研究類澱粉蛋白之調節蛋白SERF1a對阿茲海默症Amyloid-β和亨丁頓舞蹈症HttpolyQ纖維化的影響

Abstract

隨著社會高齡化的現象，現今全球已有數千萬人受到神經退化性疾病的侵襲。在各種神經退化性疾病中，特定蛋白的聚集和堆積不僅使蛋白失去原本功能，也會產生神經毒性，進而造成神經功能的喪失。此類蛋白共同的特點為聚集後會形成由交叉β折疊所構成的纖維，即所謂類澱粉蛋白。近年，small EDRK-rich factor (SERF)蛋白被發現會促進類澱粉蛋白纖維化，因此被稱為類澱粉蛋白之調節蛋白。為了更進一步了解其機制，在此篇論文中，我們致力於研究SERF1a在阿茲海默症Amyloid-β (Aβ)和亨丁頓舞蹈症HttpolyQ纖維化過程中所扮演的角色。我們利用硫代黃素-T (Thioflavin-T) 和圓二色光譜儀(circular dichroism spectroscopy)證明SERF1a加速了Aβ纖維化，而Aβ纖維量並不會因SERF1a有所差異。傅立葉轉換紅外光譜 (Fourier-transform infrared spectroscopy)和穿透式電子顯微鏡 (transmission electron microscope)的結果顯示，SERF1a會改變Aβ纖維的二級結構組成和型態。我們更進一步藉由電噴灑游離質譜法 (electrospray ionization mass spectrometry)、分析型超高速離心機(analytical ultracentrifugation)和核磁共振(nuclear magnetic resonance)發現，SERF1a是利用其N端區域與Aβ形成1:1的結合。然而，SERF1a會在改變Aβ後離開，並非成為纖維中的一部分。另外，細胞毒性測試結果顯示，SERF1a會因加速Aβ纖維化而加重Aβ對細胞的毒性。此影響可利用SERF1a抗體阻斷SERF1a與Aβ的結合來排除。在HttpolyQ的研究中，實驗室前人發現SERF1a促進HttpolyQ纖維化，並以α螺旋的區域與HttpolyQ結合。接續其研究結果，為了針對蛋白結合做更進一步的探討，我們設計了一系列HTT短胜肽，並藉由等溫滴定量熱法(isothermal titration calorimetry)和小角度X光散射(small-angle X-ray scattering)發現，SERF1a主要是與HttpolyQ的N端17個胺基酸以1:2的方式結合。除此之外，我們生產了SERF1a單株抗體，以應用在SERF1a相關的研究中。整體而言，我們證實了SERF1a以不同機制對於Aβ和HttpolyQ纖維化產生影響，以提供未來阿茲海默症和亨丁頓舞蹈症療法的新方向。

Neurodegenerative disorders have impacted millions of people worldwide with no effective cure currently. Most proteins involved in neurodegenerative diseases are prone to aggregate and deposit in the brain, leading to the loss of neuronal functions. These aggregates show fibrillary structures with a highly ordered cross β-sheet and display the characteristics of amyloid-like protein assemblies. Modifier of aggregation 4 (MOAG-4)/small EDRK-rich factor (SERF) has been identified as an amyloid modifier that promotes the aggregations of amyloidogenic proteins. To further discover the underlying mechanisms, in this dissertation, we focused on the role of SERF1a in Amyloid-β, one of the hallmarks of Alzheimer’s disease (AD), and HttpolyQ, involved in Huntington’s disease (HD), fibrillization. We first monitored SERF1a effect on Aβ fibril formation by Thioflavin T assay and far-UV circular dichroism (CD) spectroscopy combined with filter-trap assay, immunogold labeling, and partition analysis. We found that SERF1a expedited Aβ aggregation in a dose-dependent manner without affecting the fibril amount and was excluded from Aβ fibrils. Using Fourier-transform infrared spectroscopy (FTIR) and transmission electron microscope (TEM), we showed that SERF1a changed the secondary structures and the morphology of Aβ fibrils. We also investigated the complex formation of SERF1a and Aβ by photo-induced cross-linking of unmodified proteins (PICUP), electrospray ionization mass spectrometry (ESI-MS), and analytical ultracentrifugation (AUC) and the results revealed that SERF1a and Aβ mainly formed a 1:1 complex. Moreover, the NMR experiment suggested that SERF1a interacted with Aβ via its N-terminal region. Cytotoxicity assay demonstrated that SERF1a-accelerated Aβ intermediates enhanced the Aβ toxicity in neuroblastoma and the effect could be blocked by SERF1a antibody. As for HttpolyQ, based on our previous findings that SERF1a promoted HttpolyQ fibrillization and interacted with HttpolyQ, in a way different from that observed for Aβ, via its helical regions, we further investigated the interaction between SERF1a and HttpolyQ. Here, by using isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS), the HTT peptide study showed that SERF1a preferentially bound to the region containing N-terminal 17 residues of HttpolyQ in a 1:2 ratio. In addition, we produced SERF1a monoclonal antibodies for further applications in our research. Altogether, our study demonstrated that SERF1a plays a promoting role in Aβ and HttpolyQ fibrillization and offers insight into the underlying mechanisms in which SERF1a accelerates the conformational changes of Aβ and HttpolyQ to be more aggregation-prone. Our work provides a new direction for the therapeutic development of both AD and HD.