1 / 1

LUCY LY, RICHARD B. PARSONS AND BRIAN M. AUSTEN

The role of palmitoylation and farnesylation in the activation of -secretase and the release of -amyloid. LUCY LY, RICHARD B. PARSONS AND BRIAN M. AUSTEN. NEURODEGENERATION UNIT, DEPARTMENT OF BASIC MEDICAL SCIENCES, ST. GEORGES, UNIVERSITY OF LONDON, UK. INTRODUCTION.

april
Download Presentation

LUCY LY, RICHARD B. PARSONS AND BRIAN M. AUSTEN

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. The role of palmitoylation and farnesylation in the activation of -secretase and the release of -amyloid. LUCY LY, RICHARD B. PARSONS AND BRIAN M. AUSTEN NEURODEGENERATION UNIT, DEPARTMENT OF BASIC MEDICAL SCIENCES, ST. GEORGES, UNIVERSITY OF LONDON, UK INTRODUCTION METHODS & RESULTS con… METHODS & RESULTS con… • A derives from the proteolytic cleavage of the APP by - and -secretase and has been implicated to be a causative factor for AD. -secretase (BACE1) cleaves APP at the N-terminus of Abreleasing a soluble ectodomain (sAPP) and exposing a membrane-bound APP C-terminal fragment (CTF/C99), which is subsequently cleaved by -secretase [2]. • Increased levels of cholesterol are a risk factor for AD. Statins, have been shown to reduce A production by inhibiting the biosynthesis of cholesterol as well as farnesyl pyrophosphate and geranylgeranyl pyrophosphate, which play an important role in lipid modifications, such as palmitoylation and farnesylation [3]. • Palmitoylation of BACE1 with • Radiolabelled [3H]-palmatic acid. • Cerulenin prevented palmitoylation of BACE1. CVFM • prevented palmitoylation of dimeric BACE1 but not • Monomeric BACE1. • 1=untreated; 2=Cerulenin; 3=CVFM. • B.Isoprenylation of BACE1 using • radiolabelled [3H]-mevalonate. • Radiolabelling revealed a 53kDa protein, which co-purified • with BACE (lane 1). • 1=WC, non-reducing conditions; 2=WC, reducing conditions; • 3=HP BACE. Immunoprecipitation & Western blotting Figure 1. Detection of BACE. Monomeric (70kDa) and dimeric (140kDa) isoforms of BACE in his-purified and immunopurified Asp2 (arrows). 1=whole cell lysate (WC) wild-type HEK293; 2=WC Asp2; 3=His-purified (HP) HEK293; 4=HP Asp2; 5=Blank; 6=IP control; 7=IP HEK293; 8=IP Asp2. 1 2 3 4 5 6 7 8 CONCLUSION Western analysis detected BACE in Asp2 cell lines. Palmitoylation and farnesylation of BACE1 is important for its dimerisation and localisation. The reduction of Ab by farnesyl inhibitors demonstrates that lipid modification plays a role in disease pathology. BACE1 may interact with various proteins that may regulate its localisation to lipid rafts where it associates with APP. A quantification by sandwich ELISA To ascertain the role of farnesylation in the production of A, Asp2 cells were treated with FTase Inhibitor I/CVFM (344510, Calbiochem) & FTase Inhibitor II (344512, Calbiochem). Concentration of A released and remaining in the cells were measured by ELISA. FUTURE WORK AIMS Fatty acid exchange labelling This will determine and identify proteins that are palmitoylated by substituting thioester-linked palmitoyl modifications for biotinylated compounds. Biotinylated proteins will then be affinity purified using streptavidin-agarose and analysed by Western blotting [4]. Proteomics and Mass Spectrometry To determine & identify proteins that form the BACE complex, His-purified protein will be separated by 2D- electrophoresis and selected proteins will be analysed by mass spectrometry. To determine the role of palmitoylation and farnesylation in the activation of BACE1 by identifying BACE1 posttranslational profiles. In addition, identify BACE binding partners and ascertain their role in AD pathology. METHODS & RESULTS Wild-type and recombinant HEK293 cells (Asp2) were harvested and his-purified by immobilised-metal affinity chromatography. Purified lysate was then immunoprecipitated with anti-BACE and immunoblotted with anti-Myc (A4416, Sigma). Figure 2. A released into medium Figure 3. A remaining in cells CVFM, FTase Inhibitor I and FTase Inhibitor II significantly reduced the amount of A released into the medium (Figure 2) and the amount of A remaining in the cells (Figure 3), in a dose-dependent manner. REFERENCES [1] FM LaFerla, KN Green, S Oddo (2007) Intracellular amyloid- in Alzheimer’s disease. Nature 8, 499-509 [2] RB Parson, GC Price, JK Farrant, D Subramaniam, J Adeagbo-Sheikh, BM Austen (2006) Statins inhibit the dimerisation of -secretase via both isoprenoid- & cholesterol-mediated mechanisms. Biochem. J. 399, 205-14 [3] RC Drisdel, JK Alexander, A Sayeed, WN Green (2006) Assays of protein Palmitoylation. Methods 40, 127-134.

More Related