CLUES TO THE DEVELOPMENT OF THE ANTI-ATHEROSCLEROTIC HEAT SHOCK PROTEIN 27 IMMUNOTHERAPY FROM THE BIOPHYSICAL CHARACTERIZATION OF THE HSP27 IMMUNE COMPLEX

Abstract

Heat Shock Protein 27 (HSP27) is a 205-amino acid protein with novel anti-atherosclerotic properties that attenuates atherosclerosis by reducing inflammation and lowering cholesterol levels in serum and arterial plaques. Diminished serum HSP27 levels are associated with increased cardiovascular risk. Recently, we demonstrated the presence of anti-HSP27 auto-antibodies (AAB), that potentiate atheroprotection by attenuating inflammatory signaling. For example, as opposed to HSP27 alone, the combination of HSP27 plus the AAB (i.e., the immune complex) appears to dock at the cell membrane, altering the interaction with extracellular inflammatory signaling pathways (e.g., TLR4 mediated activation of NF-kB). As we are currently focusing on the development of HSP27 immunotherapeutics, this study was performed to characterize the structural features of the HSP27-AAB interaction that may be of importance to peptidomimetic design. Homology modelling and secondary structure prediction tools were used to generate design assorted forms of the HSP27 in E.coli. Various biophysical techniques including circular dichroism (CD), dynamic light scattering (DLS), isothermal titration calorimetry (ITC) were used to assess structure and function of these recombinant protein in the presence and absence of an anti-HSP27 polyclonal antibody (PAb; generated in rabbits) that was used to mimic naturally occurring AABs. In vitro, this PAb showed a high degree of fidelity with AABs. The HSP27 C-terminus includes a key conserved alpha crystalline domain with a primary beta sheet structure. The N-terminus is responsible for the formation of larger oligomers. Using ITC we determined that the interaction of HSP27 with PAB was an exothermic reaction (△H = -3.0 x 10-4 cal/mol) with a Ka of 8.9 x10-4 M-1 with an average N valve of 0.5 suggesting 2:1 binding between the PAb and HSP27. Moreover, this interaction markedly increases the beta-sheet content and complex multi-site binding profile. Our study suggests that the formation of the HSP27 immune complex involves a moderately intensity interaction which results in conformational (secondary structure) changes. These structural changes may be of importance in determining the extracellular signalling (e.g., docking with TLR4), and highlight newly exposed HSP27 motifs that will be soon be explored using candidate peptidomimetics that may be of therapeutic value for the treatment of not only atherosclerosis, but inflammation in general.