Abstract
Lethality of Plasmodium falciparum (Pf) caused malaria results from ‘cytoadherence’, which is effected by exported Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family. Several exported Pf proteins (exportome) including chaperones alongside cholesterol rich microdomains are crucial for PfEMP1 translocation to infected erythrocyte surface. An exported Hsp40 (heat shock protein 40) ‘PFA0660w’ functions as a co-chaperone of ‘PfHsp70-x’, and these co-localize to specialized intracellular mobile structures termed J-dots. Our studies attempt to understand the function of PFA0660w-PfHsp70-x chaperone pair using recombinant proteins. Biochemical assays reveal that N and C-terminal domains of PFA0660w and PfHsp70-x respectively are critical for their activity. We show the novel direct interaction of PfHsp70-x with the cytoplasmic tail of PfEMP1, and binding of PFA0660w with cholesterol. PFA0660w operates both as a chaperone and lipid binding molecule via its separate substrate and cholesterol binding sites. PfHsp70-x binds cholesterol linked PFA0660w and PfEMP1 simultaneously in vitro to form a complex. Collectively, our results and the past literature support the hypothesis that PFA0660w-PfHsp70-x chaperone pair assists PfEMP1 transport across the host erythrocyte through cholesterol containing ‘J-dots’. Since PFA0660w seems essential for parasite survival, characterization of its interaction with PfHsp70-x and J-dots may form the basis for development of future antimalarials.
- Abbreviations
- iRBC
- Infected red blood cell
- Pf
- Plasmodium falciparum
- Hsp40
- Heat shock protein 40
- Hsp70
- Heat shock protein 70
- PfEMPl
- Plasmodium falciparum erythrocyte membrane protein1
- ATS
- Acidic terminal segment
- BSA
- Bovine serum albumin
- MDH
- Malate dehydrogenase
- IPTG
- Isopropyl-1-thio-β-D galactopyranoside
- ONPG
- o-nitrophenyl-β-D-galactoside
- ONP
- ortho-nitrophenol
- Ni-NTA
- Nickel-nitrilotriacetic acid beads
- PBS
- Phosphate-buffered saline
- HRP
- Horseradish peroxidase
- SDS–PAGE
- Sodium dodecyl sulphate– polyacrylamide gel electrophoresis
- NBD
- Nucleotide-binding domain
- SBD
- Substrate binding domain