Active -Secretase Complexes Contain Only One of Each Component

Harvard University, Cambridge, Massachusetts, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 12/2007; 282(47):33985-93. DOI: 10.1074/jbc.M705248200
Source: PubMed


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.

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    • "In this assay, the proteolysis of the internally quenched peptide at the Ab40-, Ab42-, and Ab43-generating cleavage sites results in enhanced fluorescence. A calibration experiment using purified membranes containing c-secretase complex (Sato et al, 2007) showed a dose-dependent increase in fluorescence (Fig EV3F). A direct comparison of fluorescence levels generated by membrane fractions of wild-type cells versus either PS1-or PS2-deficient cells indicated a reduction in PS1 activity of 49 and 23%, respectively (Fig EV3G). "
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    ABSTRACT: Do different neurodegenerative maladies emanate from the failure of a mutual protein folding mechanism? We have addressed this question by comparing mutational patterns that are linked to the manifestation of distinct neurodegenerative disorders and identified similar neurodegeneration-linked proline substitutions in the prion protein and in presenilin 1 that underlie the development of a prion disorder and of familial Alzheimer's disease (fAD), respectively. These substitutions were found to prevent the endoplasmic reticulum (ER)-resident chaperone, cyclophilin B, from assisting presenilin 1 to fold properly, leading to its aggregation, deposition in the ER, reduction of c-secretase activity, and impaired mito-chondrial distribution and function. Similarly, reduced quantities of the processed, active presenilin 1 were observed in brains of cyclophilin B knockout mice. These discoveries imply that reduced cyclophilin activity contributes to the development of distinct neurodegenerative disorders, propose a novel mechanism for the development of certain fAD cases, and support the emerging theme that this disorder can stem from aberrant presenilin 1 function. This study also points at ER chaperones as targets for the development of counter-neurodegeneration therapies.
    The EMBO Journal 10/2015; DOI:10.15252/embj.201592042 · 10.43 Impact Factor
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    • "γ-Secretase exists on the plasma membrane as an intact complex composed of its subunit components [15,26], at a stoichiometry of presenilin:PEN-2:nicastrin:APH-1, 1:1:1:1 [27]. However, PS1 can associate intra-molecularly to form higher order complexes [28], where nicastrin, APH-1 and PEN-2 do not seem to be required for its hetero- and homodimerization [29]. "
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    ABSTRACT: Presenilin-1 (PS1) is the active component of the amyloid precursor protein cleaving γ-secretase complex. PS1 protein is a transmembrane protein containing multiple hydrophobic regions which presence in cerebrospinal fluid (CSF) has not been measured to date. This study assesses whether PS1 and other components of the γ-secretase complex are present in CSF. Here, we show that PS1 is present in ventricular post-mortem and lumbar ante-mortem CSF, and plasma as 100-150-kDa hetero-complexes containing both the N- and C-terminal fragments (NTF and CTF) of the protein. Immunoprecipitation and immunoblotting with different antibodies confirmed the identity of the PS1 species. The γ-secretase components, APH-1 (anterior pharynx-defective 1) and PEN-2 (presenilin enhancer 2), as well as presenilin-2 (PS2) fragments, co-exist within these CSF complexes, while nicastrin is not detected. These CSF-PS1 complexes differ from active γ-secretase membrane-complexes, and may represent nonspecific aggregation of the PS1 protein. Levels of PS1 complexes are increased in CSF samples from autopsy-confirmed Alzheimer's disease (AD) cases and were found to be more stable than complexes in CSF from control subjects. Despite similar levels of total PS1 in CSF from probable AD patients and cognitively normal subjects, an increased proportion of highly stable PS1 complexes were observed in AD CSF. Our data suggest that fragments of the PS1 protein present in CSF as complexes may be useful as a biomarker for AD.
    08/2013; 1(1):46. DOI:10.1186/2051-5960-1-46
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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 . "
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