S-Space College of Engineering/Engineering Practice School (공과대학/대학원) Dept. of Chemical and Biological Engineering (화학생물공학부) Theses (Ph.D. / Sc.D._화학생물공학부)
알파-시뉴클린 상호작용성 분자들의 생물학적 의의와 질환치료 및 센서 개발 응용 연구 : Biological and Therapeutic Implications of α-Synuclein Interactive Molecules and Their Application in Sensor Development
- 공과대학 화학생물공학부
- Issue Date
- 서울대학교 대학원
- α-Synuclein ; Active oligomer ; Unit-assembly ; α-Synuclein interactive molecules ; Polydiacetylene ; Amyloid-based sensor
- 학위논문 (박사)-- 서울대학교 대학원 : 공과대학 화학생물공학부, 2016. 2. 백승렬.
- α-Synuclein, amyloidogenic protein, is a major component of Lewy bodies which are commonly observed in Parkinsons disease patients. Oligomeric intermediates which are observed during amyloidogenesis of this protein are assembled into various fibrillar structures via specific pathway called unit assembly. These amyloid fibrils have different physicochemical properties corresponding to amyloid polymorphism. Furthermore, unit assembly of α-synuclein oligomers on the surface of liposome induces the formation of radiating amyloid fibrils, resulting disruption of lipid membrane structure. Therefore, it can be considered that unit assembly of oligomers on the lipid membrane is the cause of α-synuclein-mediated cellular degeneration in a molecular level.
In this study, α-synuclein interactive molecules have been investigated in order to control the oligomer-mediated cytotoxicity. First of all, mutual effect of α-synuclein and protein partners such as firefly luciferase (LUC) and glutathione peroxidase-1 (GPX-1) are investigated to unveil their involvement in the PD pathogenesis in terms of fibrillar polymorphism and cellular antioxidant defense mechanism. First of all, LUC and GPX-1 bind to α-synuclein with Kd of 8.1 μM and 17.3 nM, respectively and they are shown to accelerate the fibrillation and induce amyloid polymorphism of α-synuclein by acting as a novel template. In case of LUC-directed amyloid fibrils, altered morphological characteristics were inherited to next-generations via nucleation-dependent fibrillation process. The seed control, therefore, would be an effective means to modify amyloid fibrils with different biochemical characteristics. In addition, the LUC-directed amyloid fibrillar polymorphism also suggests that other cellular biomolecules are able to diversify amyloid fibrils, which could be self-propagated with diversified biological activities, if any, inside cells. Mutual interactions of α-synuclein and GPX-1 contributed to not only accelerated α-synuclein fibrillation but also enhancement of GPX-1 activity. Furthermore, the entrapped GPX-1 in the fibrillar meshwork of α-synuclein was protected in a latent form and its activity was fully recovered as released from the matrix. Therefore, novel defensive roles of α-synuclein and its amyloid fibrils against oxidative stress are suggested as the GPX-1 stimulator and the active depot for the enzyme, respectively.
In this study, several phenolic compounds are examined to control the effects of oligomeric species on cellular membrane and cell viability. First of all, specific oligomer which is β-sheet free, can be assembled into curly amyloid fibril (CAF) through repetitive membrane filtration and instantly transform into radiating amyloid fibril (RAF) on phosphatidylcholine liposome is defined as active oligomer. The changes of tyrosine intrinsic fluorescence of α-synuclein have shown that phenolic compounds such as resveratrol (RSV), curcumin (CUR) and (-)-epigallocatechin gallate (EGCG) directly interact with α-synuclein with Kd of 30.4 mM, 133 mM and 100 mM, respectively. According to the Coomassie Brilliant Blue-stained SDS-PAGE and CD spectroscopy, phenolic compounds have significant influences on the fibrillation of α-synuclein by either facilitating the formation of compact oligomers or accelerating the formation of amyloid fibrils with high β-sheet contents. Furthermore, phenolic compounds could effectively control the formation of CAF and RAF and then reduce the cytotoxicity of active oligomer.
Robust polydiacetylene-based colorimetric sensing material has been developed with amyloid fibrils of α-synuclein in the presence of 10,12-pentacosadiynoic acid (PCDA) by taking advantage of specific fatty acid interaction of α-synuclein and structural regularity of the self-assembled product of amyloid fibrils. PCDA facilitated not only self-oligomerization of α-synuclein but also its fibrillation into the fibrils with increased thickness. Upon UV irradiation, the PCDA-containing amyloid fibrils (AF/PCDA) turned to blue, which then became red following heat treatment. The blue-to-red color transition was also observed with other stimuli of pH and ethanol. AF/PCDA were demonstrated to be mechanically stable since not only the individual colors of blue and red but also their colorimetric transition were not affected by a number of sonications which readily disrupted the polydiacetylene (PDA) vesicles with instant loss of the colors. Therefore, AF/PCDA can be considered as a novel PDA-based colorimetric sensing material with high mechanical strength, which has potential to be employed in various areas involving advanced sensing technologies. Furthermore, litmus-type sensors capable of detecting temperature and organic solvents have been developed with α-synuclein amyloid fibrils and PCDA. The PCDA molecules localized on the amyloid fibrils through either co-incubation with monomeric α-synuclein (AF/PCDA) or simple mixing with the pre-made α-synuclein amyloid fibrils (AF+PCDA) were photopolymerized with UV to exhibit blue color on paper. The paper sensors prepared with AF/PCDA and AF+PCDA showed distinctive sensitivities toward temperature and solvents. The less tight binding of PCDA to amyloid fibrils and thus increased its molecular freedom would be responsible for the discrete sensing property. In addition, the AF+PCDA sensor was demonstrated to successfully follow ascending temperature change, which could allow the litmus-type sensor to monitor thermal history of materials. Taken together, the amyloid fibrils of α-synuclein are shown to be a decent template for PCDA to develop into the litmus-type thermochromic and solvatochromic sensors.