Abstract
Extracellular heat shock protein 70 (HSP70) is recognized by receptors on the plasma membrane, such as Toll-like receptor 4 (TLR4), TLR2, CD14, and CD40. This leads to activation of nuclear factor-kappa B (NF-κB), release of pro-inflammatory cytokines, enhancement of the phagocytic activity of innate immune cells, and stimulation of antigen-specific responses. However, the specific characteristics of HSP70 binding are still unknown, and all HSP70 receptors have not yet been described. Putative models for HSP70 complexation to the receptor for advanced glycation endproducts (RAGEs), considering both ADP- and ATP-bound states of HSP70, were obtained through molecular docking and interaction energy calculations. This interaction was detected and visualized by a proximity fluorescence-based assay in A549 cells and further analyzed by normal mode analyses of the docking complexes. The interacting energy of the complexes showed that the most favored docking situation occurs between HSP70 ATP-bound and RAGE in its monomeric state. The fluorescence proximity assay presented a higher number of detected spots in the HSP70 ATP treatment, corroborating with the computational result. Normal-mode analyses showed no conformational deformability in the interacting interface of the complexes. Results were compared with previous findings in which oxidized HSP70 was shown to be responsible for the differential modulation of macrophage activation, which could result from a signaling pathway triggered by RAGE binding. Our data provide important insights into the characteristics of HSP70 binding and receptor interactions, as well as putative models with conserved residues on the interface area, which could be useful for future site-directed mutagenesis studies.
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Acknowledgements
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), CAPES, and Propesq-UFRGS. The authors thank the Electron Microscopy Center of the Federal University of Rio Grande do Sul for their assistance with the microscopy analyses. The authors also thank Mr. Henrique Biehl for his technical assistance.
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JCFM and HV conceived and coordinated the study. DPG helped conceive the idea. MSG designed, performed, and analyzed all the experiments, as well as wrote the manuscript and prepared all the figures. RLB provided technical assistance for normal mode analyses and contributed with the writing of the manuscript. CSS and LH provided technical assistance and contributed to the preparation of Fig. 2. All authors reviewed the results and approved the final version of the manuscript.
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Grunwald, M.S., Ligabue-Braun, R., Souza, C.S. et al. Putative model for heat shock protein 70 complexation with receptor of advanced glycation end products through fluorescence proximity assays and normal mode analyses. Cell Stress and Chaperones 22, 99–111 (2017). https://doi.org/10.1007/s12192-016-0746-9
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DOI: https://doi.org/10.1007/s12192-016-0746-9