赖氨酸甲基转移酶SMYD2在BMP信号通路调控中的作用及分子机制研究

发布时间：2018-03-23 01:00

本文选题：SMYD2　切入点：BMPR2　出处：《华东师范大学》2017年博士论文　论文类型：学位论文

【摘要】：近几年的研究发现,曾经被认为主要发生于组蛋白的赖氨酸甲基化修饰,在许多非组蛋白上也广泛存在,并且越来越多的研究表明,赖氨酸甲基化修饰可能像磷酸化、乙酰化修饰一样,广泛存在并调控蛋白质结构与功能。在已经鉴定到的赖氨酸甲基化底物中,大多数是与染色质相关的核蛋白,而与信号通路相关的细胞质蛋白和膜蛋白比较少。这可能与大部分已知赖氨酸甲基转移酶主要分布于细胞核有关。因此,本研究旨在于探索非组蛋白赖氨酸甲基化修饰在调控细胞信号通路中的作用。TGF-β超家族主要由TGF-β和BMP信号分子组成,其信号通路不仅影响细胞的增殖、分化、自稳态的维持,还在胚胎发育、骨形成与重建、某些器官的形成等生命过程中起重要作用。TGF-β超家族信号通路的异常往往会伴随疾病的发生,如家族性肺动脉高血压常与Ⅱ型受体BMPR2突变相关,肿瘤的发展常伴随TGF-β通路相关蛋白的突变。SMYD家族甲基转移酶是含有一个被分割成两段的SET结构域的甲基转移酶家族,它们多数具有细胞质定位,并且修饰多种非组蛋白底物。本论文首先通过RNA干扰技术筛选SMYD家族中对TGF-β和BMP通路有影响的甲基转移酶,发现SMYD家族中SMYD2的敲低能够特异性地影响BMP通路的激活,调控SMAD1/5磷酸化水平,而不影响TGF-β通路。随后我们用shRNA敲低和CRISPR-Cas9敲除SMYD2验证了 SMYD2对BMP通路激活的特异作用。进一步研究表明敲低或者敲除SMYD2后BMP诱导的SMAD1入核减少,并且SMAD1与SMAD4的相互作用减弱,说明SMYD2能够调控SMAD1的磷酸化水平及其功能,进而调控BMP通路的活性。SMYD2酶活抑制剂能够抑制MBP诱导的SMAD1/5磷酸化,说明SMYD2对SMAD1/5磷酸化的调控依赖其甲基化酶活性。为了研究SMYD2调控BMP通路的分子机制,我们首先检测了 SMYD2与BMP通路成员的相互作用,发现SMYD2虽然与SMADs有广泛的相互作用,但只有与SMAD1的相互作用在BMP通路激活时增强;另一方面我们也检测了SMYD2是否甲基化修饰BMP通路成员,发现SMYD2能特异甲基化BMP通路特异的Ⅱ型受体BMPR2,并且甲基化位点位于BMPR2激酶活性区域。此外,我们发现在BMP信号通路激活过程中存在BMPR2甲基化。总的来说,我们的研究表明,甲基转移酶SMYD2可能通过甲基化BMPR2,从而影响其功能,进而调控BMP通路的激活。
[Abstract]:The recent study shows that, once considered lysine methylation occurs mainly in histones, also widely exist in many non histone proteins, and more and more studies show that lysine methylation may like phosphorylation, acetylation, and widespread protein structure and functional regulation have been identified. In the lysine methylation substrate, mostly associated with chromatin nuclear protein, while less cytoplasmic and membrane proteins associated with signal transduction. This may be related to most of the known lysine methyltransferase is mainly distributed in the nucleus. Therefore, the purpose of this study is to explore the non histone lysine methylation in the regulation of cell signaling pathways in the role of the.TGF- superfamily consists of TGF- beta and BMP signal molecules, its signal pathway not only affect cell proliferation, differentiation, homoeostasis, also During embryonic development, bone formation and reconstruction of some organs such as the formation of life plays an important role in.TGF- abnormal beta superfamily signaling pathway is often followed by the occurrence of diseases, such as familial pulmonary hypertension and type II receptor BMPR2 mutation is related to tumor development, often accompanied by TGF- beta pathway related protein mutation.SMYD family methyltransferase containing a SET domain is divided into two sections of the methyltransferase family, most of them have cytoplasmic localization, and modification of various non histone substrates. This paper firstly by methyl RNA interference screening SMYD family have effect on TGF- beta and BMP pathway enzymes, found the SMYD2 SMYD family the knockdown can specifically affect the activation of BMP pathway, regulates the phosphorylation level of SMAD1/5, without affecting the TGF- beta pathway. Then we use shRNA knockdown and CRISPR-Cas9 knockdown of SMYD2 proved SMYD The activation of the BMP pathway specific role 2. Further studies show that knockdown or knockout induced by BMP after SMYD2 SMAD1 into the nucleus is reduced, and the interaction between SMAD1 and SMAD4 decreased, indicating that SMYD2 can phosphorylation and functional regulation of SMAD1, and the activity of.SMYD2 is regulated by BMP pathway. SMAD1/5 inhibitors can inhibit the phosphorylation of MBP induced, it indicated that SMYD2 regulates SMAD1/5 phosphorylation dependent methyltransferase activity. In order to study the molecular mechanisms of SMYD2 regulation of the BMP pathway, we first examined the interaction between SMYD2 and members of the BMP pathway, SMYD2 found that although there are extensive interaction with the SMADs, but only the interaction with SMAD1 in enhanced activation of BMP when; on the other hand we also examined whether SMYD2 methylation of BMP pathway members found SMYD2 methylation specific BMP pathway specific type II receptor BMPR2, and methylation. In the BMPR2 kinase activity region, we also found that BMPR2 methylation existed in the activation of BMP signaling pathway. In general, our study showed that methyltransferase SMYD2 may affect its function through methylation of BMPR2, thereby regulating the activation of BMP pathway.

【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q23

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