探討膽固醇與APP蛋白間之交互作用對APP蛋白水解之影響

Abstract

阿茲海默症 (Alzheimer’s disease, AD) 是一種漸進式的神經退化疾病，其病徵之一為APP蛋白經連續性酵素水解後產生的Aβ堆積。許多研究證實膽固醇在AD的病程發展上扮演著重要的角色，但其詳細作用機轉則仍未釐清。我們初步的研究發現膽固醇與APP產生交互作用可能會影響APP在膜上的分布，進而影響其水解。 在本研究中，我們將APP上的CRAC序列以點突變的方式略作改變，試圖藉由觀察這些mutants和wild-type APP在膽固醇結合、二聚體形成、及APP processing上之差異，來釐清APP與膽固醇交互作用在APP水解中扮演的角色。結果顯示在5種APP mutants中，只有APPS622L與膽固醇的結合程度比wild-type APP低，並觀察到其C99/C83比值下降，因而推測膽固醇與APP JM/TM區段之作用可能會影響APP之水解；APPY422F與APPY422L與膽固醇結合程度的差異則可能與APP heparin domain (HBD2) 中的helix-helix結構之穩定性相關，而此結構的瓦解與否也可能會影響APP形成二聚體，進而改變APP水解路徑；至於APPY463I，其與膽固醇的結合程度較wild-type APP為高，同時dimer/monomer之比值也較高，可能是因為Y463位於helix-helix結構之外側，較為solvent accessible，不須破壞前述之helix-helix結構即可與膽固醇結合，因此推測除helix-helix結構可能對二聚體的形成有一些幫助之外，膽固醇之結合可能會有助於誘使APP形成二聚體，進而促使APP經由amyloidogenic pathway代謝。此研究發現位在近膜區的CRAC序列可能是與膽固醇產生交互作用的重要片段，而膽固醇可能可以以不同的方式影響APP形成二聚體、或是將APP帶到raft上，走向amyloidogenic pathway。

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder. One of the histopathological features of AD is the extracellular aggregation of amyloid β (Aβ) derived from amyloid precursor protein (APP) processing. Previous studies have indicated that cholesterol plays an important role in the pathogenesis of AD. However, the exactly regulatory mechanism is not yet clearly known. Our preliminary studies revealed that the interaction between cholesterol and APP might affect APP distribution, leading to altered APP processing. To elucidate the roles of cholesterol-binding in APP processing, The cholesterol- binding and dimer formation properties and processing patterns of various APP mutants (i.e., with disrupted potential cholesterol recognition amino acid consensus (CRAC) motif) generated via site-directed mutagenesis were compared with wild-type APP. Among all 5 mutants, only APPS622L appeared to have a lower cholesterol binding capacity and a lower C99/C83 ratio compared with wild-type. It is therefore suspected that the interaction between cholesterol and the APP JM/TM region may affect APP processing. The difference in cholesterol-binding capacity between APPY422F and APPY422L seems to be associated with the stability of the helix-helix structure in the heparin binding domain (HBD2) of APP, which may contribute to APP dimerization. APPY463I, on the other hand, has a higher binding capacity and a higher dimer/monomer ratio than wild-type. Since Y463 is positioned at the solvent accessible surface of the aforementioned helix-helix structure, it is possible that APPY463I is able to interact with cholesterol without disrupting its helix-helix structure. Both cholesterol-binding and a more stable helix-helix structure may contribute to the observed higher dimer/monomer ratio in the APPY463I mutant, which in turn leads to a shift towards the amyloidogenic pathway. In conclusion, results from this study indicate that the CRAC motif in the JM/TM region of APP may play a crucial role in cholesterol-APP interaction, and that cholesterol may affect APP dimerization, translocation, and processing through different kinds of mechanisms.