Eligible Student Poster 49th Lorne Conference on Protein Structure and Function 2024

Pro-inflammatory Conformational Changes in C-reactive Protein Caused by Misfolded Proteins (#321)

Abhishek Roy 1 2 3 , Johannes Zeller 2 3 4 , Steffi Shing 1 5 , James McFadyen 1 2 , Prerna Sharma 1 2 , Geoffery Pieterz 2 , Xiaowei Wang 1 3 , Steffen Eisenhardt 4 , Tracy Nero 1 5 , Micheal Parker 1 5 6 , Karlheinz Peter 1 2
  1. Baker Department of Cardiometabolic Health, The University of Melbourne, Melbourne, Victoria, Australia
  2. Atherothrombosis and Vascular Biology Laboratory, Baker Heart and Diabetes Institute, Melbourne, Victoria, Australia
  3. Molecular Imaging and Theranostics Laboratory, Baker Heart and Diabetes Institute, Melbourne, Victoria, Australia
  4. Department of Plastic and Hand Surgery, Medical Faculty of the University of Freiburg, Freiburg, Germany
  5. Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, Victoria, Australia
  6. ACRF Rational Drug Discovery Centre, St. Vincent’s Institute of Medical Research, Melbourne, Victoria, Australia

C-reactive protein is a pentameric protein (pCRP) composed of five identical subunits. The native, relatively inert pCRP is first converted into the pro-inflammatory pCRP* intermediate and then finally dissociates into its pro-inflammatory monomeric form (mCRP) upon specific interaction with phosphorylcholine lipid head groups exposed by pathogens and damaged cell membranes. Intriguingly, several studies have shown the co-localisation of pro-inflammatory CRP isoforms, i.e., pCRP*/mCRP, with amyloid beta (Aβ) plaques in the brain tissue of patients suffering from the neurodegenerative Alzheimer’s disease. However, the role of CRP in neuroinflammatory processes has not yet been fully elucidated. Hence, we performed protein docking studies using ClusPro 2.0 to predict the regions of pCRP interacting with Aβ. Aβ was found to preferentially interact with the B-face of pCRP. Interestingly, Aβ also interacts with CRP residues Glu197 and Lys123, the same residues that hold individual CRP subunits together via electrostatic bonds in the non-inflammatory pCRP. The docking results motivated us to examine the Aβ binding sites on pCRP and identify any modulation of its pentameric structure in the presence of oligomeric and fibril states of Aβ using sandwich ELISA, pseudo-native SDS-PAGE and Western blots.  The dissociation of pCRP into mCRP, as a direct result from the interaction with Aβ oligomers and fibrils, was detected in the ELISA and was significantly higher compared to the control; p = 0.0271 and p = 0.0012, respectively. SDS-PAGE and Western blot results are consistent with the ELISA data, as we observed a band corresponding to mCRP in the Aβ oligomer-pCRP and Aβ fibril-pCRP samples. In addition, pCRP dissociation was not observed when the B-face of the pentamer was unexposed (p < 0.0001), thereby validating our docking results. Based on the above-mentioned results, we obtained novel insights into Aβ-pCRP interactions. pCRP can potentially bind to Aβ via its B-face and upon interacting with Aβ, undergo a structural change towards pro-inflammatory conformations (pCRP*/mCRP). This finding provides new mechanistic insights and a new potential perspective for drug design for diseases driven by protein misfolding such as Alzheimer’s disease.