G. Majczak, S. Lilla*,M. Garay-Malpartida, J. Markovic,F.J. Medrano, G. de Nucci, J.E. Belizário
Published October 5, 2007
Genet. Mol. Res. 6 (4): 1000-1011 (2007)
About the authors
G. Majczak, S. Lilla*,M. Garay-Malpartida, J. Markovic,F.J. Medrano, G. de Nucci, J.E. Belizário
Corresponding author
J.E. Belizário
E-mail: jebeliza@usp.br
ABSTRACT
Proteolysis-inducing factor/dermcidin (PIF/DCD) is a novel human gene, located on chromosome 12, locus 12q13.1, that encodes a secreted 110-amino acid protein. Two transcripts for the protein have been identified in normal skin, breast, placenta and brain, and in various primary and metastatic tumor cells. The putative native-state structure of PIF/DCD has not been resolved. Here, we describe some biochemical features of the soluble recombinant 11-kDa protein produced in Escherichia coli. The native 11-kDa polypeptide displayed an anomalous mobility on 1% SDS-PAGE under reduced conditions and appeared as a single ~16-kDa band. Under nonreduced conditions, we detected by mass spectrometry, the presence of multiple peaks corresponding to m/z values of 21 kDa, which we confirmed as a dimeric form with a disulfide bridge between cysteine 34 of each 11-kDa monomer. The native protein exhibited an unusually high susceptibility to proteolytic attack by trypsin, and up to 13 peptides derived from its C-terminus were produced after 5 min of incubation. The secondary structure analysis of PIF/DCD native protein in aqueous solution, by circular dichroism spectroscopy, revealed regions with non-well-defined secondary structure but that acquired α-helix and β-sheet secondary structures in the presence of TFE/water mixtures and micellar and non-micellar SDS molecules. By using PONDR, DisEMBLTM, Disprot, and GlobPlotTM computational predictors, we identified a long disorder region at the N-terminus of PIF/DCD amino acid sequence. This segment (from 19-50 residues) is critical for some of its biological activities, including neuron survival. This result is coherent with successive failure of crystallization of the protein. Taken together, these data suggest that the disorder and order transition may be relevant for some biological functions of PIF/DCD.
Key words: antimicrobial peptides, intrinsically unstructured proteins, circular dichroism