Identification of two novel HSP90 proteins in Babesia orientalis: molecular characterization, and computational analyses of their structure, function, antigenicity and inhibitor interaction

Particle and Fibre Toxicology

Junlong Zhao²  Yanqin Zhou²  Min Hu²  Rui Fang²  Jinfang Hu¹  Qian Yu¹  Muhammad Sohail Sajid³  Qiqi Song¹  Qing Tao¹  Yifan Wang¹  Weichao Zhang¹  Lan He¹  Muhammad Kasib Khan³ 

[1] State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei 430070, China;Key Laboratory of Animal Epidemical Disease and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Hubei, Wuhan 430070, China;Department of Parasitology, University of Agriculture, Faisalabad 38040, Pakistan

关键词: Antigenic epitopes;    Ligand docking;    Structure and functional analysis;    Molecular characterization;    HSP90;    Babesia orientalis;   

Others  :  819466
DOI  :  10.1186/1756-3305-7-293

 received in 2014-02-12, accepted in 2014-06-17,  发布年份 2014

【 摘 要 】

Background

HSP90 protects the cells from heat stress and facilitates protein maturation and stability. The full genome sequences of piroplasms contain two putative HSP90 proteins, which are yet uncharacterized. To this end, the two putative HSP90 proteins of Babesia orientalis were identified and characterized by molecular and in silico methods.

Methods

The two putative proteins in B. orientalis genome showing homology with putative HSP90 of other piroplasms were cloned and sequenced. A computational analysis was carried out to predict the antigenic determinants, structure and function of these proteins. The interactions of two HSP90 isoforms with respective inhibitors were also examined through docking analysis.

Results

The length of BoHSP90-A gene (amplified from gDNA) was 2706 bp with one intron from position 997 to 1299 bp. This gene amplified from cDNA corresponded to full length CDS with an open reading frame (ORF) of 2403 bp encoding a 800 amino acid (AA) polypeptide with a predicted size of 91.02 kDa. The HSP90-B gene was intronless with an ORF of 2349 bp, and predicted polypeptide comprised of 797 AA with a size of 90.59 kDa. The AA sequences of these two proteins of B. orientalis were the most identical to those of B. bovis. The BoHSP90-A and BoHSP90-B were recognized as 90 kDa in the parasite lysate by the rabbit antisera raised against the recombinant BoHSP90 proteins. The anti-B. orientalis buffalo serum reacted with the rBoHSP90s expressed in E. coli, indicating that these proteins might be secreted by the parasite before entry into host cells. The overall structure and functional analyses showed several domains involved in ATPase activity, client protein binding and HSP90 dimerization. Likewise, several HSP90 inhibitors showed binding to ATP binding pockets of BoHSP90-A and BoHSP90-B, as observed through protein structure-ligand interaction analysis.

Conclusions

The two putative HSP90 proteins in B. orientalis were recognized as 90 kDa. The rBoHSP90-A and rBoHSP90-B were reacted with the B. orientalis infected buffalo serum. The computational structure and functional analyses revealed that these two proteins may have chaperonic activity. The protein structure-ligand interaction analyses indicated that these two proteins had many drug target sites.

【 授权许可】

   
2014 Khan et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Liu ZL, Zhao JL, Ma LH, Yao BA: Studies on buffalo babesiosis in Hubei province, China. Trop Anim Health Prod 1997, 29:33-36.
• [2]Liu ZL, Zhao JL, Ma LH, Yao BA: Babesia orientalis sp. nov. parasitized in buffalo Bubalus babalis in China (piroplasmida: babesiidae). Acta Vet Zootechnica Sin 1997, 28(1):84-89. in Chinese
• [3]Liu Q, Zhao JL, Zhou YQ, Liu EY, Yao BA, Fu Y: Study on some molecular characterization of Babesia orientalis. Vet Parasitol 2005, 130(3–4):191-198.
• [4]He L, Zhang Y, Zhang QL, Zhang WJ, Feng HH, Khan MK, Hu M, Zhou YQ, Zhao JL: Mitochondrial genome of Babesia orientalis, apicomplexan parasite of water buffalo (Bubalus babalis, Linnaeus, 1758) endemic in China. Parasit Vectors 2014, 7:82.
• [5]Liu ZL, Ma LH, Zhang GD, Gao XS: An investigation of babesiosis in buffaloes in Hubei province. Acta Vet Zootechnica Sin 1986, 17(1):49-54. (in Chinese)
• [6]Taipale M, Jarosz DF, Lindquist S: HSP90 at the hub of protein homeostasis: emerging mechanistic insights. Nat Rev Mol Cell Biol 2010, 11:515-528.
• [7]Smirlis D, Duszenko M, Ruiz AJ, Scoulica E, Bastien P, Fasel N, Soteriadou K: Targeting essential pathways in trypanosomatids gives insights into protozoan mechanisms of cell death. Parasit Vectors 2010, 3:107.
• [8]Sontag EM, Vonk WI, Frydman J: Sorting out the trash: the spatial nature of eukaryotic protein quality control. Curr Opin Cell Biol 2014, 26:139-146.
• [9]Röhl A, Rohrberg J, Buchner J: The chaperone Hsp90: changing partners for demanding clients. Trends Biochem Sci 2013, 38(5):253-262.
• [10]Pearl LH, Prodromou C: Structure and mechanism of the Hsp90 molecular chaperone machinery. Annu Rev Biochem 2006, 75:271-294.
• [11]Wiesgigl M, Clos J: Heat shock protein 90 homeostasis controls stage differentiation in Leishmania donovani. Mol Biol Cell 2001, 12(11):3307-3316.
• [12]Graefe SEB, Wiesgigl M, Gaworski I, Macdonald A, Clos J: Inhibition of Hsp90 in Trypanosoma cruzi induces a stress response but no stage differentiation. Eukaryot Cell 2002, 1:936-943.
• [13]Ahn HJ, Kim S, Nam HW: Molecular cloning of the 82-kDa heat shock protein (HSP90) of Toxoplasma gondii associated with the entry into and growth in host cells. Biochem Biophys Res Commun 2003, 311(3):654-659.
• [14]Banumathy G, Singh V, Pavithra SR, Tatu U: Heat shock protein 90 function is essential for Plasmodium falciparum growth in human erythrocytes. J Biol Chem 2003, 278(20):18336-18345.
• [15]Miska KB, Fetterer RH, Min W, Lillehoj HS: Heat shock protein 90 genes of two species of poultry Eimeria: expression and evolutionary analysis. J Parasitol 2005, 91(2):300-306.
• [16]Péroval M, Péry P, Labbé M: The heat shock protein 90 of Eimeria tenella is essential for invasion of host cell and schizont growth. Int J Parasitol 2006, 36(10–11):1205-1215.
• [17]Li Q, Zhou Y, Yao C, Ma X, Wang L, Xu W, Wang Z, Qiao Z: Apoptosis caused by Hsp90 inhibitor geldanamycin in Leishmania donovani during promastigote-to-amastigote transformation stage. Parasitol Res 2009, 105(6):1539-1548.
• [18]Pallavi R, Roy N, Nageshan RK, Talukdar P, Pavithra SR, Reddy R, Venketesh S, Kumar R, Gupta AK, Singh RK, Yadav SC, Tatu U: Heat shock protein 90 as a drug target against protozoan infections: biochemical characterization of HSP90 from Plasmodium falciparum and Trypanosoma evansi and evaluation of its inhibitor as a candidate drug. J Biol Chem 2010, 285(49):37964-37975.
• [19]Meyer KJ, Shapiro TA: Potent antitrypanosomal activities of heat shock protein 90 inhibitors in vitro and in vivo. J Infect Dis 2013, 208(3):489-499.
• [20]Khan MK, He L, Hussain A, Azam S, Zhang WJ, Wang LX, Zhang QL, Hu M, Zhou YQ, Zhao J: Molecular epidemiology of Theileria annulata and identification of 18S rRNA gene and ITS regions sequences variants in apparently healthy buffaloes and cattle in Pakistan. Infect Genet Evol 2013, 13:124-132.
• [21]Bonfield JK, Smith KF, Staden R: A new DNA sequence assembly program. Nucleic Acids Res 1995, 23:4992-4999.
• [22]Staden R: The Staden sequence analysis package. Mol Biotechnol 1996, 5:233-241.
• [23]Staden R, Beal KF, Bonfield JK: The staden package, 1998. Methods Mol Biol 2000, 132:115-130.
• [24]Roy A, Kucukural A, Zhang Y: I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 2010, 5(4):725-738.
• [25]Gille C: Structural interpretation of mutations and SNPs using STRAP-NT. Protein Sci 2006, 15(1):208-210.
• [26]Ali MM, Roe SM, Vaughan CK, Meyer P, Panaretou B, Piper PW, Prodromou C, Pearl LH: Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex. Nature 2006, 440(7087):1013-1037.
• [27]Arnold K, Bordoli L, Kopp J, Schwede T: The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling. Bioinformatics 2006, 22:195-201.
• [28]Laskowski RA, Watson JD, Thornton JM: Protein function prediction using local 3D templates. J Mol Biol 2005, 351:614-626.
• [29]Laskowski RA, Swindells MB: LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J Chem Inf Model 2011, 51:2778-2786.
• [30]Kolaskar AS, Tongaonkar PC: A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett 1990, 276(1–2):172-174.
• [31]Terkawi MA, Aboge G, Jia H, Goo YK, Ooka H, Yamagishi J, Nishikawa Y, Yokoyama N, Igarashi I, Kawazu SI, Fujisaki K, Xuan X: Molecular and immunological characterization of Babesia gibsoni and Babesia microti heat shock protein-70. Parasite Immunol 2009, 31(6):328-340.
• [32]Gasteiger E, Hoogland C, Gattiker A, Wilkins MR, Appel RD, Bairoch A: Protein Identification and Analysis Tools on the ExPASy Server. In The Proteomics Protocols Handbook. Edited by Walker JM. New Jersey: Humana Press; 2005:571-607.
• [33]Young JC, Moarefi I, Hartl FU: Hsp90: a specialized but essential protein-folding tool. J Cell Biol 2001, 154(2):267-273.
• [34]Chiosis G, Dickey CA, Johnson JL: A global view of Hsp90 functions. Nat Struct Mol Biol 2013, 20(1):1-4.
• [35]Wright L, Barril X, Dymock B, Sheridan L, Surgenor A, Beswick M, Drysdale M, Collier A, Massey A, Davies N, Fink A, Fromont C, Aherne W, Boxall K, Sharp S, Workman P, Hubbard RE: Structure-activity relationships in purine-based inhibitor binding to HSP90 isoforms. Chem Biol 2004, 11(6):775-785.
• [36]Barril X, Beswick MC, Collier A, Drysdale MJ, Dymock BW, Fink A, Grant K, Howes R, Jordan AM, Massey A, Surgenor A, Wayne J, Workman P, Wright L: 4-Amino derivatives of the Hsp90 inhibitor CCT018159. Bioorg Med Chem Lett 2006, 16(9):2543-2548.
• [37]Chen B, Piel WH, Gui L, Bruford E, Monteiro A: The Hsp90 family of genes in the human genome: insights into their divergence and evolution. Genomics 2005, 86(6):627-637.
• [38]Corbett KD, Berger JM: Structure of the ATP-binding domain of Plasmodium falciparum Hsp90. Proteins 2010, 78(13):2738-2744.
• [39]Meyer P, Prodromou C, Hu B, Vaughan C, Roe SM, Panaretou B, Piper PW, Pearl LH: Structural and functional analysis of the middle segment of hsp90: implications for ATP hydrolysis and client protein and cochaperone interactions. Mol Cell 2003, 11(3):647-658.
• [40]Harris SF, Shiau AK, Agard DA: The crystal structure of the carboxy-terminal dimerization domain of htpG, the Escherichia coli Hsp90, reveals a potential substrate binding site. Structure 2004, 12(6):1087-1097.
• [41]Shiau AK, Harris SF, Southworth DR, Agard DA: Structural analysis of E. Coli hsp90 reveals dramatic nucleotide-dependent conformational rearrangements. Cell 2006, 127(2):329-340.
• [42]Jhaveri K, Modi S: HSP90 inhibitors for cancer therapy and overcoming drug resistance. Adv Pharmacol 2012, 65:471-517.
• [43]Soga S, Akinaga S, Shiotsu Y: Hsp90 inhibitors as anti-cancer agents, from basic discoveries to clinical development. Curr Pharm Des 2013, 19(3):366-376.
• [44]Echeverria PC, Matrajt M, Harb OS, Zappia MP, Costas MA, Roos DS, Dubremetz JF, Angel SO: Toxoplasma gondii Hsp90 is a potential drug target whose expression and subcellular localization are developmentally regulated. J Mol Biol 2005, 350(4):723-734.
• [45]Wider D, Péli-Gulli MP, Briand PA, Tatu U, Picard D: The complementation of yeast with human or Plasmodium falciparum Hsp90 confers differential inhibitor sensitivities. Mol Biochem Parasitol 2009, 164(2):147-152.
• [46]Chatterjee M, Andrulis M, Stühmer T, Müller E, Hofmann C, Steinbrunn T, Heimberger T, Schraud H, Kressmann S, Einsele H, Bargou RC: The PI3K/Akt signaling pathway regulates the expression of Hsp70, which critically contributes to Hsp90-chaperone function and tumor cell survival in multiple myeloma. Haematologica 2013, 98(7):1132-1141.
• [47]He L, Liu Q, Quan M, Zhou DN, Zhou YQ, Zhao JL: Molecular cloning and phylogenetic analysis of Babesia orientalis heat shock protein 70. Vet Parasitol 2009, 162(3–4):183-191.
• [48]Böttger E, Multhoff G: Role of Heat Shock Proteins in Immune Modulation in Malaria. In Heat Shock Proteins of Malaria. Netherlands: Springer; 2014:119-132.