人类大脑中 KLK8 特异剪接体(Type Ⅱ)的进化遗传机制研究及其功能分析
文献类型：学位论文
| 作者 | 卢志祥 |
| 学位类别 | 博士 |
| 答辩日期 | 2009-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 宿兵 |
| 关键词 | KLK8 type Ⅱ(Neuropsin) 可变剪接 人类大脑进化 突变 |
| 其他题名 | Genetic Mechanism and Functional Characterization of the Novel Splice Form (Type Ⅱ) of KLK8 in Human Brain |
| 中文摘要 | 可变性剪接在调节基因表达和增加蛋白多样化方面具有重要的作用。它也是高等生物基因组进化中产生新蛋白的主要机制之一。人类大脑――适应性进化的产物，则更加倾向运用可变性剪接的策略来行使其高度复杂的功能。因此，鉴定出人类中枢神经系统中的特异性剪接体对于我们理解人类认知的功能进化有着相当重要的意义。 KLK8 (Kallikrein 8， 又名neuropsin)是中枢神经系统中参与大脑学习记忆的一种丝氨酸蛋白酶。以前的研究表明，这个基因的剪接形式在人和小鼠中是不同的，在人脑中表达一种特有的更长的剪接形式（type Ⅱ）。而序列分析又提示这个更长的剪接形式在灵长类中是最近起源的。本论文的研究目的：（1）揭示在人类大脑进化过程中，type Ⅱ特异剪接体产生的分子遗传机制；（2）了解type Ⅱ的蛋白异型体比原先KLK8蛋白的typeⅠ异型体有何新的功能特点。 我们发现type Ⅱ是一种人特有的剪接体，在其它非人灵长类的大脑中没有表达。运用体外剪接实验，我们证实：一个人类特有的T到A单碱基突变改变了KLK8的剪接模式，使得type Ⅱ这一崭新的剪接体在人类大脑中产生。体外突变体的剪接实验则进一步证实了这一单碱基突变是type Ⅱ表达的充要条件。此外，运用突变实验我们也证实了多个位点参与削弱KLK8原有组成性剪接位点的剪接效率。我们估计在灵长类进化过程中，新剪接位点的产生和原组成性剪接位点的削弱共同造成了KLK8剪接的变化，这就表明KLK8经过多个演化步骤最终才导致了type Ⅱ在人大脑中的表达。5’RACE，启动子区域序列分析以及启动子的活力检测则表明KLK8的转录调节在进化过程中一直在动态变化。此项研究揭示了在人类进化过程中，中枢神经系统中通过可变剪接产生新蛋白的遗传学分子机制。 除此之外，RT-PCR和western blot实验表明特异剪接体的表达是时空依赖性的，它的分泌效率也与细胞类型相关。生物化学和酶学实验则表明type Ⅱ不仅能够产生原有KLK8的活性形式蛋白，它还有type Ⅱ异型体特有的一个中间蛋白体，提示我们人类大脑中type Ⅱ蛋白的形成可能会对原有蛋白起一定的功能修饰作用。 |
| 英文摘要 | Alternative splicing is critical for the expansion of proteomic diversity and the regulation of gene expression. It is also one of the major mechanisms in the genome for the creation of new proteins during evolution. The human brain, widely accepted as a product of adaptive evolution, preferably utilizes this strategy in its functional complexity. Hence, identifying human-specific alternative splicing forms in central nervous system (CNS) is thus important for understanding the mechanisms of functional evolution of human cognition. KLK8 (Kallikrein 8, or called neuropsin) is a serine protease functioning in CNS involved in learning and memory. Previous studies show that its splicing pattern is different between human and mouse, with the longer form (type Ⅱ) only expressed in human. Sequence analysis also suggested a recent origin of type Ⅱ during primate evolution. In this study, we aim to understand 1) the mechanism of the creation of the novel form KLK8 type Ⅱduring human brain evolution, 2) the new functional character of the novel form compared to original protein isoform (KLK8 type Ⅰ). We find that the type Ⅱis absent in nonhuman primates, and is thus a human-specific splice form. With the use of an in vitro splicing assay, we show that a human-specific T to A mutation triggers the changes of splicing pattern, leading to the origin of a novel splice form in human brain. Using mutation assay, we prove that this mutation is not only necessary but also sufficient for type Ⅱ expression. In addition, using mutagenesis we also prove that multiple sites weaken KLK8’s original constitutive splicing. It is likely that both the creation of novel splice form and the weakening of constitutive splicing contribute to the splicing pattern changes during primate evolution, which suggests a mutistep process eventually leading to the origin of the typeⅡform in human. 5’RACE assay, promoter sequence analysis and promoter activity experiment indicate that transcription regulation of KLK8 is a dynamic process during evolution. Our results demonstrate a molecular mechanism for the creation of novel proteins through alternative splicing in CNS during human evolution. Additionally, RT-PCR and Western blot show that this novel form is expressed in a temporal-spatial manner and secretion efficiency is cell-type dependent. Biochemical assays and enzyme activity experiment indicate that KLK8 typeⅡnot only can produce the active form of KLK8, but also contains a type Ⅱ-specific intermediate protein form, which suggests that the emergence of type Ⅱ KLK8 in the human brain likely leads to functional modifications of KLK8. |
| 语种 | 中文 |
| 公开日期 | 2010-10-22 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6311]  |
| 专题 | 昆明动物研究所_比较基因组学 |
| 推荐引用方式 GB/T 7714 | 卢志祥. 人类大脑中 KLK8 特异剪接体(Type Ⅱ)的进化遗传机制研究及其功能分析[D]. 北京. 中国科学院研究生院. 2009. |
入库方式： OAI收割
来源：昆明动物研究所
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