Abstract
Moniliophthora perniciosa is the causal agent of witches’ broom disease of cacao. Cyclophilins have been implicated in a wide variety of cellular processes, including the response to environmental stresses, cell cycle control, regulation of calcium signaling and transcriptional control. The involvement of cyclophilins in pathogenicity was described both in pathogenic fungi toward animals and plants. In Yeast, calcineurin and cyclophilin are involved in fungal morphogenesis and virulence. There is evidence that cyclophilin interacts with calcineurin in the absence of cyclosporin A (CsA), and it has been proposed that the activity of CsA acts in the regulatory interaction between cyclophilin and calcineurin; also, fungistatic or fungicide actions have been observed in this activity. The M. perniciosa cyclophilin gene from cDNA of the fruiting body in the vector pET28a has been cloned and the protein was expressed, purified and crystallized. The cyclophilin structures in the apo and bound with CsA forms were solved at 1.85 and 1.47 Co, respectively. Comparison of structures from different organisms indicates conserved structures of cyclophilins. Small differences were found in apo and bound forms; hence, it follows that cyclophilin structures do not have accentuated modifications in the presence of the ligand. However, various hydrogen bonds between side chains of amino acids and water molecules are broken in the ligand site when CsA is bound. The germination spore assays using CsA showed a low inhibitory activity in germination, but high inhibition of the germ tube growth. These results show that cyclophilin plays an important role in the growth process, but not in the germination, hence suggesting that cyclophilin is as potential target for the fungistatic action against M. perniciosa.
Highlights
Cyclophilins are members of a highly conserved family of proteins that play a pivotal role in protein folding through enzymatic catalysis of the peptidyl-prolyl cis-trans isomerization reaction of the peptide bonds preceding proline residues [1]
M. perniciosa cyclophilin purification has shown over 95% purity based on 15% SDS-PAGE gel and the molecular mass of the purified protein estimated by gel filtration column chromatography was 21 kDa, suggesting that it acts as a monomer in the solution
The amino acid sequence alignment of M. perniciosa cyclophilin with other organisms is shown in Figure 2, where one can observe that the regions is better stage of conservation are found in most parts of the protein, comprising all regions, since the α-helix 1 until the β-strand 7, whereas the most variable regions are found in N and C-terminus
Summary
Cyclophilins are members of a highly conserved family of proteins that play a pivotal role in protein folding through enzymatic catalysis of the peptidyl-prolyl cis-trans isomerization reaction of the peptide bonds preceding proline residues [1]. The M. perniciosa cyclophilin gene was cloned, whereas the protein was expressed, purified and crystallized in apo form and in complexes with CsA. The structures were refined with one or two cyclophilin molecules in the asymmetric unit for apo and bound forms, respectively.
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