Active γ-secretase complexes contain only one of each component
ABSTRACT Gamma-secretase is an intramembrane aspartyl protease complex that cleaves type I integral membrane proteins, including the amyloid beta-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 gamma-secretase complexes, isolated by immunoprecipitation from solubilized membrane fractions and able to produce amyloid beta-peptides and amyloid beta-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 gamma-components presenilin:Pen-2:nicastrin:Aph-1 is 1:1:1:1.
- SourceAvailable from: Carmela R Abraham
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- "Under blue native conditions, the expected molecular weight of the APP monomer is ~300kDa and of the AβPP dimer is ~600kDa  . For pcDNA1 mock transfected cells, no AβPP band is seen on the blots. "
ABSTRACT: Alzheimer's disease (AD) is a progressive and fatal neurodegenerative disorder marked by memory impairment and cognitive deficits. A major component of AD pathology is the accumulation of amyloid plaques in the brain, which are comprised of amyloid beta (Aβ) peptides derived from the amyloidogenic processing of the amyloid precursor protein (AβPP) by β- and γ-secretases. In a subset of patients, inheritance of mutations in the AβPP gene is responsible for altering Aβ production, leading to early onset disease. Interestingly, many of these familial mutations lie within the transmembrane domain of the protein near the GxxxG and GxxxA dimerization motifs that are important for transmembrane interactions. As AβPP dimerization has been linked to changes in Aβ production, it is of interest to know whether familial AβPP mutations affect full-length APP dimerization. Using bimolecular fluorescence complementation (BiFC), blue native gel electrophoresis, and live cell chemical cross-linking, we found that familial Alzheimer's disease (FAD) mutations do not affect full-length AβPP dimerization in transfected HEK293 and COS7 cells. It follows that changes in AβPP dimerization are not necessary for altered Aβ production, and in FAD mutations, changes in Aβ levels are more likely a result of alternative proteolytic processing.01/2013; 2(1):15-28.
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- "Following cleavages sites are the γ-sites which produce Aβ species of 43, 42, 40, 39, 38, 37 amino acid long following the rule of trior tetrapeptide release (Takami et al., 2009; for review see Karran et al., 2011). The γ-secretase is a multiprotein complex composed of at least four proteins, Presenilin, Pen-2, Aph-1, Nicastrin, and one molecule of each is necessary and sufficient to form an active enzymatic complex (Edbauer et al., 2003; Kimberly et al., 2003; Takasugi et al., 2003; Sato et al., 2007). The α-and β-secretases are sheddases releasing the extracellular domain of APP as well as several others type I transmembrane proteins . "
ABSTRACT: Since the discovery of prion diseases, the concept has emerged that a protein could be a transmissible pathogen. As such, this transmissible pathogen agent can transfer its pathological mis-folded shape to the same but normally folded protein thus leading to the propagation of a disease. This idea is now extrapolated to several neurological diseases associated with protein mis-folding and aggregation, such as Alzheimer's disease (AD). AD is a slowly developing dementing disease characterized by the coexistence of two types of lesions: the parenchymal amyloid deposits and the intraneuronal neurofibrillary tangles (NFT). Amyloid deposits are composed of amyloid-beta peptides that derive from sequential cleavages of its precursor named amyloid protein precursor. NFT are characterized by intraneuronal aggregation of abnormally modified microtubule-associated Tau proteins. A synergistic relationship between the two lesions may trigger the progression of the disease. Thus, starting in the medial temporal lobe and slowly progressing through temporal, frontal, parietal, and occipital cortex, the spreading of NFT is well correlated with clinical expression of the disease and likely follows cortico-cortical neuronal circuitry. However, little is known about the mechanism driving the spatiotemporal propagation of these lesions ultimately leading to the disease. A growing number of studies suggest that amyloid deposits and NFT are resulting from a prion-like spreading. In the present chapter, we will develop the current hypotheses regarding the molecular and cellular mechanisms driving the development and spreading of AD lesions from the window of multivesicular endosomes/bodies and exosomes.Frontiers in Physiology 07/2012; 3:229. DOI:10.3389/fphys.2012.00229 · 3.50 Impact Factor
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- "Additional proteins might, however, be involved in the regulation of the activity or subcellular localization of the complex (Chen et al. 2006; Wakabayashi et al. 2009; He et al. 2010). The stoichiometry of the g-secretase complex is likely 1:1:1:1, based on molecular mass estimates in blue native electrophoresis (Kimberly et al. 2003), quantitative western blot analysis (Sato et al. 2007), and electron microscopy (EM) studies of the purified complex (Osenkowski et al. 2009). Thus, as there are two different PSEN genes and two different Aph1 genes (Aph1a and Aph1b) encoded in the human genome, it follows that at least four different g-secretase complexes exist (De Strooper 2003). "
ABSTRACT: Presenilins were first discovered as sites of missense mutations responsible for early-onset Alzheimer disease (AD). The encoded multipass membrane proteins were subsequently found to be the catalytic components of γ-secretases, membrane-embedded aspartyl protease complexes responsible for generating the carboxyl terminus of the amyloid β-protein (Aβ) from the amyloid protein precursor (APP). The protease complex also cleaves a variety of other type I integral membrane proteins, most notably the Notch receptor, signaling from which is involved in many cell differentiation events. Although γ-secretase is a top target for developing disease-modifying AD therapeutics, interference with Notch signaling should be avoided. Compounds that alter Aβ production by γ-secretase without affecting Notch proteolysis and signaling have been identified and are currently at various stages in the drug development pipeline.Cold Spring Harbor Perspectives in Medicine 01/2012; 2(1):a006304. DOI:10.1101/cshperspect.a006304 · 7.56 Impact Factor