title: Identification of active Plasmodium falciparum calpain to establish screening system for Pf-calpain-based drug development.
abstract: BACKGROUND
  With the increasing resistance of malaria parasites to available drugs, there is an urgent demand to develop new anti-malarial drugs. Calpain inhibitor, ALLN, is proposed to inhibit parasite proliferation by suppressing haemoglobin degradation. This provides Plasmodium calpain as a potential target for drug development. Pf-calpain, a cysteine protease of Plasmodium falciparum, belongs to calpain-7 family, which is an atypical calpain not harboring Ca2+-binding regulatory motifs. In this present study, in order to establish the screening system for Pf-calpain specific inhibitors, the active form of Pf-calpain was first identified.
  
  
  METHODS
  Recombinant Pf-calpain including catalytic subdomain IIa (rPfcal-IIa) was heterologously expressed and purified. Enzymatic activity was determined by both fluorogenic substrate assay and gelatin zymography. Molecular homology modeling was carried out to address the activation mode of Pf-calpain in the aspect of structural moiety.
  
  
  RESULTS
  Based on the measurement of enzymatic activity and protease inhibitor assay, it was found that the active form of Pf-calpain only contains the catalytic subdomain IIa, suggesting that Pf-calpain may function as a monomeric form. The sequence prediction indicates that the catalytic subdomain IIa contains all amino acid residues necessary for catalytic triad (Cys-His-Asn) formation. Molecular modeling suggests that the Pf-calpain subdomain IIa makes an active site, holding the catalytic triad residues in their appropriate orientation for catalysis. The mutation analysis further supports that those amino acid residues are functional and have enzymatic activity.
  
  
  CONCLUSIONS
  The identified active form of Pf-calpain could be utilized to establish high-throughput screening system for Pf-calpain inhibitors. Due to its unique monomeric structural property, Pf-calpain could be served as a novel anti-malarial drug target, which has a high specificity for malaria parasite. In addition, the monomeric form of enzyme may contribute to relatively simple synthesis of selective inhibitors.
authors: Laboratory of Cell & Molecular Biology, Department of Life Science, Seonam University, Namwon, Jeonbuk 590-711, Republic of Korea.
authors: Byoung Yul
authors: BY
authors: Soh
authors: Hyun-Ok
authors: HO
authors: Song
authors: Yoonji
authors: Y
authors: Lee
authors: Junghyun
authors: J
authors: Lee
authors: Kusuma
authors: K
authors: Kaewintajuk
authors: Binna
authors: B
authors: Lee
authors: Yun-Young
authors: YY
authors: Choi
authors: Jeong Hoon
authors: JH
authors: Cho
authors: Sun
authors: S
authors: Choi
authors: Hyun
authors: H
authors: Park
mesh_terms: D000595:Amino Acid Sequence; D000962:Antimalarials / Q000302:isolation & purification / Q000494:pharmacology*; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000201:enzymology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
chemical_list: D000962:Antimalarials; D011994:Recombinant Proteins; D002154:Calpain
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