结核分枝杆菌Pup-蛋白酶体系统蛋白的表达纯化研究
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摘要
结核分枝杆菌(Mycobacterium tuberculosis Mtb)中类泛素蛋白Pup (Prokaryotic ubiquitin-like protein)-蛋白酶体系统是胞内蛋白降解重要机制,在去酰胺酶Dop(Deamidase of Pup)和连接酶PafA(Proteasome accessory factor A)、ATP酶Mpa(Mycobacterial proteasome ATPase)等辅助因子的作用下,Pup可以共价标记多种功能蛋白,并介导被标记蛋白通过蛋白酶体降解,靶蛋白涉及物质中间代谢、信号通路、毒性和抗毒性因子、细胞壁和细胞膜组份等多个方面,并且与结核分枝杆菌的致病性相关,被认为是新的结核病治疗药物靶点,因此本领域研究具有深远的意义。
本文以Mtb为研究对象,优化并克隆出pup基因,同时克隆出dop、pafA和底物蛋白fabD基因,将这些基因加上用于纯化的标签构入表达载体,得到pGEX-2T-his6-pup, pET21cc-dop-his6, pET21cc-fabD-his6, pET21cc-pafA-his6, pBAD-pafA-his6重组表达质粒。通过重组蛋白技术,在大肠杆菌BL21中诱导表达出GST-His6-Pup、Dop-His6、FabD-His6和PafA-His6蛋白,并分别通过常规和变复性等方法纯化出这些蛋白,用蛋白免疫印迹等技术鉴定了这些蛋白,为建立Pup-蛋白酶体体外蛋白降解系统提供了蛋白。合成Pup的N端十肽免疫兔,得到能识别Pup蛋白且有较高效价的Pup多克隆抗体。对Mtb的非致病同源菌耻垢分枝杆菌(Mycobacterium smegmatis, Msm)的dop基因敲除进行了初步探索,为进一步研究Pup-蛋白酶体系统参与的细胞调控功能做准备。
Prokaryotic ubiquitin-like protein (Pup)-proteasome system, the essential mechanism for prokaryotic cellular protein degradation in Mycobacterium tuberculosis(Mtb).With the help of accessory factors, Dop(Deamidase of Pup), PafA(Proteasome accessory factor) and Mpa(Myco-bacterial proteasome ATPase), Pup covalently linked to the Lysε-NH2 in the target proteins and mediated the target protein degradation through the proteasome. The discovery of Pup-proteas-ome system revealed a novel mechanism of prokaryotic protein degradation, which is involved essential physiological function including the intermediary metabolism, information pathway, detoxification/virulence, cell wall and cell membrane formation and so on. Disruption of Pup-proteasome system can suppress the pathogenicity of Mtb. Therefore it is regarded as the new therapeutic target for tuberculosis. In the present paper, the progress in the study on mechanism and function of Pup-proteasome system is reviewed.
In this study, the genes of pup, dop, pafA and fabD are cloned from Mtb with gst or his6 tag for purification. With recombinant plasmids pGEX-2T-his6-pup, pET21cc-fabD-his6, pET21cc-dop-his6, pET21 cc-pafA-his6 and pBAD-pafA-his6, proteins are expressed and purified from E.coli (BL21) by nature or denature protein purification methods. These proteins are identified by western blot. Preparation of polyclonal antibody against Pup and preliminary study on dop knockout in Mycobacterium smegmatis can give us more knowledge and insight into Pup-Proteasome system and prokaryotic celluar regulatory mechanism.
本文以Mtb为研究对象,优化并克隆出pup基因,同时克隆出dop、pafA和底物蛋白fabD基因,将这些基因加上用于纯化的标签构入表达载体,得到pGEX-2T-his6-pup, pET21cc-dop-his6, pET21cc-fabD-his6, pET21cc-pafA-his6, pBAD-pafA-his6重组表达质粒。通过重组蛋白技术,在大肠杆菌BL21中诱导表达出GST-His6-Pup、Dop-His6、FabD-His6和PafA-His6蛋白,并分别通过常规和变复性等方法纯化出这些蛋白,用蛋白免疫印迹等技术鉴定了这些蛋白,为建立Pup-蛋白酶体体外蛋白降解系统提供了蛋白。合成Pup的N端十肽免疫兔,得到能识别Pup蛋白且有较高效价的Pup多克隆抗体。对Mtb的非致病同源菌耻垢分枝杆菌(Mycobacterium smegmatis, Msm)的dop基因敲除进行了初步探索,为进一步研究Pup-蛋白酶体系统参与的细胞调控功能做准备。
Prokaryotic ubiquitin-like protein (Pup)-proteasome system, the essential mechanism for prokaryotic cellular protein degradation in Mycobacterium tuberculosis(Mtb).With the help of accessory factors, Dop(Deamidase of Pup), PafA(Proteasome accessory factor) and Mpa(Myco-bacterial proteasome ATPase), Pup covalently linked to the Lysε-NH2 in the target proteins and mediated the target protein degradation through the proteasome. The discovery of Pup-proteas-ome system revealed a novel mechanism of prokaryotic protein degradation, which is involved essential physiological function including the intermediary metabolism, information pathway, detoxification/virulence, cell wall and cell membrane formation and so on. Disruption of Pup-proteasome system can suppress the pathogenicity of Mtb. Therefore it is regarded as the new therapeutic target for tuberculosis. In the present paper, the progress in the study on mechanism and function of Pup-proteasome system is reviewed.
In this study, the genes of pup, dop, pafA and fabD are cloned from Mtb with gst or his6 tag for purification. With recombinant plasmids pGEX-2T-his6-pup, pET21cc-fabD-his6, pET21cc-dop-his6, pET21 cc-pafA-his6 and pBAD-pafA-his6, proteins are expressed and purified from E.coli (BL21) by nature or denature protein purification methods. These proteins are identified by western blot. Preparation of polyclonal antibody against Pup and preliminary study on dop knockout in Mycobacterium smegmatis can give us more knowledge and insight into Pup-Proteasome system and prokaryotic celluar regulatory mechanism.
引文
1王镜岩,朱圣庚,徐长法,等.生物化学[M].北京：高等教育出版社,2002.
2朱玉贤,李毅,郑晓峰.现代分子生物学(第三版)[M].北京：高等教育出版社,1997.
3吕宪禹,孙蕾,陈小刚,等.蛋白质纯化实验方案与应用[M].北京；化学工业出版社,2010.
4李慎涛,张富春,陈振文,等.精编蛋白质科学实验指南[M].北京：科学出版社,2007.
5汪建华.融合蛋白技术的应用[J],陕西医学杂志.2004,33(12)：1128-1129.
6吴慧娟,张志刚.泛素-蛋白酶体途径及意义[J].国际病理科学与临床杂志,2006,26(1)：7-10.
7郭卉,张雪竹.泛素-蛋白酶体途径及其生物学作用的研究进展[J].现代生物医学进展,2008,8(9)：1786-1788.
8哈洛E,莱恩D.抗体技术实验指南[M].北京：科学出版社,2002.
9高媛,杨金菊,柳晓兰,等.兔抗人CXCR32B分子N端多肽多克隆抗体的制备与初步应用[J].细胞与分子免疫学杂志,2006,22(2)：223-226.
10翟中和,王喜忠,丁明孝,等.细胞生物学(第三版)[M].北京：高等教育出版社,2007.
11魏群,向本琼,崔丽华,等.分子生物学实验指导[M].北京：高等教育出版社,2007.
12陈效友,李传友,马玛,等.卡介苗菌MDP1基因敲除技术的研究[J].中华结核和呼吸杂志,2004,27(3)：183-187.
13 A Ciechanover, Intracellular protein degradation:from a vague idea thru the lysosome and the ubiquitin-proteasome system and onto human diseases and dr ug targeting[J].Cell Death and Differentiation,2005,44:5944-5967.
14 BauMeister W, Walz J, Zu F, et al. The proteasome:paradigm of a self-Compartmentalizing protease[J].Cell,1998,92(3):367-380.
15 Burns A E, Cerda-Maira F A, Wang T, et al."Depupylation" of Prokaryotic Ubiquitin-like Protein from Mycobacterial Proteasome substrates[J].Molecular Cell,2010,39(5):821-827
16 Burns K E, Pearce M J, Darwin K H, et al. Prokaryotic Ubiquitin-Like Protein Provides a Two-Part Degron to Mycobacterium Proteasome substrates[J].Journal of Bacteriology,2010,192(11): 2933-2935.
17 Burns K E, Liu W T, Boshoff H I M, et al.Proteasomal protein degradation in Mycobacteria is dependent upon a prokaryotic ubiquitin-like protein [J].Journal of Biological Chemistry, 2008,284(5):3069-3075.
18 Cerda-MairaF A, Pearce M J, Fuortes M, et al.Molecular analysis of theprokaryotic ubiquitin-like protein (Pup) conj ugation pathway in Mycobacterium tuberculosis[J]. Molecular Microbiology, 2010,77(5):1123-1135.
19 Chen X, solomon W C, Kang Y, et al.Prokaryotic Ubiquitin-Like Protein Pup is intrinsically disordered[J].Journal of molecular biology,2009,392(1):208-217.
20 Deshaies R J, Joazeiro C A P.RING domain E3 ubiquitin ligases[J].Biochemistry,2009,78(1): 399-434.
21 Darwin K H, Lin G, Chen Z, et al.Characterization of a Mycobacterium tuberculos is proteasomal ATPase homologue[J].Molecular Microbiology,2005,55(2):561-571.
22 Darwin K H, Ehrts, Gutierrez-Ramos J C, et al. The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide[J].Science,2003,302(12):1963-1966.
23 Fol M, Chauhan A, Niar N K,et al.Modulation of Mycobacterium tuberculosis proliferation by MtrA, an essential two-component response regulator[J].Molecular,2006,60(3):643-657.
24 Festa R A, McAllister F, Pearce M J, et al.Prokayrotic Ubiquitin-Like Protein (Pup) Proteome of Mycobacterium tuberculosis[J].PLos ONE,2010,5(1):e8589.
25 Festa R A, Pearce M J, Darwin K H.Characterization of the proteasome accessory factor (paf) operon in Mycobacterium tuberculosis [J] Journal of Bacteriology,2007,189(8):3044-3050.
26 Gandotra s, schnappinger D, Monteleone M, et al.In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice[J].Nature medicine,2007,13(12):1515-1520.
27 Gandotra s, Lebron M B, Ehrt s.The Mycobacterium tuberculosis proteasome active site threonine is essential for persistence yet dispensable for replication and resistance to Nitric Oxidea[J].PLos Pathogens,2010,6(8):e1001040.
28 Guth E, Tho mMen M, Weber-Ban E.Mycobacterial ubiquitin-like protein ligase PafA follows a two-step reaction pathway with a phosphorylated Pup intermediate[J] Journal of Biological Chemistry,2011,286(6):4213-4219.
29 Ginsberg A M, spigelman M. Challenges in tuberculosis dr ug research and development[J].Nature medicine,2007,13(3):290-294.
30 Honer Zu Bentrup K, Miczak A, Swenson D L, et al. Characterization of Activity and Expression of Isocitrate Lyase in Mycobacterium aviuM and Mycobacterium tuberculosis [J] Journal of Bacteriology,1999,181(23):7661-7667.
31 Hu G, Lin G, Wang M, et al. Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate[J].Molecular Microbiology,2006,59(5): 1417-1428.
32 Hershko, Ciechanover.The ubiquitin system [J].Nature medicine,2000,6(10):1073-1081.
33 Harth G, Horwitz MA.Export of Recombinant Mycobacterium tuberculosis superoxide Dismutase Is Dependent upon Both Information in the Protein and Mycobacterial Export Machinery. A model for studying export of leaderless proteins by pathogenicmycobacteria[J]Journal of Biological Chemistry,1999,274(7):4281-4292
34 Imkamp F, Rosenberger T, striebel F, et al.Deletion of dop in Mycobacterium smegmatis abolishes pupylation of protein substrates in vivo[J].Molecular Microbiology,2010,75(3):744-754.
35 Imkamp F, striebel F, sutter M, et al.Dop functions as a depupylase in the prokaryotic ubiquitin-like modification pathway[J].EMBO Rep,2010,11(10):791-797.
36 Iyer L, Burro ughs A M, Aravind L. Unraveling the biochemistry and provenance of pupylation:a prokaryotic analog of ubiquitination[J].Biology Direct,2008,3(6150):45-52.
37 Kremer L, Nampoothiri KM, Lesjean s, et al.Biochemical Characterization of Acyl Carrier Protein (AcpM) and Malonyl-CoA:AcpM Transacylase (mtFabD), Two Major Components of Mycobacterium tuberculosis Fatty Acid synthase II[J].Journal of Biological Chemistry,2001, 276(30):27967-27974.
38 Knipfer N, Shrader T E.Inactivation of the 20s proteasome in Mycobacterium smegmatis[J]. Molecular microbiology,1997,25(2):375-383.
39 Lin G, Hu G, Tsu C, Tsu C, et al. Mycobacterium tuberculosis prcBA genes encode a gated proteasome[J].Molecular microbiology,2006,59(5):1405-1416.
40 Lowe J, stock D, Jap B, et al. Crystal structure of the 20s proteasome from the archaeon T. acidophiluM at 3.4 A resolution[J].Science,1995,268(5210):533-539.
41 Lin G, Tsu C, Dick L, et al. Distinct specificities of Mycobacterium tuberculosis and Mammalian Proteasomes for N-Acetyl Tripeptide substrates[J].Journal of Biological Chemistry,2008, 283(49):34423-34431.
42 Li D, Li H L, Wang T, et al.structural basis for the assembly and gate closure mechanisms of the Mycobacterium tuberculosis 20s proteasome[J].The EMBO Journal,2010,29(12):2037-2047.
43 Lamichhane G, Raghunand T R, Morroson N E, et al.Deletion of a Mycobacterium tuberculosis Proteasomal ATPase Homologue Gene Produces a slow-growing stain that persistFs in host tissues[J].The Journal of infectious diseases,2006,194(9):1233-2140.
44 Li G, Li D Y, Carvalho L P s, et al.Inhibitors selective for mycobacterial versus huMan proteasomes[J].Nature,2009,464(1):621-626.
45 Lin G, Li D Y, Chidawanyika, et al.Fellutamide B is a potent inhibitor of the Mycobacterium tuberculosis proteasome[J].Archives of Biochemistry and Biopyhsics,2010,501:214-220
46 Mitchison D A.Tuberculosis hits back[J].Nature,2004,427(22):295.
47 Movahedzadeh F, smith DA, Norman RA, et al.The Mycobacterium tuberculosis inol gene is essential for growth and virulence[J].Molecular Microbiology,2004,51(4):1003-1014
48 Nathan C, shiloh M U.Reactive oxygen and nitrogen intermediates in the relationship between ma mMalian hosts and microbial pathogens[J].Proc Natl Acad sci UsA,2000,97(16):8841-8848.
49 Pearce M J, Arora P, Festa R A, et al.Identification of substrates of the Mycobacterium tuberculosis proteasome[J].The EMBO Journal,2006,25(22):5423-5432.
50 Pearce M J, Mintseris J, Ferreyra J, et al. Ubiquitin-like protein involved in the proteasome pathway of Mycobacterium tuberculosis[J].Science,2008,322(5904):1104-1107.
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2朱玉贤,李毅,郑晓峰.现代分子生物学(第三版)[M].北京：高等教育出版社,1997.
3吕宪禹,孙蕾,陈小刚,等.蛋白质纯化实验方案与应用[M].北京；化学工业出版社,2010.
4李慎涛,张富春,陈振文,等.精编蛋白质科学实验指南[M].北京：科学出版社,2007.
5汪建华.融合蛋白技术的应用[J],陕西医学杂志.2004,33(12)：1128-1129.
6吴慧娟,张志刚.泛素-蛋白酶体途径及意义[J].国际病理科学与临床杂志,2006,26(1)：7-10.
7郭卉,张雪竹.泛素-蛋白酶体途径及其生物学作用的研究进展[J].现代生物医学进展,2008,8(9)：1786-1788.
8哈洛E,莱恩D.抗体技术实验指南[M].北京：科学出版社,2002.
9高媛,杨金菊,柳晓兰,等.兔抗人CXCR32B分子N端多肽多克隆抗体的制备与初步应用[J].细胞与分子免疫学杂志,2006,22(2)：223-226.
10翟中和,王喜忠,丁明孝,等.细胞生物学(第三版)[M].北京：高等教育出版社,2007.
11魏群,向本琼,崔丽华,等.分子生物学实验指导[M].北京：高等教育出版社,2007.
12陈效友,李传友,马玛,等.卡介苗菌MDP1基因敲除技术的研究[J].中华结核和呼吸杂志,2004,27(3)：183-187.
13 A Ciechanover, Intracellular protein degradation:from a vague idea thru the lysosome and the ubiquitin-proteasome system and onto human diseases and dr ug targeting[J].Cell Death and Differentiation,2005,44:5944-5967.
14 BauMeister W, Walz J, Zu F, et al. The proteasome:paradigm of a self-Compartmentalizing protease[J].Cell,1998,92(3):367-380.
15 Burns A E, Cerda-Maira F A, Wang T, et al."Depupylation" of Prokaryotic Ubiquitin-like Protein from Mycobacterial Proteasome substrates[J].Molecular Cell,2010,39(5):821-827
16 Burns K E, Pearce M J, Darwin K H, et al. Prokaryotic Ubiquitin-Like Protein Provides a Two-Part Degron to Mycobacterium Proteasome substrates[J].Journal of Bacteriology,2010,192(11): 2933-2935.
17 Burns K E, Liu W T, Boshoff H I M, et al.Proteasomal protein degradation in Mycobacteria is dependent upon a prokaryotic ubiquitin-like protein [J].Journal of Biological Chemistry, 2008,284(5):3069-3075.
18 Cerda-MairaF A, Pearce M J, Fuortes M, et al.Molecular analysis of theprokaryotic ubiquitin-like protein (Pup) conj ugation pathway in Mycobacterium tuberculosis[J]. Molecular Microbiology, 2010,77(5):1123-1135.
19 Chen X, solomon W C, Kang Y, et al.Prokaryotic Ubiquitin-Like Protein Pup is intrinsically disordered[J].Journal of molecular biology,2009,392(1):208-217.
20 Deshaies R J, Joazeiro C A P.RING domain E3 ubiquitin ligases[J].Biochemistry,2009,78(1): 399-434.
21 Darwin K H, Lin G, Chen Z, et al.Characterization of a Mycobacterium tuberculos is proteasomal ATPase homologue[J].Molecular Microbiology,2005,55(2):561-571.
22 Darwin K H, Ehrts, Gutierrez-Ramos J C, et al. The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide[J].Science,2003,302(12):1963-1966.
23 Fol M, Chauhan A, Niar N K,et al.Modulation of Mycobacterium tuberculosis proliferation by MtrA, an essential two-component response regulator[J].Molecular,2006,60(3):643-657.
24 Festa R A, McAllister F, Pearce M J, et al.Prokayrotic Ubiquitin-Like Protein (Pup) Proteome of Mycobacterium tuberculosis[J].PLos ONE,2010,5(1):e8589.
25 Festa R A, Pearce M J, Darwin K H.Characterization of the proteasome accessory factor (paf) operon in Mycobacterium tuberculosis [J] Journal of Bacteriology,2007,189(8):3044-3050.
26 Gandotra s, schnappinger D, Monteleone M, et al.In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice[J].Nature medicine,2007,13(12):1515-1520.
27 Gandotra s, Lebron M B, Ehrt s.The Mycobacterium tuberculosis proteasome active site threonine is essential for persistence yet dispensable for replication and resistance to Nitric Oxidea[J].PLos Pathogens,2010,6(8):e1001040.
28 Guth E, Tho mMen M, Weber-Ban E.Mycobacterial ubiquitin-like protein ligase PafA follows a two-step reaction pathway with a phosphorylated Pup intermediate[J] Journal of Biological Chemistry,2011,286(6):4213-4219.
29 Ginsberg A M, spigelman M. Challenges in tuberculosis dr ug research and development[J].Nature medicine,2007,13(3):290-294.
30 Honer Zu Bentrup K, Miczak A, Swenson D L, et al. Characterization of Activity and Expression of Isocitrate Lyase in Mycobacterium aviuM and Mycobacterium tuberculosis [J] Journal of Bacteriology,1999,181(23):7661-7667.
31 Hu G, Lin G, Wang M, et al. Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate[J].Molecular Microbiology,2006,59(5): 1417-1428.
32 Hershko, Ciechanover.The ubiquitin system [J].Nature medicine,2000,6(10):1073-1081.
33 Harth G, Horwitz MA.Export of Recombinant Mycobacterium tuberculosis superoxide Dismutase Is Dependent upon Both Information in the Protein and Mycobacterial Export Machinery. A model for studying export of leaderless proteins by pathogenicmycobacteria[J]Journal of Biological Chemistry,1999,274(7):4281-4292
34 Imkamp F, Rosenberger T, striebel F, et al.Deletion of dop in Mycobacterium smegmatis abolishes pupylation of protein substrates in vivo[J].Molecular Microbiology,2010,75(3):744-754.
35 Imkamp F, striebel F, sutter M, et al.Dop functions as a depupylase in the prokaryotic ubiquitin-like modification pathway[J].EMBO Rep,2010,11(10):791-797.
36 Iyer L, Burro ughs A M, Aravind L. Unraveling the biochemistry and provenance of pupylation:a prokaryotic analog of ubiquitination[J].Biology Direct,2008,3(6150):45-52.
37 Kremer L, Nampoothiri KM, Lesjean s, et al.Biochemical Characterization of Acyl Carrier Protein (AcpM) and Malonyl-CoA:AcpM Transacylase (mtFabD), Two Major Components of Mycobacterium tuberculosis Fatty Acid synthase II[J].Journal of Biological Chemistry,2001, 276(30):27967-27974.
38 Knipfer N, Shrader T E.Inactivation of the 20s proteasome in Mycobacterium smegmatis[J]. Molecular microbiology,1997,25(2):375-383.
39 Lin G, Hu G, Tsu C, Tsu C, et al. Mycobacterium tuberculosis prcBA genes encode a gated proteasome[J].Molecular microbiology,2006,59(5):1405-1416.
40 Lowe J, stock D, Jap B, et al. Crystal structure of the 20s proteasome from the archaeon T. acidophiluM at 3.4 A resolution[J].Science,1995,268(5210):533-539.
41 Lin G, Tsu C, Dick L, et al. Distinct specificities of Mycobacterium tuberculosis and Mammalian Proteasomes for N-Acetyl Tripeptide substrates[J].Journal of Biological Chemistry,2008, 283(49):34423-34431.
42 Li D, Li H L, Wang T, et al.structural basis for the assembly and gate closure mechanisms of the Mycobacterium tuberculosis 20s proteasome[J].The EMBO Journal,2010,29(12):2037-2047.
43 Lamichhane G, Raghunand T R, Morroson N E, et al.Deletion of a Mycobacterium tuberculosis Proteasomal ATPase Homologue Gene Produces a slow-growing stain that persistFs in host tissues[J].The Journal of infectious diseases,2006,194(9):1233-2140.
44 Li G, Li D Y, Carvalho L P s, et al.Inhibitors selective for mycobacterial versus huMan proteasomes[J].Nature,2009,464(1):621-626.
45 Lin G, Li D Y, Chidawanyika, et al.Fellutamide B is a potent inhibitor of the Mycobacterium tuberculosis proteasome[J].Archives of Biochemistry and Biopyhsics,2010,501:214-220
46 Mitchison D A.Tuberculosis hits back[J].Nature,2004,427(22):295.
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