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Westmeyer, Gil Gregor (2006): Identification of a BACE dimer and characterization of its biochemical and enzymatic properties. Dissertation, LMU München: Faculty of Medicine
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Abstract

The deposition amyloid β peptide in the brains of patients is a hallmark of Alzheimer’s disease and is thought to play a major pathogenetic role in the development of the demential symptoms of this severe illness. The amyloid β peptide is generated from the β-amyloid precursor protein (APP) by cleavage of the “β-site APP-cleaving enzyme” (BACE) followed by cleavage of the "gamma-secretase”. Whereas it has recently been discovered that the gamma-secretase is a multi-protein complex, it has not yet been investigated whether under native conditions, BACE functions in association with other proteins. The present work thus studied BACE by means of blue native gel electrophoresis and found that native BACE has a molecular weight of 140 kDa, whereas BACE under denaturing conditions has a molecular weight of 70 kDa which is only half of its native mass. Co-immunoprecipitation experiments with differently tagged full-length BACE constructs subsequently showed that this higher molecular weight species of BACE corresponds to a BACE homodimer. In contrast, a BACE ectodomain, lacking the C-terminus and the transmembrane domain, is a monomer. A consecutive domain analysis revealed that both the C-terminus and the transmembrane domain of BACE are dispensable for dimerization. In line with this, it could be shown that the ectodomain of BACE can dimerize if it is attached to the membrane by a GPI anchor. In terms of the cellular localization of the dimerization process, it could furthermore be demonstrated that retention of BACE in the ER by addition of a KKXX-motif does not prevent dimerization. This suggests that dimerization can occur prior to full maturation of BACE which takes place in the Golgi apparatus. In addition, kinetic analyses of the purified native BACE dimer revealed a higher affinity and turnover rate for an APP-like substrate in comparison to the monomeric soluble BACE ectodomain. This suggests a putative function of dimerization in improving enzymatic efficiency. The implication of these findings for our understanding of the Amyloid-β synthesis as well as for a putatively alternative therapeutic strategy are discussed.