碱化修饰甲状腺素运载蛋白显著增强对淀粉样β蛋白聚集的抑制作用

doi:10.11949/0438-1157.20230144

摘要/Abstract

摘要：

阿尔茨海默症与淀粉样β蛋白（amyloid-β protein，Aβ）的纤维化聚集有关，因此开发高效的抗Aβ聚集的抑制剂是防治阿尔茨海默症的策略之一。研究发现，甲状腺素运载蛋白（transthyretin，TTR）可通过疏水作用抑制Aβ单体之间的聚集，但抑制作用需要较高蛋白浓度（150 μg·ml^-1）。为了提高TTR抑制Aβ聚集的作用，本研究利用乙二胺修饰TTR表面的羧基，生成碱化甲状腺素运载蛋白（TTR-B），以增加其与带负电荷的Aβ之间的静电相互作用。研究表明，修饰度最高的TTR-B3（平均每分子TTR上38.9%的羧基被转换成氨基）对Aβ聚集的抑制能力显著提高，在较低的浓度下（15 μg·ml^-1）即可有效缓解Aβ对细胞的毒性（使细胞活性从78%提高至>90%），浓度仅为TTR的10%。与碱化人血清白蛋白（HSA-BF）相比，TTR-B3用量为HSA-BF的75%时即可使AD线虫延长相同的寿命，在HSA-BF用量的45%时即可完全清除AD线虫体内的Aβ斑块。结果表明TTR-B3是一种高效的Aβ聚集抑制剂。

关键词: 阿尔茨海默症, 蛋白质, 淀粉样β蛋白, 甲状腺素运载蛋白, 聚集, 生物医学工程

Abstract:

Alzheimer's disease (AD) is considered to be closely associated with the fibrillar aggregation of amyloid-β protein (Aβ). Therefore, development of efficient inhibitors against Aβ fibrillization is one of the strategies to combat AD. Previous studies have found the inhibition effect of transthyretin (TTR) on Aβ fibrillization through hydrophobic interactions, but it only showed high inhibitory potency at quite high concentrations (150 μg·ml^-1). To elevate the potency of TTR on inhibiting Aβ aggregation, this work used the basification method by modifying TTR with ethylenediamine via its reaction with the carboxyl groups on the protein surface. The TTR basification could increase the electrostatic interactions between basified TTR (TTR-B) and the negatively charged Aβ. Studies have shown that TTR-B3 with the highest degree of modification (on average 38.9% of the carboxyl groups on TTR per molecule are converted to amino groups) has a significantly improved ability to inhibit Aβ aggregation. The potency of TTR-B3 was much more efficient than native TTR. TTR-B3 alleviated the toxicity of Aβ aggregates and increased the cultured cell viability from 78% to >90% at 15 μg·ml^-1, which was only 10% of native TTR. As compared to basified human serum albumin (HSA-BF), TTR-B3 also worked at significantly lower dosages. TTR-B3 extended the same lifespan of AD nematodes at 75% of HSA-BF, and completely scavenged Aβ plaques in AD nematodes at 45% of HSA-BF. The results indicated that TTR-B3 is a highly effective inhibitor of Aβ aggregation.

Key words: Alzheimer's disease, protein, amyloid-?β protein, transthyretin, aggregation, biomedical engineering

中图分类号:

TQ 460.1

侯文起, 孙彦, 董晓燕. 碱化修饰甲状腺素运载蛋白显著增强对淀粉样β蛋白聚集的抑制作用[J]. 化工学报, 2023, 74(5): 2100-2110.

Wenqi HOU, Yan SUN, Xiaoyan DONG. Basification modification of transthyretin significantly enhances inhibitory effect on amyloid-β protein aggregation[J]. CIESC Journal, 2023, 74(5): 2100-2110.

图/表 11

图1 TTR-B的制备(灰色片段及下划线标记为TTR疏水区域;加粗标记为可能被修饰的酸性氨基酸残基)

Fig.1 Preparation of TTR-B

图2 TTR纯化的结果分析

Fig.2 Analysis of purification results of TTR

图3 TTR-B的表征

Fig.3 Characterization of TTR-B

表1 TTR和TTR-B的物理化学性质

Table 1 Physicochemical properties of TTR and TTR-B

样品	羧基数目	氨基数目	修饰度/%	Zeta电势（pH 7.4）/mV
TTR	18	9	0	0.017
TTR-B1	16	11	11.1±0.6	1.64±0.02
TTR-B2	13	14	27.8±1.7	3.51±0.05
TTR-B3	11	16	38.9±1.1	4.60±0.12

图4 不同浓度TTR和TTR-B对Aβ40聚集的影响(单独Aβ40或加入不同浓度抑制剂在37℃培养160 h后，测定Aβ40的相对ThT荧光强度；Aβ40浓度为25 μmol·L-1；单独Aβ40培养160 h后的荧光为100%)

Fig.4 Effect of different concentration of TTR/TTR-B on Aβ40 aggregation

图5 不同浓度TTR和TTR-B3对Aβ40聚集动力学的影响

Fig.5 Effect of different concentration of TTR/TTR-B3 on Aβ40 aggregation kinetics

表2 不同条件下Aβ40聚集动力学的延滞期

Table 2 Lag phase time （Tlag） of Aβ40 aggregation kinetics in different conditions

样品	T_lag/h
单独Aβ40	35.9±0.7
Aβ40+1 μmol·L^-1 TTR	43.1±0.3
Aβ40+5 μmol·L^-1 TTR	59.5±0.3
Aβ40+10 μmol·L^-1 TTR	65.2±0.4
Aβ40+1 μmol·L^-1 TTR-B3	82.1±0.2
Aβ40+5 μmol·L^-1 TTR-B3	112.4±0.2
Aβ40+10 μmol·L^-1 TTR-B3	-

图6 不同浓度TTR和TTR-B3对Aβ40聚集体形貌的影响

Fig.6 AFM images of 25 μmol·L-1 Aβ40 incubated without or with different concentrations of TTR/TTR-B3 for 160 h

图7 TTR和TTR-B3与Aβ40共培养的圆二色光谱

Fig.7 Far-UV circular dichroism spectra of 25 μmol·L-1 Aβ40 incubated in the absence and presence of different concentrations TTR/TTR-B3 at 160 h

图8 MTT细胞活性实验[仅用PBS缓冲液(100 mmol·L-1,pH 7.4)处理的细胞活力设定为100%;***表示p<0.001]

Fig.8 MTT cell viability of SH-SY5Y cell

图9 线虫实验(CL2006为AD突变型线虫,N2为健康线虫)

Fig.9 Nematode test

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