Cell Stress and Chaperones

, Volume 16, Issue 4, pp 389–401 | Cite as

Plasmodium falciparum encodes a single cytosolic type I Hsp40 that functionally interacts with Hsp70 and is upregulated by heat shock

  • Melissa Botha
  • Annette N. Chiang
  • Patrick G. Needham
  • Linda L. Stephens
  • Heinrich C. Hoppe
  • Simone Külzer
  • Jude M. Przyborski
  • Klaus Lingelbach
  • Peter Wipf
  • Jeffrey L. Brodsky
  • Addmore Shonhai
  • Gregory L. Blatch
Original Paper

Abstract

Heat shock protein 70 (Hsp70) and heat shock protein 40 (Hsp40) function as molecular chaperones during the folding and trafficking of proteins within most cell types. However, the Hsp70–Hsp40 chaperone partnerships within the malaria parasite, Plasmodium falciparum, have not been elucidated. Only one of the 43 P. falciparum Hsp40s is predicted to be a cytosolic, canonical Hsp40 (termed PfHsp40) capable of interacting with the major cytosolic P. falciparum-encoded Hsp70, PfHsp70. Consistent with this hypothesis, we found that PfHsp40 is upregulated under heat shock conditions in a similar pattern to PfHsp70. In addition, PfHsp70 and PfHsp40 reside mainly in the parasite cytosol, as assessed using indirect immunofluorescence microscopy. Recombinant PfHsp40 stimulated the ATP hydrolytic rates of both PfHsp70 and human Hsp70 similar to other canonical Hsp40s of yeast (Ydj1) and human (Hdj2) origin. In contrast, the Hsp40-stimulated plasmodial and human Hsp70 ATPase activities were differentially inhibited in the presence of pyrimidinone-based small molecule modulators. To further probe the chaperone properties of PfHsp40, protein aggregation suppression assays were conducted. PfHsp40 alone suppressed protein aggregation, and cooperated with PfHsp70 to suppress aggregation. Together, these data represent the first cellular and biochemical evidence for a PfHsp70–PfHsp40 partnership in the malaria parasite, and furthermore that the plasmodial and human Hsp70–Hsp40 chaperones possess unique attributes that are differentially modulated by small molecules.

Keywords

Aggregation ATPase Codon harmonisation Heat shock protein Malaria Molecular chaperone 

Abbreviations

BSA

Bovine serum albumin

DAPI

4′6-Diamidino-2-phenylindole

DMSO

Dimethyl sulphoxide

Hsp40

Heat shock protein 40

Hsp70

Heat shock protein 70

HRP

Horseradish peroxidase

IPTG

Isopropyl-1-thio-β-d-galactopyranoside

LB

Luria–Bertani media

MDH

Malate dehydrogenase

Ni-NTA

Nickel-nitrilotriacetic acid beads

PBS

Phosphate-buffered saline

PMSF

Phenyl methyl sulphonyl fluoride

SDS–PAGE

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis

TRITC

Tetramethyl rhodamine isothiocyanate

Supplementary material

12192_2010_250_MOESM1_ESM.doc (46 kb)
Fig. S1Protein sequence alignment of PfHsp40, Pfj1 and type I Hsp40 proteins of eukaryotic and prokaryotic origin. Alignments were performed using type I Hsp40 protein sequences from Homo sapiens (Hdj2, NP_001530.1), Saccharomyces cerevisiae (Ydj1, CAA95937.1), E. coli (DnaJ, P08622.3) and P. falciparum (PfHsp40, PF14_0359/NP_702248.1; and Pfj1, PFD0462w/NP_702750.1). The N-terminal extensions of PfHsp40 and Pfj1, and the C-terminal extension of Pfj1 are shaded in red. The highly conserved J domain is shaded in blue with the conserved HPD, KFK and QKRAA motifs indicated in black boxes. The GF region is shaded in green with the conserved DIF motif highlighted in a black box (Cajo et al. [2006] J. Biol. Chem. 281: 12436–12444). The zinc-finger motifs of the zinc-binding domain (yellow shading) are similarly indicated in black boxes. Residues in bold and indicated by a downward arrow are proposed to be involved in substrate binding (Li and Sha [2005] Biochem. J. 386: 453–460). The CAAX-box motifs of PfHsp40, Ydj1 and Hdj2 are shaded in grey. The region of PfHsp40 used for the generation of peptide-based antibodies is shaded in purple. Conserved identical and similar residues are indicated in the consensus line by asterisks and dots, respectively. The alignment was performed using ClustalW (version 1.83; Chenna et al. [2003] Nucleic Acids Res. 31: 3497–3500) (DOC 46 kb)
12192_2010_250_MOESM2_ESM.doc (883 kb)
Fig. S2Heterologous expression and purification of PfHsp40. Recombinant His6-PfHsp40 was expressed in E. coli XL1-Blue (pQPfHsp40), and purified by nickel affinity chromatography under denaturing conditions. The different stages of the purification process were examined by 10% SDS–PAGE (upper panels). Lane 1, E. coli XL1-Blue (pQPfHsp40) whole cell lysate fraction, 16 h post-induction; 2, E. coli XL1-Blue (pQPfHsp40) soluble fraction after sonication and treatment with urea and PEI; 3, unbound protein fraction (flow-through); 4, wash fraction using 150 mM imidazole; 5, elution fraction using 1 M imidazole, showing recovery of purified recombinant His6-PfHsp40 at 49 kDa. The presence of the His6-PfHsp40 protein in the various samples was verified by western blot analysis using anti-His tag antibodies (lower panels). This experiment was repeated at least three times (DOC 883 kb)

References

  1. Acharya P, Kumar R, Tatu U (2007) Chaperoning a cellular upheaval in malaria: heat shock proteins in Plasmodium falciparum. Mol Biochem Parasitol 153:85–94PubMedCrossRefGoogle Scholar
  2. Akide-Ndunge OB, Tambini E, Giribaldi G, McMillan PJ, Müller S, Arese P, Turrini F (2009) Co-ordinated stage-dependent enhancement of Plasmodium falciparum antioxidant enzymes and heat shock protein expression in parasites growing in oxidatively stressed or G6PD-deficient red blood cells. Malar J 8:113PubMedCrossRefGoogle Scholar
  3. Angov E, Hiller CJ, Kincad R, Lyon JA (2008) Heterologous protein expression is enhanced by harmonizing the codon usage frequencies of the target gene with those of the expression host. PLoS ONE 3:e2189PubMedCrossRefGoogle Scholar
  4. Botha M, Pesce E-R, Blatch GL (2007) The Hsp40 proteins of Plasmodium falciparum: regulating chaperone power in the parasite and the host. Int J Biochem Cell Biol 39:1781–1803PubMedCrossRefGoogle Scholar
  5. Brodsky JL, Bracher A (2007) Nucleotide exchange factors for Hsp70 molecular chaperones. In: Blatch GL (ed) Networking of chaperones by co-chaperones. Austin: Landes Bioscience; New York: Springer Science+Business Media; 26–37Google Scholar
  6. Cheetham ME, Caplan AJ (1998) Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function. Cell Stress Chaperones 3:28–36PubMedCrossRefGoogle Scholar
  7. Cheetham ME, Jackson AP, Anderton BH (1994) Regulation of 70-kDa heat-shock-protein ATPase activity and substrate binding by human DnaJ-like proteins, HSJ1a and HSJ1b. Eur J Biochem 226:99–107PubMedCrossRefGoogle Scholar
  8. Chiang AN, Valderramos JC, Balachandran R, Chovatiya RJ, Mead BP, Schneider C, Bell SL, Klein MG, Huryn DM, Chen XS, Day BW, Fidock DA, Wipf P, Brodsky JL (2009) Select pyrimidinones inhibit the propagation of the malarial parasite, Plasmodium falciparum. Bioorg Med Chem 17:1527–1533PubMedCrossRefGoogle Scholar
  9. Crabb BS, Cooke BM, Reeder JC, Waller RF, Caruana SR, Davern KM, Wickham ME, Brown GV, Coppel RL, Cowman AF (1997) Targeted gene disruption shows that knobs enable malaria-infected red cells to cytoadhere under physiological shear stress. Cell 89:287–296PubMedCrossRefGoogle Scholar
  10. de Koning-Ward TF, Gilson PR, Boddey JA, Rug M, Smith BJ, Papenfuss AT, Sanders PR, Lundie RJ, Maier AG, Cowman AF, Crabb BS (2009) A newly discovered protein export machine in malaria parasites. Nature 459:945–949PubMedCrossRefGoogle Scholar
  11. Famin O, Ginsburg H (2003) The treatment of Plasmodium falciparum-infected erythrocytes with chloroquine leads to accumulation of ferriprotoporphyrin IX bound to particular parasite proteins and to the inhibition of the parasite’s 6-phosphogluconate dehydrogenase. Parasite 10:39–50PubMedGoogle Scholar
  12. Fewell SW, Smith CM, Lyon MA, Dumitrescu TP, Wipf P, Day B, Brodsky JL (2004) Small molecule modulators of endogenous and co-chaperone-stimulated Hsp70 ATPase activity. J Biol Chem 279:51131–51140PubMedCrossRefGoogle Scholar
  13. Flaherty KM, DeLuca-Flaherty C, McKay DB (1990) Three-dimensional structure of the ATPase fragment of a 70-K heat shock cognate protein. Nature 346:623–628PubMedCrossRefGoogle Scholar
  14. Guiguemde WA, Anang A, Shelat AA et al (2010) Chemical genetics of Plasmodium facliparum. Nature 465:311–315PubMedCrossRefGoogle Scholar
  15. Hennessy F, Nicoll WS, Zimmermann R, Cheetham ME, Blatch GL (2005) Not all J domains are created equal: implications for the specificity of Hsp40-Hsp70 interactions. Protein Sci 14:1697–1709PubMedCrossRefGoogle Scholar
  16. Hiller NL, Bhattacharjee S, van Ooij C, Liolios K, Harrison T, Lopez-Estrano C, Haldar K (2004) A host-targeting signal in virulence proteins reveals a secretome in malarial infection. Science 306:1934–1937PubMedCrossRefGoogle Scholar
  17. Jiang J, Maes EG, Taylor AB, Wang L, Hinck AP, Lafer EM, Sousa R (2007) Structural basis of the J cochaperone binding and regulation of Hsp70. Mol Cell 28:1–12CrossRefGoogle Scholar
  18. Kabani M, Martineau CN (2008) Multiple Hsp70 isoforms in the eukaryotic cytosol: mere redundancy or functional specificity? Curr Genomics 9:338–348PubMedCrossRefGoogle Scholar
  19. Kappes B, Suetterlin BW, Hofer-Warbinek R, Humar R, Franklin RM (1993) Two major phosphoproteins of Plasmodium falciparum are heat shock proteins. Mol Biochem Parasitol 59:83–94PubMedCrossRefGoogle Scholar
  20. Külzer S, Rug M, Brinkman K, Cannon P, Cowman A, Lingelbach K, Blatch GL, Maier AG, Przyborski JM (2010) Parasite encoded Hsp40 proteins define novel mobile structures in the cytosol of the P. falciparum infected erythrocyte. Cell Microbiol 12:1398–1420PubMedCrossRefGoogle Scholar
  21. Kumar N, Koski G, Harada M, Aikawa M, Zheng H (1991) Induction and localization of Plasmodium falciparum stress proteins related to the heat shock protein 70 family. Mol Biochem Parasitol 48:47–58PubMedCrossRefGoogle Scholar
  22. Kumar A, Tanveer A, Biswas S, Ram EVSR, Gupta A, Kumar B, Habib S (2010) Nuclear-encoded DnaJ homologue of Plasmodium falciparum interacts with replication ori of the apicoplast genome. Mol Microbiol 75:942–956PubMedCrossRefGoogle Scholar
  23. Lambros C, Vandeberg JP (1979) Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 65:418–420PubMedCrossRefGoogle Scholar
  24. Landry SJ (2003) Structure and energetics of an allele-specific genetic interaction between dnaJ and dnaK: correlation of nuclear magnetic resonance chemical shift perturbations in the J-domain of Hsp40/DnaJ with binding affinity for the ATPase domain of Hsp70/DnaK. Biochemistry 42:4926–4936PubMedCrossRefGoogle Scholar
  25. Le Roch KG, Zhou Y, Blair PL, Grainger M, Moch JK, Haynes JD, De La Vega P, Holder AA, Batalov S, Carucci DJ, Winzeler EA (2003) Discovery of gene function by expression profiling of the malaria parasite lifecycle. Science 301:1487–1488CrossRefGoogle Scholar
  26. Li J, Qian X, Sha B (2009) Heat shock protein 40: structural studies and their functional implications. Protein Pept Lett 16:606–612PubMedCrossRefGoogle Scholar
  27. Lu Z, Cyr DM (1998) Protein folding activity of Hsp70 is modified differentially by the Hsp40 co-chaperones Sis1 and Ydj1. J Biol Chem 273:27824–27830PubMedCrossRefGoogle Scholar
  28. Maier AG, Rug M, O’Neill MT, Brown M, Chakravorty S, Szestak T, Chesson J, Wu Y, Hughes K, Coppel RL, Newbold C, Beeson JG, Craig A, Crabb BS, Cowman AF (2008) Exported proteins required for virulence and rigidity of Plasmodium falciparum-infected human erythrocytes. Cell 134:48–61PubMedCrossRefGoogle Scholar
  29. Marti M, Good RT, Rug M, Knuepfer E, Cowman AF (2004) Targeting malaria virulence and remodeling proteins to the host erythrocyte. Science 306:1930–1933PubMedCrossRefGoogle Scholar
  30. Matambo TS, Odunuga OO, Boshoff A, Blatch GL (2004) Overproduction, purification, and characterization of the Plasmodium falciparum heat shock protein 70. Protein Expr Purif 33:214–222PubMedCrossRefGoogle Scholar
  31. McNamara C (2006) Rhodes University MSc thesisGoogle Scholar
  32. Miller LH, Baruch DI, Marsh K, Doumbo OK (2002) The pathogenic basis of malaria. Nature 415:673–679PubMedCrossRefGoogle Scholar
  33. Misra G, Ramachandran R (2009) Hsp70-1 from Plasmodium falciparum: protein stability, domain analysis and chaperone activity. Biophys Chem 142:55–64PubMedCrossRefGoogle Scholar
  34. Nicoll WS, Botha M, Mcnamara C, Schlange M, Pesce ER, Boshoff A, Ludewig MH, Zimmermann R, Cheetham ME, Chapple JP, Blatch GL (2007) Cytosolic and ER J-domains of mammalian and parasitic origin can functionally interact with DnaK. Int J Biochem Cell Biol 39:736–751PubMedCrossRefGoogle Scholar
  35. Pavithra SR, Banumathy G, Joy O, Singh V, Tatu U (2004) Recurrent fever promotes Plasmodium falciparum development in human erythrocytes. J Biol Chem 279:46692–46699PubMedCrossRefGoogle Scholar
  36. Pavithra SR, Kumar R, Tatu U (2007) Systems analysis of chaperone networks in the malarial parasite Plasmodium falciparum. PLoS Comput Biol 3:1701–1715PubMedCrossRefGoogle Scholar
  37. Pesce E-R, Acharya P, Tatu U, Nicoll WS, Shonhai A, Hoppe HC, Blatch GL (2008) The Plasmodium falciparum heat shock protein 40, Pfj4, associates with heat shock protein 70 and shows similar heat induction and localisation patterns. Int J Biochem Cell Biol 40:2914–2926PubMedCrossRefGoogle Scholar
  38. Pesce E-R, Cockburn IL, Goble JL, Stephens LL, Blatch GL (2010) Malaria heat shock proteins: drug targets that chaperone other drug targets. Infect Disord Drug Targets 10:147–157PubMedGoogle Scholar
  39. Ramya TNC, Surolia NN, Surolia A (2006) 15-Deoxyspergualin modulates Plasmodium falciparum heat shock protein function. Biochem Biophys Res Commun 348:585–592PubMedCrossRefGoogle Scholar
  40. Rosser MFN, Cyr DM (2007) Do Hsp40s act as chaperones or co-chaperones? In: Blatch GL (ed) Networking of Chaperones by Co-Chaperones. Austin: Landes Bioscience; New York: Springer Science+Business Media, 38–51Google Scholar
  41. Rüdiger S, Schneider-Mergener J, Bukau B (2001) Its substrate specificity characterizes the DnaJ co-chaperone as a scanning factor for the DnaK chaperone. EMBO J 20:1042–1050PubMedCrossRefGoogle Scholar
  42. Schnaider T, Soti C, Cheetham ME, Miyata Y, Yahara I, Csermely P (2000) Interaction of the human DnaJ homologue, HSJ1b with the 90 kDa heat shock protein, Hsp90. Life Sci 67:1455–1465PubMedCrossRefGoogle Scholar
  43. Shonhai A (2010) Plasmodial heat shock proteins: targets for chemotherapy. FEMS Immunol Med Microbiol 58:61–74PubMedCrossRefGoogle Scholar
  44. Shonhai A, Boshoff A, Blatch GL (2005) Plasmodium falciparum heat shock protein 70 is able to suppress the thermosensitivity of an Escherichia coli DnaK mutant strain. Mol Genet Genomics 274:70–78PubMedCrossRefGoogle Scholar
  45. Shonhai A, Boshoff A, Blatch GL (2007) The structural and functional diversity of Hsp70 proteins from Plasmodium falciparum. Protein Sci 16:1803–1818PubMedCrossRefGoogle Scholar
  46. Shonhai A, Botha M, de Beer TAP, Boshoff A, Blatch GL (2008) Structure-function study of Plasmodium falciparum heat shock protein 70 using three dimensional modeling and in vitro analysis. Protein Pept Lett 15:1117–1125PubMedCrossRefGoogle Scholar
  47. Silva MD, Cooke BM, Guillotte M, Buckingham DW, Sauzet JP, Le Scanf C, Contamin H, David P, Mercereau-Puijalon O, Bonnefoy S (2005) A role for the Plasmodium falciparum RESA protein in resistance against heat shock demonstrated using gene disruption. Mol Microbiol 56:990–1003PubMedCrossRefGoogle Scholar
  48. Snow RW, Guerra CA, Noor AM, Myint H, Hay SI (2005) The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature 434:214–217PubMedCrossRefGoogle Scholar
  49. Stolinski LA, Eisenmann DM, Arndt KM (1997) Identification of RTF1, a novel gene important for TATA site selection by TATA box-binding protein in Saccharomyces cerevisiae. Mol Cell Biol 17:4490–4500PubMedGoogle Scholar
  50. Walsh P, Bursac D, Law YC, Cry D, Lithgow T (2004) The J-protein family: modulating protein assembly, disassembly and translocation. EMBO Rep 5:567–571PubMedCrossRefGoogle Scholar
  51. Wang T-F, Chang J, Wang C (1993) Identification of the peptide binding domain of hsc70. 18-kilodalton fragment located immediately after ATPase domain is sufficient for high affinity binding. J Biol Chem 268:26049–26051PubMedGoogle Scholar
  52. Watanabe J (1997) Cloning and characterization of heat shock protein DnaJ homologues from Plasmodium falciparum and comparison with ring infected erythrocyte surface antigen. Mol Biochem Parasitol 88:253–258PubMedCrossRefGoogle Scholar
  53. Westhoff B, Chapple JP, van der Spuy J, Höhfeld J, Cheetham ME (2005) HSJI is a neuronal shuttling factor for the sorting of chaperone clients to the proteasome. Curr Biol 15:1058–1064PubMedCrossRefGoogle Scholar
  54. Wisén S, Bertelsen EB, Thompson AD, Patury S, Ung PM-U, Chang L, Evans CG, Walter GM, Wipf P, Carlson HA, Brodsky JL, Zuiderweg ER, Gestwicki J (2010) Binding of a small molecule at a protein-protein interface regulates the chaperone activity of Hsp70-Hsp40. ACS Chem Biol 5:611–622PubMedCrossRefGoogle Scholar
  55. Wittung-Stafshede P, Guidry J, Horne BE, Landry SJ (2003) The J domain of Hsp40 couples ATP hydrolysis to substrate capture in Hsp70. Biochemistry 42:4937–4944PubMedCrossRefGoogle Scholar
  56. Wright CM, Chovatiya RJ, Jameson NE, Turner DM, Zhu G, Werner S, Huryn DM, Pipas JM, Day BW, Wipf P, Brodsky JL (2008) Pyrimidinone-peptoid hybrid molecules with distinct effects on molecular chaperone function and cell proliferation. Bioorg Med Chem 16:3291–3301PubMedCrossRefGoogle Scholar

Copyright information

© Cell Stress Society International 2010

Authors and Affiliations

  • Melissa Botha
    • 1
  • Annette N. Chiang
    • 2
  • Patrick G. Needham
    • 2
  • Linda L. Stephens
    • 1
  • Heinrich C. Hoppe
    • 3
  • Simone Külzer
    • 4
  • Jude M. Przyborski
    • 4
  • Klaus Lingelbach
    • 4
  • Peter Wipf
    • 5
  • Jeffrey L. Brodsky
    • 2
  • Addmore Shonhai
    • 6
  • Gregory L. Blatch
    • 1
  1. 1.Biomedical Biotechnology Research Unit, Department of Biochemistry, Microbiology and BiotechnologyRhodes UniversityGrahamstownSouth Africa
  2. 2.Department of Biological SciencesUniversity of PittsburghPittsburghUSA
  3. 3.Council for Scientific and Industrial ResearchPretoriaSouth Africa
  4. 4.Department of Parasitology, Faculty of BiologyPhilipps University MarburgMarburgGermany
  5. 5.Department of ChemistryUniversity of PittsburghPittsburghUSA
  6. 6.Department of Biochemistry and MicrobiologyUniversity of ZululandKwadlangezwaSouth Africa

Personalised recommendations