1 and ?and2;2; the other is specific to the last 20 amino acids of PS1 (from Santa Cruz, data not shown). other components is required for full maturation of NCT. strong class=”kwd-title” Keywords: Alzheimers disease, gamma-secretase, presenilin, Pen-2, APP -amyloid peptide (A) is usually produced from a large amyloid precursor protein (APP) by -secretase and -secretase; the latter cleaves APP within its transmembrane domain at multiple sites in a sequential manner: Phentolamine HCl first at -cleavage at A49, rapidly followed by -cleavage at A46 and -cleavage at A40/42 (Xu, 2009).. According to the amyloid cascade hypothesis, the ratio of long A vs short A, A42/A40, is usually a key factor in the development and pathogenesis of AD (Zhang and Xu, 2007). Mutations in the presenilin (PS) protein, which functions as the catalytic core of the -secretase complex, have been found either to increase the total A load or increase the A42/A40 ratio (Borchelt et al., 1996; De Strooper and Annaert, 2000). Therefore, understanding the molecular nature of the -secretase complex and its biological function with respect to processing of APP and A formation is critical for understanding AD pathology. A functional -secretase complex consists of presenilin (PS1 or PS2) and three other transmembrane proteins: nicastrin (NCT), anterior pharynx defective 1 (Aph-1), and PS enhancer 2 (PEN-2) (Dries and Yu, 2008). Presenilins are believed to be nine-pass transmembrane proteins that undergo endoproteolytic processing between the 6th and 7th transmembrane domains resulting in a 28 kDa N-terminal fragment (PSN) and a 17 kDa C-terminal fragment (PSC) (Thinakaran et al., 1996). The discoveries that knockout of both PS1 and PS2 results in the abolishment of -secretase activity (De Strooper et al., 1998; Herreman et al., 2000; Zhang et al., 2000) and that two conserved aspartate residues in the 6th and 7th transmembrane domains of PS have been identified as essential for -secretase activity (Kimberly et al., 2000; Wolfe et al., 1999) suggest that PS bear the -secretase active site. NCT has been suggested to function as the substrate receptor (Shah et al., 2005). Using siRNA technology, studies suggested that Aph-1 is required for stabilization of the PS1 endoproteolysis products PS1N and PS1C (Francis et al., 2002; Lee et al., 2002; Steiner et al., 2002) and that Pen-2 is required for endoproteolysis of PS1 (Luo et al., 2003; Takasugi et al., 2003). However, data from our current study, using knockout cell lines and siRNA Phentolamine HCl technology, indicate Pen-2 is usually dispensable for the endoproteolysis of PS1. Our study also revealed several other interesting findings that contribute to a better understanding of the role of each -secretase component in the assembly and functional activity of the -secretase complex. Methods Cell culture Mouse embryonic fibroblast (MEF) cells obtained from PS1/PS2-KO (Herreman et al., 2000), NCT-KO (Li et al., 2003), APH-KO (Ma et al., 2005), and wild type MEFs were cultured in Dulbeccos altered Eagles medium (DMEM) made up of 10% fetal bovine serum. Immunoprecipitation and Western blotting Immunoprecipitation and Western blotting were carried out as explained previously (Zhao et al., 2004). siRNA treatment Both siRNAs and delivery reagent were purchased from Qiagen (Valencia, CA, USA), and treatment of cells with siRNAs was carried out according to the manufacturers instruction. Materials Proteasome inhibitor MG132 was purchased from Peptides International (Louisville, KY, USA). -secretase inhibitors compound E and L685, 458 were from EMD Chemicals (Gibbstown, NJ, USA). Polyclonal antibodies against components of -secretase were raised or purchased as follows: Anti-PS1N and anti-PS1C were raised N-terminal (residues 27-50) and C-terminal (residues 307-321) peptides of PS1 as explained previously (Xu et al., 2002; Zhao et al., 2004); Ab14, a PS1N-specific antibody used in a previous study (Luo et al., 2003) was received as a gift from Dr. Huaxi Xu (Sanford-Burnham Medical Research Phentolamine HCl Institute, San Diego, CA, USA); Commercial anti-PS1N (N-19) and anti-PS1C (C-20) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Anti-NCT from Sigma-Aldrich (St. Louis, MO, USA); Polyclonal antibodies anti-APH1aL and anti-PEN-2N and the monoclonal antibody 6E10 against the first 17 amino acids of A were from Covance (Emeryville, CA, USA). The other anti-PEN-2 antibody used, anti-PEN2C, was raised against a peptide with corresponding PEN-2 residues from 86 to 101; A commercial anti-PEN-2 raised against the full length of PEN-2 was from Santa Cruz. Results Pen-2, NCT, and Phentolamine HCl Aph-1 are not required for PS endoproteolytic processing, but are required for stabilizing the endoproteolytic products of PS Rabbit Polyclonal to PKCB1 To achieve knockdown of Pen-2, the wild type MEFs stably expressing human Swedish mutant APP were treated with Pen-2 siRNA. As.