A new mechanism for ROS generation from mitochondria
Heat shock protein-47 (HSP47) is present in the endoplasmic reticulum of cells and performs several functions regarding cartilage and bone formation, autophagy, and fibrosis. Post-translocational modifications (PTMs) have become the main focus of scientists and researchers due to their roles in protein and cellular signaling. Electron and X-ray irradiation can induce different changes in the protein via PTMs that lead to significant physiological and pathophysiological effects in the cells and tissues.
In a new study, Dr. Hiroko Indo and Dr. Hiromu Ito, and led by Professor Hideyuki Majima from Kagoshima University explored the effects of irradiation on HSP47 protein and its outcomes in human neuroblastoma SK-N-SH cells. Two types of irradiation including electron and X-ray were used with varying doses. Electron irradiation was carried out at doses of 0, 1, 3, and 10 Gy, and X-ray irradiation was carried out with 0, 1, 3, 10, and 15 Gy, both at room temperature. These radiations induced different mechanisms due to PTMs of HSP47 protein. The original research article is now published in the Archives of biochemistry and biophysics. Dr. Keiichi Nakagawa at The University of Tokyo Hospital, and Dr. Luksana Chaiswing from the University of Kentucky also contributed to the study.
The research team found the generation of ROS, lipid peroxidation, and increased HSP47 levels after irradiation of human neuroblastoma SK-N-SH cells with specific doses of electron and X-ray irradiation. All these changes were significant after exposure from 3 and 10 Gy electron irradiation and 15 Gy X-ray irradiation, as compared to lower doses (Fig. 1–3). An increase in myristoylation, farnesylation, and levels of mitochondrial HSP47 fraction was apparent after PTMs by 10 Gy electron and 15 Gy X-ray irradiations (Fig. 4-7). Anti-HSP47 antibodies coexisted with anti-myristoylation and anti-farnesylation antibodies when analyzed by immunoprecipitation study indicating close physical interaction between these molecules. HSP47 overexpression and transfection were also investigated, which showed elevated mitochondrial ROS and HSP47 production (Fig. 8). From their studies, the authors concluded that HSP47 plays an important role in mitochondrial function.
Several important findings were observed using elegant molecular and cellular experiments. For example, the authors carried out translocation of HSP47 study after exposure to electron and X-ray irradiation. After irradiation of the human neuroblastoma SK-N-SH cell line, a significant increase in ROS levels was observed with 3 and 10 Gy electron irradiation. However, a significant increase in ROS levels was also observed with 10 and 15 Gy X-ray irradiation. An increase in ROS levels was observed with fluorescent dye HPF. Lipid peroxidation was significantly increased on exposure to 10 Gy electron and 10 and 15 Gy X-ray irradiation. Lipid peroxidation was observed microscopically using HNE staining. Quantitative analysis of expression of HSP47 using microscopic detection was observed, which showed significantly higher expression with 3 and 10 Gy electron-irradiated cells and 15 Gy X-ray-irradiated cells, as compared to other radiations.
The levels and localization of HSP47 were also observed after 10 Gy electron and 15 Gy X-ray irradiation, which showed identical effects. Levels of myristoylation and farnesylation were also increased after a similar dose of radiation. After overexpression and transfection of HSP47 induced by 10 Gy electron and 15 Gy X-ray irradiation, mitochondrial ROS release and levels of HSP47 fraction in mitochondria were increased.
A unique finding was observed when two immunoprecipitation tests were performed with immobilized cells in which anti-myristoylation and anti-farnesylation antibodies were analyzed for bounding protein with anti-HSP47 antibody, using western blotting. Both the myristoyl group and farnesyl group were bound to HSP47.
In the bottom line, the study by Professor Hideyuki J. Majima and his colleagues demonstrated that optimum doses of electron and X-ray irradiation have profound physiologic and pathologic effects on the mitochondrial HSP47 protein, which affects the cell and tissue functioning in several ways (Fig. 8).
Indo, H. P., Ito, H., Nakagawa, K., Chaiswing, L., & Majima, H. J. (2021). Translocation of HSP47 and generation of mitochondrial reactive oxygen species in human neuroblastoma SK-N-SH cells following electron and X-ray irradiation. Archives of biochemistry and biophysics, 703, 108853. https://doi.org/10.1016/j.abb.2021.108853Go To Archives of biochemistry and biophysics