Satoru Funamoto

Spatial and temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis.

We have investigated the mechanisms of leading edge formation in chemotaxing Dictyostelium cells. We demonstrate that while phosphatidylinositol 3-kinase (PI3K) transiently translocates to the plasma membrane in response to chemoattractant stimulation and to the leading edge in chemotaxing cells, PTEN, a negative regulator of PI3K pathways, exhibits a reciprocal pattern of localization. By uniformly localizing PI3K along the plasma membrane, we show that chemotaxis pathways are activated along the lateral sides of cells and PI3K can initiate pseudopod formation, providing evidence for a direct instructional role of PI3K in leading edge formation. These findings provide evidence that differential subcellular localization and activation of PI3K and PTEN is required for proper chemotaxis.

Signaling pathways controlling cell polarity and chemotaxis.

Many important biological processes, including chemotaxis (directional cell movement up a chemoattractant gradient), require a clearly established cell polarity and the ability of the cell to respond to a directional signal. Recent advances using Dictyostelium cells and mammalian leukocytes have provided insights into the biochemical and molecular pathways that control chemotaxis. Phosphoinositide 3-kinase plays a central and possibly pivotal role in establishing and maintaining cell polarity by regulating the subcellular localization and activation of downstream effectors that are essential for regulating cell polarity and proper chemotaxis. This review outlines our present understanding of these pathways.

gamma-Secretase: successive tripeptide and tetrapeptide release from the transmembrane domain of beta-carboxyl terminal fragment.

Amyloid β protein (Aβ), a pathogenic molecule associated with Alzheimers disease, is produced by γ-secretase, which cleaves the β-carboxyl terminal fragment (βCTF) of β-amyloid precursor protein in the middle of its transmembrane domain. How the cleavage proceeds within the membrane has long been enigmatic. We hypothesized previously that βCTF is cleaved first at the membrane–cytoplasm boundary, producing two long Aβs, Aβ48 and Aβ49, which are processed further by releasing three residues at each step to produce Aβ42 and Aβ40, respectively. To test this hypothesis, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) to quantify the specific tripeptides that are postulated to be released. Using CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxyl-1-propanesulfonate)-reconstituted γ-secretase system, we confirmed that Aβ49 is converted to Aβ43/40 by successively releasing two or three tripeptides and that Aβ48 is converted to Aβ42/38 by successively releasing two tripeptides or these plus an additional tetrapeptide. Most unexpectedly, LC-MS/MS quantification revealed an induction period, 3–4 min, in the generation of peptides. When extrapolated, each time line for each tripeptide appears to intercept the same point on the x-axis. According to numerical simulation based on the successive reaction kinetics, the induction period exists. These results strongly suggest that Aβ is generated through the stepwise processing of βCTF by γ-secretase.

Potent amyloidogenicity and pathogenicity of Aβ43

The amyloid-β peptide Aβ42 is known to be a primary amyloidogenic and pathogenic agent in Alzheimers disease. However, the role of Aβ43, which is found just as frequently in the brains of affected individuals, remains unresolved. We generated knock-in mice containing a pathogenic presenilin-1 R278I mutation that causes overproduction of Aβ43. Homozygosity was embryonic lethal, indicating that the mutation involves a loss of function. Crossing amyloid precursor protein transgenic mice with heterozygous mutant mice resulted in elevated Aβ43, impairment of short-term memory and acceleration of amyloid-β pathology, which accompanied pronounced accumulation of Aβ43 in plaque cores similar in biochemical composition to those observed in the brains of affected individuals. Consistently, Aβ43 showed a higher propensity to aggregate and was more neurotoxic than Aβ42. Other pathogenic presenilin mutations also caused overproduction of Aβ43 in a manner correlating with Aβ42 and with the age of disease onset. These findings indicate that Aβ43, an overlooked species, is potently amyloidogenic, neurotoxic and abundant in vivo.

Active gamma-secretase complexes contain only one of each component.

γ-Secretase is an intramembrane aspartyl protease complex that cleaves type I integral membrane proteins, including the amyloid β-protein precursor and the Notch receptor, and is composed of presenilin, Pen-2, nicastrin, and Aph-1. Although all four of these membrane proteins are essential for assembly and activity, the stoichiometry of the complex is unknown, with the number of presenilin molecules present being especially controversial. Here we analyze functional γ-secretase complexes, isolated by immunoprecipitation from solubilized membrane fractions and able to produce amyloid β-peptides and amyloid β-protein precursor intracellular domain. We show that the active isolated protease contains only one presenilin per complex, which excludes certain models of the active site that require aspartate dyads formed between two presenilin molecules. We also quantified components in the isolated complexes by Western blot using protein standards and found that the amounts of Pen-2 and nicastrin were the same as that of presenilin. Moreover, we found that one Aph-1 was not co-immunoprecipitated with another in active complexes, evidence that Aph-1 is likewise present as a monomer. Taken together, these results demonstrate that the stoichiometry of γ-components presenilin:Pen-2:nicastrin:Aph-1 is 1:1:1:1.

Equimolar Production of Amyloid β-Protein and Amyloid Precursor Protein Intracellular Domain from β-Carboxyl-terminal Fragment by γ-Secretase

We showed previously that cells expressing wild-type (WT) β-amyloid precursor protein (APP) or coexpressing WTAPP and WT presenilin (PS) 1/2 produced APP intracellular domains (AICD) 49-99 and 50-99, with the latter predominating. On the other hand, the cells expressing mutant (MT) APP or coexpressing WTAPP and MTPS1/2 produced a greater proportion of AICD-(49-99) than AICD-(50-99). In addition, the expression of amyloid β-protein (Aβ) 49 in cells resulted in predominant production of Aβ40 and that of Aβ48 leads to preferential production of Aβ42. These observations suggest that ϵ-cleavage and γ-cleavage are interrelated. To determine the stoichiometry between Aβ and AICD, we have established a 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized γ-secretase assay system that exhibits high specific activity. By using this assay system, we have shown that equal amounts of Aβ and AICD are produced from β-carboxyl-terminal fragment (C99) by γ-secretase, irrespective of WT or MTAPP and PS1/2. Although various Aβ species, including Aβ40, Aβ42, Aβ43, Aβ45, Aβ48, and Aβ49, are generated, only two species of AICD, AICD-(49-99) and AICD-(50-99), are detected. We also have found that M233T MTPS1 produced only one species of AICD, AICD-(49-99), and only one for its counterpart, Aβ48, in contrast to WT and other MTPS1s. These strongly suggest thatϵ-cleavage is the primary event, and the produced Aβ48 and Aβ49 rapidly undergo γ-cleavage, resulting in generation of various Aβ species.

γ-Secretase associated with lipid rafts: multiple interactive pathways in the stepwise processing of β-carboxyl terminal fragment

Background: Intramembranous cleavages of β-carboxyl-terminal fragment (βCTF) by γ-secretase generate amyloid β-protein (Aβ). Results: Three- to six-residue peptides are released successively along with Aβ generation by lipid raft-associated γ-secretase. Conclusion: γ-Secretase cleaves βCTF through multiple interactive pathways for stepwise successive processing to generate Aβ. Significance: This cleavage model provides insights into the precise molecular mechanism of Aβ generation. γ-Secretase generates amyloid β-protein (Aβ), a pathogenic molecule in Alzheimer disease, through the intramembrane cleavage of the β-carboxyl-terminal fragment (βCTF) of β-amyloid precursor protein. We previously showed the framework of the γ-secretase cleavage, i.e. the stepwise successive processing of βCTF at every three (or four) amino acids. However, the membrane integrity of γ-secretase was not taken into consideration because of the use of the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized reconstituted γ-secretase system. Here, we sought to address how the membrane-integrated γ-secretase cleaves βCTF by using γ-secretase associated with lipid rafts. Quantitative analyses using liquid chromatography-tandem mass spectrometry of the βCTF transmembrane domain-derived peptides released along with Aβ generation revealed that the raft-associated γ-secretase cleaves βCTF in a stepwise sequential manner, but novel penta- and hexapeptides as well as tri- and tetrapeptides are released. The cropping of these peptides links the two major tripeptide-cleaving pathways generating Aβ40 and Aβ42 at several points, implying that there are multiple interactive pathways for the stepwise cleavages of βCTF. It should be noted that Aβ38 and Aβ43 are generated through three routes, and γ-secretase modulator 1 enhances all the three routes generating Aβ38, which results in decreases in Aβ42 and Aβ43 and an increase in Aβ38. These observations indicate that multiple interactive pathways for stepwise successive processing by γ-secretase define the species and quantity of Aβ produced.

Phosphoinositides Suppress γ-Secretase in Both the Detergent-soluble and -insoluble States

γ-Secretase is an aspartic protease that hydrolyzes type I membrane proteins within the hydrophobic environment of the lipid bilayer. Using the CHAPSO-solubilized γ-secretase assay system, we previously found that γ-secretase activity was sensitive to the concentrations of detergent and phosphatidylcholine. This strongly suggests that the composition of the lipid bilayer has a significant impact on the activity of γ-secretase. Recently, level of secreted β-amyloid protein was reported to be attenuated by increasing levels of phosphatidylinositol 4,5-diphosphate (PI(4,5)P2) in cultured cells. However, it is not clear whether PI(4,5)P2 has a direct effect on γ-secretase activity. In this study, we found that phosphoinositides directly inhibited CHAPSO-solubilized γ-secretase activity. Interestingly, neither phosphatidylinositol nor inositol triphosphate altered γ-secretase activity. PI(4,5)P2 was also found to inhibit γ-secretase activity in CHAPSO-insoluble membrane microdomains (rafts). Kinetic analysis of β-amyloid protein production in the presence of PI(4,5)P2 suggested a competitive inhibition. Even though phosphoinositides are minor phospholipids of the membrane, the concentration of PI(4,5)P2 within the intact membrane has been reported to be in the range of 4–8 mm. The presence of PI(4,5)P2-rich rafts in the membrane has been reported in a range of cell types. Furthermore, γ-secretase is enriched in rafts. Taking these data together, we propose that phosphoinositides potentially regulate γ-secretase activity by suppressing its association with the substrate.

Substrate ectodomain is critical for substrate preference and inhibition of γ-secretase

Understanding the substrate recognition mechanism of γ-secretase is a key step for establishing substrate-specific inhibition of amyloid β-protein (Aβ) production. However, it is widely believed that γ-secretase is a promiscuous protease and that its substrate-specific inhibition is elusive. Here we show that γ-secretase distinguishes the ectodomain length of substrates and preferentially captures and cleaves substrates containing a short ectodomain. We also show that a subset of peptides containing the CDCYCxxxxCxCxSC motif binds to the amino terminus of C99 and inhibits Aβ production in a substrate-specific manner. Interestingly, these peptides suppress β-secretase-dependent cleavage of APP, but not that of sialyltransferase 1. Most importantly, intraperitoneal administration of peptides into mice results in a significant reduction in cerebral Aβ levels. This report provides direct evidence of the substrate preference of γ-secretase and its mechanism. Our results demonstrate that the ectodomain of C99 is a potent target for substrate-specific anti-Aβ therapeutics to combat Alzheimer’s disease.

Enzymatic Characteristics of I213T Mutant Presenilin-1/γ-Secretase in Cell Models and Knock-in Mouse Brains FAMILIAL ALZHEIMER DISEASE-LINKED MUTATION IMPAIRS γ-SITE CLEAVAGE OF AMYLOID PRECURSOR PROTEIN C-TERMINAL FRAGMENT β

Presenilin (PS)/γ-secretase-mediated intramembranous proteolysis of amyloid precursor protein produces amyloid β (Aβ) peptides in which Aβ species of different lengths are generated through multiple cleavages at the γ-, ζ-, and ϵ-sites. An increased Aβ42/Aβ40 ratio is a common characteristic of most cases of familial Alzheimer disease (FAD)-linked PS mutations. However, the molecular mechanisms underlying amyloid precursor protein proteolysis leading to increased Aβ42/Aβ40 ratios still remain unclear. Here, we report our findings on the enzymatic analysis of γ-secretase derived from I213T mutant PS1-expressing PS1/PS2-deficient (PS–/–) cells and from the brains of I213T mutant PS1 knock-in mice. Kinetics analyses revealed that the FAD mutation reduced de novo Aβ generation, suggesting that mutation impairs the total catalytic rate of γ-secretase. Analysis of each Aβ species revealed that the FAD mutation specifically reduced Aβ40 levels more drastically than Aβ42 levels, leading to an increased Aβ42/Aβ40 ratio. By contrast, the FAD mutation increased the generation of longer Aβ species such as Aβ43, Aβ45, and >Aβ46. These results were confirmed by analyses of γ-secretase derived from I213T knock-in mouse brains, in which the reduction of de novo Aβ generation was mutant allele dose-dependent. Our findings clearly indicate that the mechanism underlying the increased Aβ42/Aβ40 ratio observed in cases of FAD mutations is related to the differential inhibition of γ-site cleavage reactions, in which the reaction producing Aβ40 is subject to more inhibition than that producing Aβ42. Our results also provide novel insight into how enhancing the generation of longer Aβs may contribute to Alzheimer disease onset.Presenilin (PS)/gamma-secretase-mediated intramembranous proteolysis of amyloid precursor protein produces amyloid beta (Abeta) peptides in which Abeta species of different lengths are generated through multiple cleavages at the gamma-, zeta-, and epsilon-sites. An increased Abeta42/Abeta40 ratio is a common characteristic of most cases of familial Alzheimer disease (FAD)-linked PS mutations. However, the molecular mechanisms underlying amyloid precursor protein proteolysis leading to increased Abeta42/Abeta40 ratios still remain unclear. Here, we report our findings on the enzymatic analysis of gamma-secretase derived from I213T mutant PS1-expressing PS1/PS2-deficient (PS(-/-)) cells and from the brains of I213T mutant PS1 knock-in mice. Kinetics analyses revealed that the FAD mutation reduced de novo Abeta generation, suggesting that mutation impairs the total catalytic rate of gamma-secretase. Analysis of each Abeta species revealed that the FAD mutation specifically reduced Abeta40 levels more drastically than Abeta42 levels, leading to an increased Abeta42/Abeta40 ratio. By contrast, the FAD mutation increased the generation of longer Abeta species such as Abeta43, Abeta45, and >Abeta46. These results were confirmed by analyses of gamma-secretase derived from I213T knock-in mouse brains, in which the reduction of de novo Abeta generation was mutant allele dose-dependent. Our findings clearly indicate that the mechanism underlying the increased Abeta42/Abeta40 ratio observed in cases of FAD mutations is related to the differential inhibition of gamma-site cleavage reactions, in which the reaction producing Abeta40 is subject to more inhibition than that producing Abeta42. Our results also provide novel insight into how enhancing the generation of longer Abetas may contribute to Alzheimer disease onset.