficiently sorted between these divergent downstream membrane pathways, highlighting the occurrence of specific molecular sorting of GPCRs after endocytosis (12).Recycling of various integral membrane proteins can occur by default, essentially by bulk membrane flow in the absence of lysosomal sorting determinants (13). There is increasing evidence that various GPCRs, such as the β2AR, require distinct cytoplasmic determinants to recycle efficiently (14). In addition to requiring a cytoplasmic sorting determinant, sequence-dependent recycling of the β2AR differs from default recycling in its dependence on an intact actin cytoskeleton and its regulation by the conserved endosomal sorting protein Hrs (hepatocyte growth factor receptor substrate) (11, 14). Compared with the present knowledge regarding protein complexes that mediate sorting of GPCRs to lysosomes (15, 16), however, relatively little is known about the biochemical basis of sequence-directed recycling or its regulation.The β2AR-derived recycling sequence conforms to a canonical PDZ (PSD-95/Discs Large/ZO-1) protein-binding determinant (henceforth called PDZbd), and PDZ-mediated protein association(s) with this sequence appear to be primarily responsible for its endocytic sorting activity (17-20). Fusion of this sequence to the cytoplasmic tail of the δOR effectively re-routes endocytic trafficking of engineered receptors from lysosomal to recycling pathways, establishing the sufficiency of the PDZbd to function as a transplantable sorting determinant (18). The β2AR-derived PDZbd binds with relatively high specificity to the NHERF/EBP50 family of PDZ proteins (21, 22). A well-established biochemical function of NHERF/EBP50 family proteins is to associate integral membrane proteins with actin-associated cytoskeletal elements. This is achieved through a series of protein-interaction modules linking NHERF/EBP50 family proteins to ERM (ezrin-radixin-moesin) family proteins and, in turn, to actin filaments (23-26). Such indirect actin connectivity is known to mediate other effects on plasma membrane organization and function (23), however, and NHERF/EBP50 family proteins can bind to additional proteins potentially important for endocytic trafficking of receptors (23, 25). Thus it remains unclear if actin connectivity is itself sufficient to promote sequence-directed recycling of GPCRs and, if so, if such connectivity recapitulates the normal cellular regulation of sequence-dependent recycling. In the present study, we took advantage of the modular nature of protein connectivity proposed to mediate β2AR recycling (24, 26), and extended the opioid receptor fusion strategy used successfully for identifying diverse recycling sequences in GPCRs (27-29), to address these fundamental questions.Here we show that the recycling activity of the β2AR-derived PDZbd can be effectively bypassed by linking receptors to ERM family proteins in the absence of the PDZbd itself. Further, we establish that the protein connectivity network can be further simplified by fusing receptors to an interaction module that binds directly to actin filaments. We found that bypassing the PDZ-mediated interaction using either domain is sufficient to mimic the ability of the PDZbd to promote efficient, actin-dependent recycling of receptors. Hrs-dependent regulation, however, which is characteristic of sequence-dependent recycling of wild-type receptors, was recapitulated only by the fused PDZbd and not by the proposed downstream interaction modules. These results support a relatively simple architecture of protein connectivity that is sufficient to mimic the core recycling activity of the β2AR-derived PDZbd, but not its characteristic cellular regulation. Given that an increasing number of GPCRs have been shown to bind PDZ proteins that typically link directly or indirectly to cytoskeletal elements (17, 27, 30-32), the present results also suggest that actin connectivity may represent a common biochemical principle underlying sequence-dependent recycling of various GPCRs.     

Detecting Morphologically Distinct Oligomeric Forms of ��-Synuclein

Neuropathologic and genetics studies as well as transgenic animal models have provided strong evidence linking misfolding and aggregation of α-synuclein to the progression of Parkinson disease (PD) and other related disorders. A growing body of evidence implicates various oligomeric forms of α-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death. Although numerous different oligomeric forms of α-synuclein have been identified in vitro, it is not known which forms are involved in PD or how, when, and where different forms contribute to the progression of PD. Reagents that can interact with specific aggregate forms of α-synuclein would be very useful not only as tools to study how different aggregate forms affect cell function, but also as potential diagnostic and therapeutic agents for PD. Here we show that a single chain antibody fragment (syn-10H scFv) isolated from a phage display antibody library binds to a larger, later stage oligomeric form of α-synuclein than a previously reported oligomeric specific scFv isolated in our laboratory. The scFv described here inhibits aggregation of α-synuclein in vitro, blocks extracellular α-synuclein-induced toxicity in both undifferentiated and differentiated human neuroblastoma cell lines (SH-SY5Y), and specifically recognizes naturally occurring aggregates in PD but not in healthy human brain tissue.Parkinson disease (PD)² is the second most common neurodegenerative disorder of the elderly, affecting more than 500,000 people in the United States (1), with 50,000 new cases reported each year at an annual cost estimated at 10 billion dollars per year. Pathologically, PD is characterized by the progressive loss of dopaminergic neurons in the substantia nigra and formation of fibrillar cytoplasmic inclusions known as Lewy bodies and Lewy neurites (2, 3). The protein α-synuclein has been strongly linked to PD (4, 5) and other related neurodegenerative disorders (6, 7) by several lines of evidence. 1) It is the major component of the hallmark Lewy body aggregates associated with PD. 2) Mutations (A53T, A30P, and E46K, where A30P is human A30P α-synuclein; A53T is human A53T α-synuclein; E46K is human E46K α-synuclein) or multiplication in the α-synuclein gene have been linked to familial PD (8–10). 3) Overexpression of α-synuclein in transgenic mice and Drosophila has been shown to induce the formation of PD-like pathological phenotypes and behavior, although the animal models do not in general replicate neuronal loss patterns (11, 12).α-Synuclein is a small protein (14 kDa) expressed mainly in brain tissues and is primarily localized at the presynaptic terminals of neurons (13). The primary structure of α-synuclein consists of three distinct regions. The N-terminal region of α-synuclein includes the mutation sites associated with familial PD (A53T, A30P, and E46K) and contains six imperfectly conserved repeats (KTKEGV) that may facilitate protein-protein binding. This repeat section is predicted to form amphipathic α-helices, typical of the lipid-binding domain of apolipoproteins (14). The central region, non-amyloid component, is extremely hydrophobic and includes a 12-residue stretch (VTGVTAVAQKTV) that is essential for aggregation (15). The C-terminal region is enriched with acidic glutamate and aspartate residues and is responsible for the chaperone function of α-synuclein (16).α-Synuclein normally exists as an unfolded protein, but it can adopt several different folded conformations depending on the environment, including small aggregates or oligomers, spherical and linear protofibrils, as well as the fibrillar structure found in Lewy bodies (14, 15). A growing body of evidence implicates the oligomeric forms of α-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death (16–18). Several different oligomeric forms of α-synuclein including spherical, annular (19), pore-like (20), and dopamine-stabilized structures have been identified in vitro (21).α-Synuclein is considered a cytosolic protein, and consequently its pathogenic effect was assumed to be limited to the cytoplasm of single cells. However, recent studies have suggested that α-synuclein also has extracellular pathogenic effects (22–25). α-Synuclein was detected in blood plasma and cerebrospinal fluid in both monomeric and oligomeric forms (22–25), and the presence of significantly elevated levels of oligomeric species of α-synuclein has been reported extracellularly in plasma and cerebrospinal fluid samples from patients with PD (23). Furthermore, various studies have shown that aggregated α-synuclein added extracellularly to the culture medium is cytotoxic (26–32).Despite all these studies, it is still not clear how the various aggregate forms of α-synuclein are involved in the progression of PD. Therefore, reagents that can interact with specific aggregate forms of α-synuclein would be very useful not only for fundamental studies of how α-synuclein aggregates affect cell function but also as potential diagnostic and therapeutic agents for PD.Recently, we reported inhibition of both aggregation and extracellular toxicity of α-synuclein in vitro by a single chain variable domain antibody fragment (scFv) that specifically recognized an oligomeric form of α-synuclein (32). In this study, we describe a second scFv (syn-10H) that binds a larger later stage oligomeric form of α-synuclein than the previously reported scFv. The syn-10H scFv neutralizes α-synuclein-induced toxicity in both undifferentiated and differentiated SH-SY5Y human neuroblastoma cell line and inhibits α-synuclein aggregation in vitro. The syn-10H scFv reacts specifically with homogenized PD brain tissue but does not cross-react with similarly treated samples taken from Alzheimer disease (AD) or healthy brain samples. Such scFvs therefore have potential value as diagnostic reagents to identify the presence of specific oligomeric species in PD tissue and fluid samples. The scFvs also have value as therapeutic agents as they can be used either extracellularly or expressed intracellularly (intrabodies) to prevent formation of toxic aggregates in vivo whether inside or outside of cells. Intrabodies have been used efficiently to neutralize toxic effects of different pathogenic agents, including α-synuclein (33–36). Moreover, immunization studies in mouse models of PD have shown that extracellular antibodies can reduce accumulation of intracellular aggregates of α-synuclein (37), thereby providing precedent for the use of scFvs in potential passive vaccination strategies for treating PD.