caveolin-1, suggesting that only the enzymatic activity of �-secretase issubject to the regulation by caveolin-1. We also showed that overexpres-sion of caveolin-1 can attenuate �-secretase-mediated proteolysis of APPand Notch and that PS1 and NCT co-localize with caveolin-1 in HEK293cells. Conclusions: Our results clearly suggest that caveolin-1 is an im-portant endogenous regulator of �-secretase.

P3-335 UPSTREAM OF N-RAS (UNR) IS INVOLVED INTRANSLATIONAL CONTROL OF ADAM10PROTEIN EXPRESSION

Sven Lammich1, Sonja Zilow1, Dominik Bull1, Ann-Katrin Ludwig1,Claudia Prinzen2, Falk Fahrenholz2, Helene Jacquemin-Sablon3,Christian Haass1, 1Ludwig Maximilians University, Adolf ButenandtInstitute, Munich, Germany; 2Johannes Gutenberg University, Institutefor Biochemistry, Mainz, Germany; 3INSERM U889, Gref, UniversityBordeaux, Bordeaux, France. Contact e-mail: slammich@med.uni-muenchen.de

Background: The amyloid beta peptide (A�) is derived by proteolyticprocessing of the amyloid precursor protein (APP) by the beta-secretaseBACE1 and gamma-secretase. In contrast to this amyloidogenic process-ing, APP is predominantly cleaved by the alpha-secretase within the A�

domain and this precludes the formation of A�. We and other researchgroups could show that BACE1 protein expression is regulated by the5’untranslated region (UTR) of the BACE1 mRNA, however little isknown about the regulation of alpha-secretase. Similar to the 5’UTR ofBACE1, the 5’UTR of ADAM10 consists of 444 nucleotides with aGC-content of 70% and two upstream open reading frames. We hypothe-size that ADAM10, the major anti-amyloidogenic alpha-secretase, is alsoregulated by its 5’UTR and postulate that ADAM10 and BACE1 areregulated on a posttranscriptional level by 5’UTR binding proteins. Meth-ods: We performed a large mutagenesis analysis to identify regions withinthe 5’UTR of ADAM10 which are important for translational regulation.To identify possible binding candidates we performed a UTR-databasesearch. Results: We found that ADAM10 expression is regulated by its5’UTR. In the presence of the 5’UTR we observed a significant reductionof ADAM10 protein levels in HEK293 cells. mRNA levels were notaffected. Deletions from the 3’ end of the 5’UTR led to an even strongerinhibition of ADAM10 expression. However, a stepwise deletion of thefirst 259 nucleotides from the 5’end resulted in a strong increase inADAM10 protein expression. Moreover we show that RNA binding pro-teins exist which bind specifically and selectively to either the 5’UTR ofADAM10 or BACE1. Using Electophoretic Mobility Shift Assays weidentified UNR, a cytosolic RNA binding protein, as one binding partner ofthe ADAM10 5’UTR. Deleting the two well conserved binding sites withinthe UTR abolished the binding of recombinant UNR to the 5’UTR ofADAM10 and resulted in an increase of ADAM10 expression after tran-sient transfection. Conclusions: In this sudy we demonstrate that thecytoplasmic protein UNR is able to bind to the 5’UTR of ADAM10 andthat UNR might be involved in translational regulation of ADAM10protein expression.

P3-336 PURIFICATION AND COCRYSTALLIZATION OFNICASTRIN ECTODOMAIN

Guoqing Lin1, Seong-Hun Kim2, Brenda Leung1, Akiko Koide1,Shohei Koide1, Sangram S. Sisodia1, 1University of Chicago, Chicago,IL, USA; 2University of Florida, Gainesville, FL, USA. Contact e-mail:glin@bsd.uchicago.edu

Background: Nicastrin (NCT) is an essential component of gamma-secre-tase, a multiprotein complex that catalyzes intramembranous cleavage ofAPP, Notch and other substrates. Shah, Yu and colleagues have reportedthat the large ectodomain of NCT functions as a receptor that recognizesand captures substrate to facilitate proteolysis. At present, very little in-formation has emerged pertaining to the domains through which the com-ponents within the complex interact, or the structural basis for NCT

recognition and binding to substrates. Methods: We have employed aco-crystallization strategy that provides a superb methodology to facilitatecrystal growth and improve crystal quality for nicastrin structural studies.Results: We purified nicastrin ectodomain (NCTect) from the medium ofa mammalian cell line that stably expresses NCTect, and deglycosylatedthis subject to remove N-glycans. Using a phage library that displays alarge number of potential binders with desired diversities, we performedmultiple rounds of affinity selection against NCTect. Eight independentFab fragments have been successfully identified that tightly bind NCTect.The binding strength was determined by surface plasmon resonance bio-sensor, with dissociation constants in the lower nanomolar range. Conclu-sions: The nicastrin and Fab fragment form stable complexes which arenow being isolated and purified for crystallization trials.

P3-337 S-PALMITOYLATION TARGETS BACE-1 TOLIPID RAFTS

Xavier Meckler1, Kulandaivelu S. Vetrivel1, Ying Chen1, Tong Li2,Nabil G. Seidah3, Robert Vassar4, Philip C. Wong2, Masaki Fukata5,Gopal Thinakaran1, 1University of Chicago, Chicago, IL, USA; 2TheJohn Hopkins University School of Medicine, Baltimore, MD, USA;3Clinical Research Institute of Montreal, Montreal, QC, Canada;4Northwestern University Feinberg School of Medicine, Chicago, IL,USA; 5National Institute for Longevity Sciences, Aichi, Japan. Contacte-mail: xmeckler@bsd.uchicago.edu

Background: Several studies have previously highlighted the importanceof cholesterol-rich lipid raft membrane microdomains in the processing ofamyloid precursor protein (APP) by �- and �-secretases to release Alzhei-mer’s disease-associated amyloid peptides (A�). Biochemical fractionationand antibody co-patching methods indicate that a significant fraction of the�-secretase BACE-1 is targeted to lipid rafts. However, the moleculardeterminants responsible for BACE-1 targeting to lipid rafts are stillunknown. Methods: Radiolabeled palmitic acid labeling, biochemical lipidrafts-isolation, Western blot, antibody co-patching, immunocytochemistryResults: Here, we show that BACE-1 undergoes post-translational S-palmitoylation at four cysteine residues within the transmembrane andcytosolic domains. Substitution of these 4 cysteines to alanines was suffi-cient to abolish BACE-1 S-palmitoylation and targeting to lipid raft do-mains. However, wild type and palmitoylation-defective BACE-1 appearto have similar subcellular distribution in stably transfected cell lines. Wealso identified a subset of DHHC-containing palmitoyl-transferases thatwere able to mediate BACE-1 S-palmitoylation. Conclusions: Our studiesindicate that S-palmitoylation is required for BACE-1 targeting to lipidrafts. As S-palmitoylation is a reversible post-translational modification,DHHC-containing palmitoyl-transferase activities might mediate dynamicmodulation of BACE-1 association to lipid rafts.

P3-338 ACE PROTEIN AND ENZYME ACTIVITY LEVELSIN BRAIN AND CSF IN ALZHEIMER’S DISEASE:INFLUENCE OF DISEASE SEVERITY AND ACE-1GENOTYPE

Scott Miners, Emma Ashy, Seth Love, Patrick Kehoe, BristolUniversity, Bristol, United Kingdom. Contact e-mail:Scott.Miners@Bristol.ac.uk

Background: Several observations point to a role for angiotensin-convert-ing enzyme (ACE) in the pathogenesis of Alzheimer’s disease (AD): ACEcleaves human amyloid beta (A�) in vitro, the level and activity of ACEare reportedly increased in AD, and there is significant association betweenvariations in the ACE gene and AD. Objectives: We have measured ACEactivity and protein levels in CSF and frontal cortex from post-mortem ADcases and matched controls, and analyzed the relationships to Braak tanglestage, parenchymal A� load and the ACE-1 I/D polymorphism. Methods:Frozen frontal cortex from 121 cases of AD and 60 controls (all charac-terized for Braak tangle stage, ACE-1 I/D polymorphism and frontalparenchymal A� load was homogenized and used for sandwich ELISA of

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