Article

Length and overall sequence of the PEN-2 C-terminal domain determines its function in the stabilization of presenilin fragments.

Adolf-Butenandt-Institute, Department of Biochemistry, Laboratory for Alzheimer's and Parkinson's Disease Research, Ludwig-Maximilians-University, Munich, Germany.
Journal of Neurochemistry (Impact Factor: 4.24). 08/2005; 94(1):57-62. DOI: 10.1111/j.1471-4159.2005.03165.x
Source: PubMed

ABSTRACT Gamma-secretase is an aspartyl protease complex that catalyzes the intramembrane cleavage of a subset of type I transmembrane proteins including the beta-amyloid precursor protein (APP) implicated in Alzheimer's disease. Presenilin (PS), nicastrin (NCT), anterior pharynx defective (APH-1) and presenilin enhancer-2 (PEN-2) constitute the active gamma-secretase complex. PEN-2, the smallest subunit, is required for triggering PS endoproteolysis. Stabilization of the resultant N- and C-terminal fragments, which carry the catalytically active site aspartates, but not endoproteolysis itself, requires the C-terminal domain of PEN-2. To functionally dissect the C-terminal domain we created C-terminal deletion mutants and mutagenized several evolutionary highly conserved residues. The PEN-2 mutants were then probed for functional complementation of a PEN-2 knockdown, which displays deficient PS1 endoproteolysis and impaired NCT maturation. Progressive truncation of the C-terminus caused increasing loss of function. This was also observed for an internal deletion mutant as well as for C-terminally tagged PEN-2 with a twofold elongated C-terminal domain. Interestingly, only simultaneous, but not individual substitution of the highly conserved D90, F94, P97 and G99 residues with alanine interfered with PEN-2 function. All loss of function mutants identified allowed PS1 endoproteolysis, but failed to stably associate with the resultant PS1 fragments, which like the PEN-2 loss of function mutants underwent proteasomal degradation. However, complex formation of the PEN-2 mutants with PS1 fragments could be recovered when proteasomal degradation was blocked. Taken together, our data suggest that the PS-subunit stabilizing function of PEN-2 depends on length and overall sequence of its C-terminal domain.

0 Bookmarks
 · 
53 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: One of the most critical pathological features of Alzheimer's disease (AD) is the accumulation of β-amyloid (Aβ) peptides that form extracellular senile plaques in the brain. Aβ is derived from β-amyloid precursor protein (APP) through sequential cleavage by β- and γ-secretases. γ-secretase is a high molecular weight complex minimally composed of four components: presenilins (PS), nicastrin, anterior pharynx defective 1 (APH-1), and presenilin enhancer 2 (PEN-2). In addition to APP, γ-secretase also cleaves many other type I transmembrane (TM) protein substrates. As a crucial enzyme for Aβ production, γ-secretase is an appealing therapeutic target for AD. Here, we summarize current knowledge on the structure and function of γ-secretase, as well as recent progress in developing γ-secretase targeting drugs for AD treatment.
    Frontiers in Cellular Neuroscience 01/2014; 8:427. · 4.18 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The 19-transmembrane γ-secretase complex generates the amyloid β-peptide (Aβ) of Alzheimer's disease (AD) by intramembrane proteolysis of the β-amyloid precursor protein (APP). This complex is comprised of presenilin, Aph1, nicastrin and Pen-2. The exact function and mechanism of the highly conserved Pen-2 subunit remains poorly understood. Using systematic mutagenesis, we confirm and extend understanding of which key regions and specific residues play roles in various aspects of γ-secretase function, including maturation, localization and activity, but not processivity. In general, mutations: 1) within the first half of transmembrane domain (TMD) 1 of Pen-2 decreased PS1 endoproteolysis and γ-secretase proteolytic activity; 2) within the second half of TMD1 increased proteolytic activity; 3) within the cytosolic loop region decreased proteolytic activity; 4) within TMD2 decreased PS1 endoproteolysis; 5) within the first half of TMD2 decreased proteolytic activity; and 6) within C-terminal residues decreased proteolytic activity. Specific mutational effects included: N33A in TMD1 causing an increase in γ-secretase complexes at the cell surface and a modest decrease in stability; and the previously unreported I53A mutation in the loop region reducing stability 10-fold and proteolytic activity by half. In addition, we confirm that minor PS1 endoproteolysis can occur in the complete absence of Pen-2. Together, these data suggest that rather than solely being a catalyst for γ-secretase endoproteolysis, Pen-2 may also stabilize the complex prior to PS1 endoproteolysis, allowing time for full assembly and proper trafficking.
    Biochemistry 06/2014; · 3.19 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The 19-transmembrane, multi-subunit γ-secretase complex generates the amyloid β-peptide (Aβ) of Alzheimer's disease (AD) by an unusual intramembrane proteolysis of the β-amyloid precursor protein (APP). The complex, which similarly processes many other type 1 transmembrane substrates, is composed of presenilin, Aph1, nicastrin, and presenilin enhancer (Pen-2), all of which are necessary for proper complex maturation and enzymatic activity. Obtaining a high-resolution atomic structure of the intact complex would greatly aid the rational design of compounds to modulate activity but is a very difficult task. A complementary method is to generate structures for each individual subunit to allow one to build a model of the entire complex. Here, we describe a method by which recombinant human Pen-2 can be purified from bacteria to >95% purity at milligram quantities per liter, utilizing a maltose binding protein tag to both increase solubility and facilitate purification. Expressing the same construct in mammalian cells, we show that the large N-terminal MBP tag on Pen-2 still permits incorporation into the complex and subsequent presenilin-1 endoproteolysis, nicastrin glycosylation and proteolytic activity. These new methods provide valuable tools to study the structure and function of Pen-2 and the γ-secretase complex. This article is protected by copyright. All rights reserved.
    Journal of Neurochemistry 05/2014; · 4.24 Impact Factor

Full-text (2 Sources)

Download
3 Downloads
Available from
Dec 8, 2014