人类神经生长抑制因子的结构、性质与功能

【作者】 蔡斌；

【导师】 黄仲贤；

【作者基本信息】 复旦大学 ， 无机化学， 2006， 博士

【摘要】 人类神经生长抑制因子（human neuronal growth inhibitory factor，hGIF）属于金属硫蛋白家族，又称金属硫蛋白-3（metallothionein-3，MT3）。它由68个氨基酸组成，包含20个保守的半胱氨酸。和其它金属硫蛋白成员一样，hGlF能结合7份二价d¹⁰金属离子诸如Zn²⁺、Cd²⁺，形成两个相对独立的结构域，每个结构域分别包裹着一个金属硫簇，结构域之间由一段3个氨基酸组成的linker联接，整个分子呈哑铃形状。hGIF主要在中枢神经系统表达，有研究显示大脑皮层拥有大量富含游离锌的神经元，hGIF的分布同这些神经元的分布基本一致，暗示hGIF可能参与游离Zn²⁺的细胞内传递与调控。老年痴呆症是一种神经退行性疾病，病人脑提取液中hGIF的表达水平明显低于正常人，这导致神经元不受约束的生长，过度消耗了营养，最终使神经元调亡；此外，对动物模型的研究显示，当大脑受到损伤后hGIF的表达量出现明显变化。这些研究说明hGIF可能与神经元的生长调控和修复有关。尽管关于hGIF抑制神经元生长的机理以及它的靶分子尚且不清楚，种种迹象表明多种因素共同参与调节其生理活性。本实验室已有多年研究金属硫蛋白的历史，曾经在大肠杆菌中构建了金属硫蛋白的高效表达体系，并且优化了纯化步骤，获得很高的蛋白产量；通过基因工程技术，本实验室还曾经构建了一系列hGIF的突变体，并且详细研究了结构与蛋白稳定性、反应性之间的关系。在此基础上，为进一步阐明hGIF结构、性质和功能之间的关系，我们全面而系统的考察了hGIF一系列位点上的突变对神经元生长抑制活性的影响。这些突变体包括：1)针对肽链N端β结构域（1-30）保守的Thr5设计的突变体（AT5、T5A、T5S）；2)针对肽链N端β结构域保守的CPCP序列的突变（P7S／PgA）3)围绕保守的Glu4位点的突变（E4W、△E4、P3S／E4R）；4)NO反应的酸碱催化位点的突变（E23K）；5)围绕肽链C端α结构域（34-68）的EAAEAE插入段设计的突变体（△55-60、E58K、E55／58／60Q、G53／54A）：6)对linker进行改造（△31-34、△32-33、KKS-SP）；7)结构域间的转换杂合（△34-36、βMT3-βMT3、βMT3-αMT1）；8)单结构域（β-domain、α-domain）等。活性测试研究表明，1)β结构域对hGIF的神经元抑制活性是必须的，其中hGIF独有的保守T（5）CPCP（9）序列尤其重要。2)我们还发现，Thr5侧链上的羟基基团同生理活性密切相关，人为地除去羟基将导致突变体生物活性严重丧失。参考突变体蛋白结构和反应性的结果，我们认为一方面Thr5的羟基可能通过形成分子内氢键帮助稳定hGIF的结构，另一方面Thr5也是一个潜在地磷酸化位点。3)再次，虽然保留了TCPCP序列，但是E23K突变体完全失去了神经元生长抑制功能。以往的研究指出，E23K除了金属硫簇的暴露程度提高，其光谱学性质和反应性均和野生型hGIF相似。尽管E23K丧失生理活性的原因有待进一步研究，我们的结果说明除了TCPCP序列，β结构域中的其它残基也可以影响hGIF执行其功能。4)我们还发现，在α结构域中，改变E（55）AAEAE（60）插入段的电荷分布，或者增加它形成α螺旋的倾向，并不太多地影响整个突变体蛋白的生物活性。然而，EAAEAE插入段缺失干扰了蛋白的正常活性。对α结构域的结构解析表明，EAAEAE插入段在hGIF蛋白中是一段非常松散、不受约束的无规卷曲，它使得α结构域的动力学性质更加活泼。进一步，α结构域也通过结构域间的相互作用，影响到β结构域，间接的改变了β结构域的动力学性质和其它相关性质，从而干扰了hGIF蛋白的生理活性。5)最后，在hGIF中linker的部分或完全缺失对活性没有太大的影响，可是，当用SP（甲壳纲动物MT的linker）取代KKS（哺乳动物MT的linker）作为linker时，突变体KKS-SP的生物活性就基本丧失了。从进化的角度看，这也是自然界为什么选用保守的KKS作为linker的一个原因。有研究指出，在酵母双杂交实验中hGIF能与Rab3A蛋白相互作用，并且hGIF和GDP／Rab3A的结合具有特异性（K_D=2.6μM）。Rab3A属于Ras家族，能够结合GTP，并具有GTP水解酶活性。当Rab3A与GTP结合时，GTP／Rab3A形成具有活性的蛋白构象，通过和下游蛋白相互作用引发一系列过程，调节神经元突触囊泡的运输；当Rab3A将结合的GTP水解成GDP后，GDP／Rab3A结构的构象发生变化，蛋白失去活性。同时，Rab3A和GDP或GTP的这种结合也受到多种蛋白的调控，包括GEPs（guanine nucleotide exchange proteins）、GAPs（GTPase-activating proteins）和GDIs（GDP dissociation inhibitors）等。目前为止，对Rab3A和hGIF之间关系的了解还非常少。为了探索它们之间的相互作用，我们运用分子克隆构建了GST-Rab3A融合蛋白表达系统质粒，蛋白在大肠杆菌中表达，纯化后蛋白分子量经MS验证正确无误。随后，我们对hGIF和Rab3A的相互作用进行了初步的研究。GIF（即MT3）和金属硫蛋白Ⅰ和Ⅳ（MT1，MT4）都属于金属硫蛋白家族，它们的一级序列具有很高的同源性，可是，只有GIF表现出神经生长抑制活性。GIF的这种生理活性是否同它金属硫簇的结构、性质有关呢?为此，我们考察了hGIF金属硫簇的结构、性质，并同人类MT1g（hMT1g）、小鼠MT4（mMT4）进行了比较。结果表明，hMT1g和mMT4的金属硫簇具有相似的结构和稳定性，而相比之下，hGIF金属硫簇结构有所差异，且稳定性降低；其次，hGIF和hMT1g、mMT4之间金属硫簇结构和稳定性的差异，导致其和巯基试剂的反应性也受到影响；最后，不同蛋白传递金属离子至EDTA的能力依次为hGIF＞hMT1g＞mMT4。总体来看，hGIF在溶液中结构比hMT1g和mMT4更加松散。我们认为，hGIF的这种性质是几个因素共同作用的结果：hGIF相对较多的负电荷，EAAEAE插入段，CPCP保守序列，以及结构域之间的相互作用等，都可能对hGIF的金属硫簇结构和性质产生影响，并进一步影响其功能。总之，通过构建突变体蛋白、神经元培养和活性考察、结构性质研究等一系列手段，我们研究了可能影响hGIF神经元生长抑制活性的各结构基元和特定的氨基酸残基，认为hGIF的生理活性受到多种因素调控，包括Trh5羟基基团、CPCP保守序列、结构域间的相互作用、linker影响、金属硫簇的结构性质等等。更多还原

【Abstract】 Human neuronal growth inhibitory factor （hGIF）, also named as metallothionein-3 （MT3）, is a member of metallothionein （MT） family. This protein consists of 68 amino acid residues, including 20 conserved cysteine residues similar to the other members of MT family. Moreover, human GIF can bind 7 divalent metal ions such as Zn²⁺ and Cd²⁺, and forms two relatively independent domains, each wrapping around a metal-thiolate cluster. There is a 3-amino-acid linker between these two domains, and the whole molecule presents a dumbbell-like shape. Human GIF is mainly expressed in the central nerves system. In the brain cortex, the distribution of zinc-rich neurons is in accordance with that of human GIF protein, suggesting that human GIF may participate in transport and homeostasis of cellular zinc ions. Alzheimer’s disease （AD） is a neuron degenerative disease. It was reported that the level of human GIF in the brain extract of patients with AD is significantly lower than that of healthy people, which can stimulate overgrowth of neuron cells and finally lead to neuron death because of exhaustion of neurotrophic factor. Moreover, animal models show that the expression level of GIF obviously alters after brain injury, indicating that human GIF may take part in the process of neuron growth and regeneration. Although the mechanism of neuron inhibitory activity of human GIF is unclear and the target of human GIF is uncertain, more and more evidences indicate that the bioactivity of human GIF is regulated by several factors cooperatively.This lab has studied the structural-functional relationship of MTs for many years: An MT expression system in E coli with high yield has been established, and the purification process has been optimized; a series of human GIF mutants was generated by molecular biology method, and their structure, stability and reactivity were investigated in detail. To further clarify the connections between protein structure, biochemical properties and bioactivity, we have conducted a systematical study on the effects of mutations at different sites of human GIF on the inhibitory activity toward neurons. These mutants include: 1) Thr5 insert mutants in the β-domain, i.e. ΔT5, T5S, T5A; 2) mutant focuses on the conserved CPCP motif in theβ-domain, i.e. P7S/P9A; 3) Glu4 mutants in the β-domain, including E4W, ΔE4 and P3S/E4R; 4) mutant focuses on the acid-base catalytic site of nitrosylation in the β-domain, i.e. E23K; 5) the hexapeptide insert mutants in the α-domain of hGIF, including Δ55-60, E58K, E55/58/60Q and G53/54A etc.; 6) the linker mutants, including Δ31-34, Δ32-33 and KKP-SP; 7) domain hybridized mutants, i.e. Δ34-36, βGIF-βGIF, and βGIF-α-MT1; 8) single domain, i.e. β-domain and α-domain of human GIF.Results of the neuron culture assay proved that 1) the β-domain is indispensable for the neuronal inhibitory activity of human GIF, and the conserved CPCP motif in the β-domain is particularly important. 2) We also found out that the hydroxyl group in Thr5 is critical for the bioactivity. Deleting the hydroxyl group leads to almost completely loss of bioactivity of the T5A mutant. Based on the structure and reactivity information, we conclude that the hydroxyl group in Thr5 may stabilize the solution structure of human GIF by forming intra-molecular hydrogen bond. On the other hand, Thr5 can also serve as a potential phosphorylation site. 3) The mutant E23K loses its bioactivity, although it keeps the critical TCPCP motif. It was pointed out that except the increasing solvent accessibility of the metal-thiolate clusters, the spectroscopic properties and the reactivity of E23K are similar to those of human GIF. It can be inferred that besides the TCPCP motif, other residues in the β-domain also contribute to the bioactivity of human GIF. 4) As we observed, changing charge distribution or increasing α-helix inclination of the EAAEAE insert in the α-domain has little effect on the bioactivity of human GIF. However, deleting this hexapeptide will impair seriously the bioactivity of human GIF. Structural analysis of the α-domain of human GIF reveals that the EAAEAE insert in the α-domain lies on an extremely flexible loop with no constraint by the metal-thiolate cluster, leading to enhancement of dynamics in the α-domain of human GIF. The increased dynamics of the α-domain will affect the properties of the β-domain through inter-domain interaction, and therefore the bioactivity of human GIF β-domain is regulated by the α-domain. 5) Finally, it is observed that the absence of linker between two domains does not significantly alter the bioactivity of mutants （in the Δ31-34 and Δ32-33 mutants ofhGIF）. However, replacement of KKS （mammalian MT linker） with SP （crustacean MT linker） causes total loss of the bioactivity. The mechanism for this phenomenon is not sure, but from the point of view of evolution, this may provide a reason for why mammalian MTs choose KKS as the linker.It was reported that human GIF can interact with Rab3A in the two-hybrid-yeast screen. Moreover, human GIF binds to GDP/Rab3A specifically （K_d - 2.6 μM）. Rab3A belongs to Ras family. It can bind GTP and has GTP hydrolyzing activity. When Rab3A forms GTP/Rab3A, it can interact with downward targets to regulate trafficking of synaptic vesicle. After GTP is hydrolyzed to GDP, the GDP/Rab3A is inactive. The GDP/GTP exchange rate in Rab3A is controlled by several proteins, including guanine nucleotide exchange proteins （GEPs）, GTPase-activating proteins （GAPs）, and GDP dissociation inhibitors （GDIs）. Till now little is known about the interaction between Rab3A and human GIF. To study the mechanism of their interaction in vitro, we cloned Rab3A cDNA into GST fusion protein expression plasmid, and the GST-Rab3A fusion protein was expressed in E coli. After purification the molecular weight of GST-Rab3A is verified by mass spectroscopy, and the interaction between human GIF and Rab3A was investigated.GIF, MT1, and MT4 all belong to MT family. They exhibit high sequence identity, while only GIF has the unique neuronal growth inhibitory activity. Is the bioactivity of human GIF related to the structure and properties of its metal-thiolate clusters? Therefore we investigated the structure and properties of metal-thiolate clusters in human GIF, and compared with those of clusters in human MT1 and mouse MT4. The results show that human MT1 and mouse MT4 have similar cluster structure and stability, while the clusters in human GIF have different structure and lower stability. The ability of human GIF clusters to interact with thiol regents is also affected by the alteration of cluster structure and stability. Furthermore, the metal transfer ability of the three proteins follows human GIF > human MT1 > mouse MT4. These results indicate that the solution structure of human GIF is much looser than those of the other two MTs. Several cooperative factors may be attributed to the effect: the overall more negative charge of human GIF, the EAAEAE insert, the CPCP motif, and theinter-domain interaction et al.In summary, we have studied the structure-reactivity-function relationship of human GIF using chemical and biological tools, including protein engineering, spectroscopic characterization, and primary neuron culture assay and so on. The neuronal growth inhibitory activity of human GIF is regulated by multiple factors cooperatively, including the hydroxyl group in Thr5, the conservative CPCP motif, the inter-domain interaction, the linker, and the metal-thiolate clusters etc.更多还原