Characterization of an atypical gamma-secretase complex from hematopoietic origin.

Gamma-secretase is a widely expressed multisubunit enzyme complex which is involved in the pathogenesis of Alzheimer disease and hematopoietic malignancies through its aberrant processing of the amyloid precursor protein (APP) and Notch1, respectively. While gamma-secretase has been extensively studied, there is a dearth of information surrounding the activity, composition, and function of gamma-secretase expressed in distinct cellular populations. Here we show that endogenous gamma-secretase complexes of hematopoietic origin are distinct from epithelial derived gamma-secretase complexes. Hematopoietic gamma-secretase has reduced activity for APP and Notch1 processing compared to epithelial gamma-secretase. Characterization of the active complexes with small molecule affinity probes reveals that hematopoietic gamma-secretase has an atypical subunit composition with significantly altered subunit stoichiometry. Furthermore, we demonstrate that these discrete complexes exhibit cell-line specific substrate selectivity suggesting a possible mechanism of substrate regulation. These data underscore the need for studying endogenous gamma-secretase to fully understand of the biology of gamma-secretase and its complexity as a molecular target for the development of disease therapeutics.

Moderate reduction of gamma-secretase attenuates amyloid burden and limits mechanism-based liabilities.

Although gamma-secretase is recognized as a therapeutic target for Alzheimer's disease, side effects associated with strong inhibition of this aspartyl protease raised serious concerns regarding this therapeutic strategy. However, it is not known whether moderate inhibition of this enzyme will allow dissociation of beneficial effects in the CNS from mechanism-based toxicities in the periphery. We tested this possibility by using a series of mice with genetic reduction of gamma-secretase (levels ranging from 25 to 64% of control mice). Here, we document that even 30% reduction of gamma-secretase can effectively ameliorate amyloid burden in the CNS. However, global reduction of this enzyme below a threshold level increased the risk of developing squamous cell carcinoma as well as abnormal proliferation of granulocytes in a gamma-secretase dosage-dependent manner. Importantly, we demonstrate that there exists a critical gamma-secretase level that reduces the risk of amyloidosis in the CNS and limits tumorigenesis in epithelia. Our findings suggest that moderate inhibition of gamma-secretase represents an attractive anti-amyloid therapy for Alzheimer's disease.

When monomers are preferred: a strategy for the identification and disruption of weakly oligomerized proteins.

Oligomerization is important for the structure and function of many proteins, but frequently complicates their characterization. It is often desirable to obtain the protein in monomeric form. Here, we report a strategy that allows the generation of monomers from weakly associated oligomers but does not require knowledge of the three-dimensional structure of the protein. The dynamics of protein association are used in solution NMR spectroscopy to identify regions of the polypeptide chain that are likely to be responsible for the interaction. Protein sequence analysis further refines the selection, as conserved sites with moderate hydrophobicity are targeted for modification. Gel filtration and activity assays straightforwardly reveal the consequences of the change and are used to screen for the desired mutants. The strategy is demonstrated for the Rac1 binding domain of plexin-B1. A monomeric variant is generated which preserves the Rac1 binding activity and the wild-type protein structure.

Gender- and age-dependent gamma-secretase activity in mouse brain and its implication in sporadic Alzheimer disease.

Alzheimer disease (AD) is an age-related disorder. Aging and female gender are two important risk factors associated with sporadic AD. However, the mechanism by which aging and gender contribute to the pathogenesis of sporadic AD is unclear. It is well known that genetic mutations in gamma-secretase result in rare forms of early onset AD due to the aberrant production of Abeta42 peptides, which are the major constituents of senile plaques. However, the effect of age and gender on gamma-secretase has not been fully investigated. Here, using normal wild-type mice, we show mouse brain gamma-secretase exhibits gender- and age-dependent activity. Both male and female mice exhibit increased Abeta42ratioAbeta40 ratios in aged brain, which mimics the effect of familial mutations of Presenilin-1, Presenlin-2, and the amyloid precursor protein on Abeta production. Additionally, female mice exhibit much higher gamma-secretase activity in aged brain compared to male mice. Furthermore, both male and female mice exhibit a steady decline in Notch1 gamma-secretase activity with aging. Using a small molecule affinity probe we demonstrate that male mice have less active gamma-secretase complexes than female mice, which may account for the gender-associated differences in activity in aged brain. These findings demonstrate that aging can affect gamma-secretase activity and specificity, suggesting a role for gamma-secretase in sporadic AD. Furthermore, the increased APP gamma-secretase activity seen in aged females may contribute to the increased incidence of sporadic AD in women and the aggressive Abeta plaque pathology seen in female mouse models of AD. In addition, deceased Notch gamma-secretase activity may also contribute to neurodegeneration. Therefore, this study implicates altered gamma-secretase activity and specificity as a possible mechanism of sporadic AD during aging.

Pen2 and presenilin-1 modulate the dynamic equilibrium of presenilin-1 and presenilin-2 gamma-secretase complexes.

gamma-Secretase is known to play a pivotal role in the pathogenesis of Alzheimer disease through production of amyloidogenic Abeta42 peptides. Early onset familial Alzheimer disease mutations in presenilin (PS), the catalytic core of gamma-secretase, invariably increase the Abeta42:Abeta40 ratio. However, the mechanism by which these mutations affect gamma-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 Abeta42:Abeta40 ratio observed in cell and animal models. We have developed a series of small molecule affinity probes that allow us to characterize active gamma-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 Abeta42:Abeta40 ratios. These data suggest that perturbations to gamma-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 Abeta42:Abeta40 ratio.

Dual roles of the transmembrane protein p23/TMP21 in the modulation of amyloid precursor protein metabolism.

BACKGROUND: Alzheimer's disease (AD) is characterized by cerebral deposition of beta-amyloid (Abeta) peptides. Abeta is released from ectodomain cleaved amyloid precursor protein (APP) via intramembranous proteolysis by gamma-secretase, a complex consisting of presenilin and a few other proteins. p23/TMP21, a member of the p24 family type I transmembrane proteins, was recently identified as a presenilin complex component capable of modulating gamma-secretase cleavage. The p24 family proteins form oligomeric complexes and regulate vesicular trafficking in the early secretory pathway, but their role in APP trafficking has not been investigated. RESULTS: Here, we report that siRNA-mediated depletion of p23 in N2a neuroblastoma and HeLa cells produces concomitant knockdown of additional p24 family proteins and increases secretion of sAPP. Furthermore, intact cell and cell-free Abeta production increases following p23 knockdown, similar to data reported earlier using HEK293 cells. However, we find that p23 is not present in mature gamma-secretase complexes isolated using an active-site gamma-secretase inhibitor. Depletion of p23 and expression of a familial AD-linked PS1 mutant have additive effects on Abeta42 production. Knockdown of p23 expression confers biosynthetic stability to nascent APP, allowing its efficient maturation and surface accumulation. Moreover, immunoisolation analyses show decrease in co-residence of APP and the APP adaptor Mint3. Thus, multiple lines of evidence indicate that p23 function influences APP trafficking and sAPP release independent of its reported role in gamma-secretase modulation. CONCLUSION: These data assign significance to p24 family proteins in regulating APP trafficking in the continuum of bidirectional transport between the ER and Golgi, and ascribe new relevance to the regulation of early trafficking in AD pathogenesis.