PINK1/Parkin-Ub复合物的构建、表达与纯化

Construction，Expression，and Purification of PINK1/Parkin Ub Complex

摘要

PINK1 是定位于线粒体外膜的丝氨酸/苏氨酸蛋白激酶，在氧化损伤或膜电位崩溃时从线粒体基质转位至外 膜，经自磷酸化激活后催化泛素(Ub)生成 pSer65-Ub，通过解除 Parkin 的自抑制构象以启动线粒体自噬，其功能异常 与帕金森氏症(Parkinson’s disease，PD)等神经退行性疾病密切相关．PINK1 招募活化 Parkin 涉及“磷酸化-构象变构- 泛素化”级联反应，在细胞内受到精确调控，但此关键途径的分子机制尚未阐明．本研究创新性地构建基于循环排列 增强型绿色荧光蛋白(cpEGFP)的 PINK1 活性分子探针 pParkin-cpEGFP-pUb，通过将拟磷酸化突变的 Parkin(S65D) 与 Ub(S65D)分别融合至 cpEGFP 的 N 端和 C 端，利用激活态 PINK1 结合探针两端以诱导 cpEGFP 中 β-桶状结构重 组并重新发出绿色荧光的原理，实现复合物形成的实时监测．本研究分别表达赤拟谷盗虫源 PINK1(TcPINK1)激活突变体 TcPINK1^L552R (135-570)及探针蛋白，经亲和层析和分子筛层析纯化后，两者以 2∶1 摩尔比共孵育，经 Superose 6 凝胶过滤层析显示复合物洗脱峰(13.3 mL)显著早于单一组分(15.1 mL 和 14.9 mL)，Western-blot 证实峰内同时含 His 标签的 PINK1 激酶和麦芽糖结合蛋白(MBP)标签的探针组分．通过 0.075%戊二醛在 4 ℃交联 10 min 稳定复合物构 象，负染电镜(Talos L120C)显示颗粒呈直径 15~20 nm 均一构象，成功获得了 PINK1 与探针的稳定复合物，并为后续 研究 PINK1 激活与招募 Parkin 的分子机制提供了基于基因编码的荧光检测工具．

Abstract

PTEN-induced putative kinase 1(PINK1)is a serine/threonine protein kinase. It is localized to the mito- chondrial outer membrane，but translocates from the mitochondrial matrix to the outer membrane upon oxidative dam- age or a collapse in the membrane potential. After autophosphorylation-mediated activation，PINK1 catalyzes the phosphorylation of ubiquitin(Ub) to generate pSer65-Ub，which releases the E3 ubiquitin-protein ligase(Parkin) from an autoinhibited conformation to initiate mitophagy. Dysfunctions in this process are strongly implicated in the pathogenesis of neurodegenerative disorders，including Parkinson’s disease. The recruitment and activation of Parkin by PINK1 involves a “phosphorylation-conformational change-ubiquitination” cascade that is stringently regulated. However，the molecular mechanisms underlying the control of this critical pathway remain poorly understood. This study adopted a novel methodology by utilizing circularly permuted enhanced green fluorescent protein(cpEGFP)to create a PINK1 activity-sensing molecular probe，pParkin-cpEGFP-pUb，by fusing the phosphomimetic Parkin (S65D)and Ub(S65D)to the N- and C-termini of cpEGFP. The activated PINK1 bound to both ends of the probe， inducing a β-barrel restructuring of the cpEGFP and restoring green fluorescence，thereby enabling the real-time moni- toring of complex formation. We expressed the hyperactive Tribolium castaneum PINK1 mutant (TcPINK1L552R，residues 135-570)along with the probe. After purification via affinity and molecular sieve chromatography，co-incubation at a molar ratio of 2∶1 yielded a complex with a distinct elution peak(13.3 mL)upon Superose 6 gel filtration，much ear- lier than the individual components(TcPINK1^L552R：15.1 mL；probe：14.9 mL). Western blot confirmed the presence of His-tagged PINK1 and maltose binding protein(MBP)-tagged probe within the complex corresponding to the peak. Chemical cross-linking，achieved by treating with 0.075% glutaraldehyde for 10 min at 4 ℃，resulted in particles with uniform conformations(15 — 20 nm in diameter) ，which were visualized using negative-stain electron micros- copy(Talos L120C). A stable PINK1-probe complex was obtained，which provides a gene-encoded fluorescent tool for detecting the molecular mechanisms underlying the activation and recruitment of Parkin by PINK1 in further studies.

关键词

帕金森氏症 / PINK1/Parkin 通路 / 循环排列荧光蛋白

Key words

Parkinson’s disease / PINK1/Parkin pathway / circularly permuted fluorescent protein

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基金资助

国家自然科学基金资助项目(31400645)