组氨酸为活性中心的蛋白质酪氨酸磷酸酶和细胞自噬标记物的研究
摘要
蛋白质的磷酸化与去磷酸化调控着人体重要的生物学功能,与许多人类的疾病密切相关。本论文主要以一类新型的蛋白质酪氨酸磷酸酶——T细胞受体信号通路抑制因子1和2(STS-1/2)为目标,对它们的生物学功能进行探索。本研究克隆了STS-1/2的催化结构域,并在大肠杆菌表达系统中表达出融合蛋白,通过柱层析色谱技术,分离纯化得到了高纯度的融合蛋白。通过体外酶促反应动力学分析,确定了STS-2的最适酶促反应条件为pH=4.0,NaCl浓度为0。应用带有GST标签的融合蛋白模拟体内的二聚体结构,发现了STS-2对STS-1的磷酸酶活性具有一定的抑制作用。本文证明了以组氨酸为活性中心的STS-1/2具有蛋白质酪氨酸磷酸酶的活性,并且发现两种蛋白间存在相互抑制的作用。另外,本论文还对小鼠mLC3b蛋白在细胞自嗜的标记物的应用上进行了研究,我们克隆、表达了小鼠mLC3b蛋白,通过与绿色荧光蛋白形成嵌合体,我们对mLC3b在细胞中进行定位。结果表明,当在哺乳动物细胞中表达的时候,mLC3b分布在细胞质中;当诱导细胞自嗜现象产生的时候,mLC3b则发生聚集并与细胞自嗜小体发生结合。研究表明,mLC3b可以作为标记细胞自嗜的标记物在生物学研究中广泛应用。
Protein phosphatases play important functions in cell growth, division and differentiation. They counteract the activities of protein kinase thereby controlling protein phosphorylation and cell signaling pathways. Among protein phosphatases, there are 107 protein tyrosine phosphatases. As crucial regulators of signal transduction, dysfunction of these enzymes causes many human diseases.
STS-1 and STS-2 form a sub-family of protein tyrosine phosphatase. STS-1 was initially identified as a suppressor of the T cell receptor signaling pathway. STS-1 and STS-2 have multi-functional structures consisting of UBA domain, SH3 domain, PGM domain, and dimmerization domain. The UBA domain is involved in protein ubiquitination and regulates degradation of STS1 and STS2. The SH3 domain initiates interactions with proline-rich segments of signaling proteins. The dimmerization domain is responsible for formation of STS-1 and STS-2 dimmers. PGM domain is homologous to phosphataseglycerol mutase which catalyzes conversion of 2-phosphoglycerol to 3-phosphoglycerol. Recently, the PGM domain of STS-1 was found to have protein tyrosine phosphatase activity. It dephosphorylates para-nitrophenolphosphate (p-NPP) in biochemical assays and reduces tyrosine phosphorylation of the epidermal growth factor receptor in transfected cells. However, if the PGM domain of STS-2 has similar activity is not known. Based on the high sequence homology of STS-1 and STS-2, we hypothesize that STS-2 is also a protein phosphatase.
To confirm our hypothesis, we expressed three fragments containing the PGM domains of STS-1 and STS-2 as non-fusion proteins as well as GST fusion proteins in E. coli cells. These proteins were purified to near homogeneity by using chromatographic procedures. We first analyzed the phosphatase activities of purified non-fusion proteins by using pNPP as a substrate under various conditions. In contrast to STS-1 which displays optimal activity at neutral pH, STS-2 strongly prefer acidic pH. Furthermore, while increase in ionic strength reduces activity of STS-2, this has no effects on STS-1. Overall, STS-1 is more active with a kcat value of 20/second versus 0.1/second for STS-2. We then compared with activity obtained with GST-fusion proteins. Interestingly, while fusion of STS-1 with GST increases its phosphatase activity, GST-STS-2 is much less active then STS-2 alone. Since GST intends to induce protein dimmer formation, the data suggest that dimmerization distinctly regulates activity of STS-1 and STS2.
We further analyzed the activity of STS-1 and STS-2 with a recombinant protein substrate designated GST-JAKS and extracts of Jurkat cells which were stimulated with pervanadate to stimulate tyrosine phosphorylation. Both STS-1 and STS-2 effectively dephosphorylated tyrosine phosphorylated proteins with optimal pH 7 for the former and pH4 for the latter. Interestingly, they appeared to selectively dephosphorylate proteins in the Jurkat cell extracts. This indicates different substrate specificity.
Finally, we investigated the interaction of STS-1 and STS-2 by analyzing STS-1 activity in the presence of STS-2. The data demonstrated that STS-2 significantly inhibits STS-1. This represents a novel mechanism for the regulation of these enzymes. Together, our study demonstrated that STS-2 is a protein tyrosine phosphatase with distinct feature from STS-1. STS-1 and STS2 may have different biological functions.
Autophagy is a catabolic process which causes degradation of a cell’s own components through the lysosomal machinery. It plays an important role in cell growth and development. There are different methods for detection of autophagy. In this study, we cloned a chimeric protein containing the mouse microtubule-associated protein light chain 3 beta (LC3b) and enhanced green fluorescent protein (EGFP). When expressed in mammalian cells, the EGFP-mLC3b chimeric protein is diffused in the entire cytosol. Upon treatment of the transfected cells with reagents which induce autophagy, EGFP-mLC3b is localized to punctate intracellular structures resembling autophagosomes. Therefore, EGFP-mLC3b can serve as a marker of autophagy. Our study thus produced a valuable tool for further study of autophagy.
Protein phosphatases play important functions in cell growth, division and differentiation. They counteract the activities of protein kinase thereby controlling protein phosphorylation and cell signaling pathways. Among protein phosphatases, there are 107 protein tyrosine phosphatases. As crucial regulators of signal transduction, dysfunction of these enzymes causes many human diseases.
STS-1 and STS-2 form a sub-family of protein tyrosine phosphatase. STS-1 was initially identified as a suppressor of the T cell receptor signaling pathway. STS-1 and STS-2 have multi-functional structures consisting of UBA domain, SH3 domain, PGM domain, and dimmerization domain. The UBA domain is involved in protein ubiquitination and regulates degradation of STS1 and STS2. The SH3 domain initiates interactions with proline-rich segments of signaling proteins. The dimmerization domain is responsible for formation of STS-1 and STS-2 dimmers. PGM domain is homologous to phosphataseglycerol mutase which catalyzes conversion of 2-phosphoglycerol to 3-phosphoglycerol. Recently, the PGM domain of STS-1 was found to have protein tyrosine phosphatase activity. It dephosphorylates para-nitrophenolphosphate (p-NPP) in biochemical assays and reduces tyrosine phosphorylation of the epidermal growth factor receptor in transfected cells. However, if the PGM domain of STS-2 has similar activity is not known. Based on the high sequence homology of STS-1 and STS-2, we hypothesize that STS-2 is also a protein phosphatase.
To confirm our hypothesis, we expressed three fragments containing the PGM domains of STS-1 and STS-2 as non-fusion proteins as well as GST fusion proteins in E. coli cells. These proteins were purified to near homogeneity by using chromatographic procedures. We first analyzed the phosphatase activities of purified non-fusion proteins by using pNPP as a substrate under various conditions. In contrast to STS-1 which displays optimal activity at neutral pH, STS-2 strongly prefer acidic pH. Furthermore, while increase in ionic strength reduces activity of STS-2, this has no effects on STS-1. Overall, STS-1 is more active with a kcat value of 20/second versus 0.1/second for STS-2. We then compared with activity obtained with GST-fusion proteins. Interestingly, while fusion of STS-1 with GST increases its phosphatase activity, GST-STS-2 is much less active then STS-2 alone. Since GST intends to induce protein dimmer formation, the data suggest that dimmerization distinctly regulates activity of STS-1 and STS2.
We further analyzed the activity of STS-1 and STS-2 with a recombinant protein substrate designated GST-JAKS and extracts of Jurkat cells which were stimulated with pervanadate to stimulate tyrosine phosphorylation. Both STS-1 and STS-2 effectively dephosphorylated tyrosine phosphorylated proteins with optimal pH 7 for the former and pH4 for the latter. Interestingly, they appeared to selectively dephosphorylate proteins in the Jurkat cell extracts. This indicates different substrate specificity.
Finally, we investigated the interaction of STS-1 and STS-2 by analyzing STS-1 activity in the presence of STS-2. The data demonstrated that STS-2 significantly inhibits STS-1. This represents a novel mechanism for the regulation of these enzymes. Together, our study demonstrated that STS-2 is a protein tyrosine phosphatase with distinct feature from STS-1. STS-1 and STS2 may have different biological functions.
Autophagy is a catabolic process which causes degradation of a cell’s own components through the lysosomal machinery. It plays an important role in cell growth and development. There are different methods for detection of autophagy. In this study, we cloned a chimeric protein containing the mouse microtubule-associated protein light chain 3 beta (LC3b) and enhanced green fluorescent protein (EGFP). When expressed in mammalian cells, the EGFP-mLC3b chimeric protein is diffused in the entire cytosol. Upon treatment of the transfected cells with reagents which induce autophagy, EGFP-mLC3b is localized to punctate intracellular structures resembling autophagosomes. Therefore, EGFP-mLC3b can serve as a marker of autophagy. Our study thus produced a valuable tool for further study of autophagy.
引文
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[1] Hotchkiss RS, Strasser A, McDunn JE, Swanson PE., Cell death. [J]. N Engl J Med. 2009, 16, 1570-1583
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[2] Andersen, J.N., Mortensen, O.H., Peters, G.H., Drake, P.G., Iversen, L.F., Olsen,O.H., Jansen, P.G., Andersen, H.S., Tonks, N.K., and Moller, N.P. Structural and evolutionary relationships among protein tyrosine phosphatasedomains. [J]. Mol. Cell. Biol. 2001. 21, 7117–7136.
[3] Tonks NK. Protein tyrosine phosphatases: from genes, to function, to disease. [J].Nat Rev Mol Cell Biol, 2006. 7(11):833-846.
[4] Aricescu, A.R., Siebold, C., Choudhuri, K., Chang, V.T., Lu, W., Davis, S.J., vander Merwe, P.A., and Jones, E.Y., Structure of a tyrosine phosphatase adhesive interaction reveals a spacer-clamp mechanism. [J]. Science. 2007. 317, 1217–1220.
[5] Tabernero L, Aricescu AR, Jones EY, Szedlacsek SE. Protein tyrosine phosphatases: structure-function relationships. [J]. FEBS J. 2008. 275, 867-882.
[6] Zhang, Z.Y. Protein tyrosine phosphatases: structure and function, substrate specificity, and inhibitor development. [J]. Annu. Rev. Pharmacol.Toxicol. 2002. 42, 209–234.
[7] Barford, D., Das, A.K., and Egloff, M.P. The structure and mechanism of protein phosphatases: insights into catalysis and regulation. [J]. Annu. Rev. Biophys. Biomol. Struct. 1998. 27, 133–164
[8] Stuckey JA, Schubert HL, Fauman EB, Zhang ZY, Dixon JE & Saper MA. Crystal structure of Yersinia protein tyrosine phosphatase at 2.5 A ? and the complex with tungstate. [J]. Nature. 1994. 370, 571–575.
[9] Frangioni JV, Beahm PH, Shifrin V, Jost CA & Neel BG. The nontransmembrane tyrosine phosphatase PTP-1B localizes to the endoplasmic reticulum via its 35 amino acid C-terminal sequence. [J]. Cell, 1992. 68, 545–560.
[10] Pulido R, Zuniga A, Ullrich A. PTP-SL and STEP protein tyrosine phosphatases regulate the activation of the extracellular signal-regulated kinases ERK1 and ERK2 by association through a kinase interaction motif. [J]. EMBO. J. 1998. 17, 7337–7350.
[11] Large-Scale Structural Analysis of the Classical Human Protein Tyrosine Phosphatome Alastair J. Barr, Emilie Ugochukwu, Wen Hwa Lee, Oliver N.F. King, Panagis Filippakopoulos, Ivan Alfano, Pavel Savitsky, Nicola A. Burgess-Brown, Susanne Mu¨ller, and Stefan Knapp. [J]. Cell. 2009. 136, 352–363,
[12] Villa F, Deak M, Bloomberg GB, Alessi DR & van Aalten DM Crystal structure of the PTPL1 ? FAP-1 human tyrosine phosphatase mutated in colorectal cancer: evidence for a second phosphotyrosine substrate recognition pocket. [J]. J. Biol Chem. 2005. 280, 8180–8187.
[13] Puius YA, Zhao Y, Sullivan M, Lawrence DS, Almo SC & Zhang ZY Identification of a second aryl phosphate-binding site in protein-tyrosine phosphatase 1B: a paradigm for inhibitor design. [J]. Proc Natl Acad Sci USA.1997. 94, 13420–13425.
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