摘要
c-Myc是一类高度保守的、含螺旋环螺旋结构的蛋白,通过与Max蛋白结合形成异源二聚体而结合E-box,而调控基因的转录。c-Myc是一个具有多重功能的蛋白,它可以调节细胞周期,生长,代谢,分化,凋亡,转化,基因组不稳定性以及血管生成。同样c-Myc在生理及病理过程中如细胞分化、干细胞自我更新与分化、脂肪细胞的分化及肿瘤的发生等过程中发挥着重要作用。
SCP1是RNA聚合酶Ⅱ的C端结构域多肽的小磷酸酯酶Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase1。SCP1(又名NLI-IF, CTDSP1)是一个从果蝇到哺乳动物中都高度保守的磷酸酯酶,能够通过识别底物上保守的SP序列而去磷酸化,调控磷酸化的靶蛋白。已报道SCP1能够在非神经细胞中抑制神经基因的表达；能够调控RNA聚合酶的活性而抑制转录。同时也能够稳定Snail蛋白的表达,而在乳腺癌里促进细胞的迁移。尽管近年来SCP1作为肿瘤抑制因子的生物功能得到了广泛的研究,但其抑制肿瘤发生的具体分子机制及其新的生物学功能仍然有待于进一步研究。
在本论文中,我们运用生物信息学、细胞生物学、分子生物学的技术筛选鉴定了调控c-Myc蛋白去磷酸化的一种新的磷酸酯酶即SCP1。本课题重点研究了c-Myc蛋白去磷酸化的修饰和其稳定性,从生化、细胞、分子水平上研究了SCP1调控c-Myc蛋白去磷酸化的具体分子机制及新的生物学功能；同时也研究了发现了SCP1定位细胞膜和其细胞膜定位的分子机制。
第一部分主要分成以下几章：
一、SCP1调控c-Myc62位苏氨酸的去磷酸化修饰
我们通过磷酸酯酶库筛选出新的调控c-Myc蛋白Ser62的去磷酸化的磷酸酯酶SCP1,并通过体内,体外去磷酸化实验确定了SCP1能够去c-Myc蛋白Ser62的磷酸化。
二、c-Myc的HLH结构和SCP1的C端相互作用的分子机制研究
我们通过体内体,外实验证明了c-Myc和SCP1能够结合,并且在内源蛋白也能够相互作用,通过SCP1的缺失突变体,我们进一步研究证明SCP1的C末端能够和c-Myc蛋白相互作用。同时我也对c-Myc蛋白进行了缺失突变,发现c-Myc的C端同样对和SCP1的相互作用至关重要。
三、SCP1的磷酸化修饰调控c-Myc影响其蛋白稳定性
Ser62磷酸化对c-Myc蛋白的稳定性至关重要,我们通过过表达和Knockdown实验证明,SCP1的去磷酸化修饰导致c-Myc蛋白表达量减弱。而通过之前的实验结果,我们进一步发现SCP1的C末端对其蛋白酶活性至关重要。通过点突变实验,发现SCP1的Thr251和Ser252是影响其酶活性关键氨基酸。
四、SCP1介导c-Myc蛋白的去磷酸化并影响肝癌细胞增殖
通过对SCP1在小鼠组织表达分布,我们挑选肝细胞和肝癌细胞系发现SCP1和c-Myc的表达是负相关的关系。因此我们进一步在相对高表达SCP1细胞系中Knock Down SCP1,通过CHX Pulse chase实验,发现c-Myc表达明显增多：在低表达SCP1的细胞系中过表达SCP1,发现c-Myc蛋白的磷酸化减少,同时通过MTT实验发现存活的细胞也显著减少。综上所述,阐述了SCP1在肝癌中的抑癌基因作用。
第二部分主要分成以下几章：
一、SCP1细胞膜定位的研究
通过免疫荧光实验发现SCP1能够定位细胞膜,并通过生化实验分离细胞膜,从过表达和内源检测SCP1,确定其能定位细胞膜,并通过缺失突变确定SCP1的N端31-55氨基酸是决定其定位细胞膜。
二、SCP1棕榈酰化的研究
通过生物信息学分析,分析出C44,C45和C47可能是潜在的棕榈酰化修饰位点而定位细胞膜。首先通过点突变和棕榈酰化抑制剂,间接证明SCP1可能发生棕榈酰化,进而我们通过ABE实验和同位素标记实验直接证明,SCP1确实发生棕榈酰化修饰,而使SCPl定位于细胞膜。
综上所述,我们运用磷酸酯酶库、通过细胞生物学生化及分子生物学技术、技术成功地筛选并鉴定了调控c-Myc蛋白去磷酸化的一种新的磷酸酯酶SCP1,首次发现了SCP1蛋白对c-Myc蛋白水平的负调控机制。对于进一步理解c-Myc蛋白稳定性及生物功能提供了新的线索和思路。本课题着重研究了SCP1对c-Myc蛋白稳定性的调控,发现磷酸酯酶SCP1能够通过磷酸化依赖的途径,调控促转录因子c-Myc的去磷酸化,在蛋白水平上抑制c-Myc调控的下游基因的表达,在细胞水平上,SCP1抑制了肝癌细胞的增殖。c-Myc在肿瘤发生当中扮演着重要的角色,.发现SCP1对c-Myc蛋白水平的负调控,对于理解肿瘤的发生具有重要意义,可以为治疗肿瘤及其他疾病提供新的靶点。此外,发现并证实SCP1通过棕榈酰化修饰定位于细胞膜,而且这是迄今第一个有文献报道的丝氨酸苏氨酸磷酸酯酶定位与细胞膜的研究,预示SCP1有更多的调控机制来影响肿瘤细胞增殖的进程。
c-Myc is a highly conserved, B/HLH/Zip domain containing oncoprotein. It can regulate its downstream genes transcription through forming a heterologous dimer. c-Myc has multiple functions in human cells, such as regulate cell cycle, proliferation, metabolism, apoptosis, transforming, genomic instability and angiogenesis. In addition, c-Myc also plays an important role in cell differentiation, stem cell self-renew, adipocyte differentiation and tumor genesis.
SCP1is Carboxy-terminal domain RNA polymerase Ⅱ polypeptide a small phosphatase1. It is also called NLI-IF, SCP1, and the phosphatase very highly conserved from flies to mammals that can recognize specific SP phosphorylation site in its target. It has been reported that, SCP1can repress neuronal gene expression in non-neuronal cells. And it can also perform transcript suppression by regulate RNA polymerase activity. Meanwhile, SCP1can also promote cell migration by stable Snail protein. Although recently, some studies have shown that SCP1has a tumor suppressior function. The detailed mechanism and its new biological function have not been revealed.
In this paper, we used technics including bioinformatics, cell biology and molecular biology to screen the new phosphatase regulates c-Myc S62dephosphorylation. We find that SCP1can dramatically dephosphorylate c-Myc S62and promote its protein instability. At the same time, we find that besides its nucleus localization, SCP1also localizates in the cell membrane, and the molecular mechanism is also discussed.
The chapters in part one:
I. SCP1regulates c-Myc Ser62dephosphorylation
We took advantage of a phosphatase library to screen a potential phosphatase that can dephosphorylate Ser62of c-Myc, and used a Ser62phosphorylation antibody to test the phosphorylation status. Then we screened that SCP1can notably dephosphorylates the c-Myc. Then we conformed the result by a series of in vivo and in vitro dephosphorylation assays.
Ⅱ. the HLH domain of c-Myc can bind the c-tail of SCP1
We used in vivo and in vitro assays to show that overexpressed c-Myc and SCP1can be interation each other, as is the endogenous proteins. Through the construct of SCP1deletion mutant, we find out that c-Myc can interact with the c-tail of SCP1. Meanwhile, the deletion mutant of c-Myc showed that the c-term of c-Myc is also important for its interaction with SCP1.
Ⅲ. SCP1regulates c-Myc instability by dephosphorylation
It has been reported that phosphorylation of Ser62is important for its protein stability. Both overexpression and knockdown assay showed that, SCP1can promter c-Myc protein instability through specifically dephosphorylation of c-Myc Ser62. In addition to, we discovered that C-tail of SCP1is of great importance to its phosphatase activity. And Tyr251and Ser252is the critical site.
Ⅳ SCP1suppress liver tumor cell proliferation by promote c-Myc Ser62dephosphorylation We tested SCP1protein expression in mouse organs, and it's highly expresstion in liver. Then we chose normal mouse liver cell and human liver tumor cell and find significant negative correlation between SCP1and c-Myc. What is more, as the expression of SCP1decreases, liver tumor cells proliferation increases.
The chapters in part two:
Ⅰ. the cell membrane localization of SCP1
Unexpected that, we find SCP1localizates on the cell membrane through immunofluorescence assay. And we tested endogenous SCP1and overexpressed SCP1through cell fraction assay, and confirm our findings. The following cell fraction and immunofluorescence of the deletion mutant of SCP1showed that the N-terminal31-55animo acid is critical for its cell membrane localization.
Ⅱ. Palmitoylation of SCP1
Bioinformatics analysis suggested that C44, C45and C47are the potential palmitoylation sites which cause SCP1membrane localization. First, we use point mutation of SCP1and palmitoylation inhibitor to indirectly show the possibility that SCPl can be palmitoylated. Then, to test this hypothesis we used to directly prove palmitoylation by ABE assay and3H palmitoylation assay.
In a word, after we screen the phosphatase library and use the approaches of Cell Biology, Biochemistry and Molecular Biology, we successfully identified SCP1as a new phosphatase that regulate dephosphorylation of c-Myc. We first discover that tumor suppressor SCP1works as a negative regulator of c-Myc protein, which provide new clues and ideas for further understanding the protein instability and biology function of c-Myc. We mainly focused on the regulation of the c-Myc protein instability by SCP1.SCP1is discovered, as a tumor suppressor, dephosphorylate c-Myc through a phosphorylation-dependent pathway. The transcription activity of c-Myc is suppressed on protein levels by SCP1, which restrain the proliferation of liver cancer on cell levels. c-Myc plays an important role in tumorgenesis. It's the significance of discovering SCP1as a negative regulator of c-Myc on protein level that provide a new target for the treatment of cancer and other diseases. Furthermore, the discovery of SCPllocaliaztes in the cell membrane through palmitoylation, that is the first reported investigation about the relationship between serine and threonine phosphatase and membrane localization, which indicates that SCP1influence cellular regulation through more mechanism.
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