The protective role of protein L-isoaspartyl (D-aspartate) O-methyltransferase for maintenance of mitochondrial morphology in A549 cell

ABSTRACT

Purpose: The increasing amounts of evidence with abnormal aging process have been involved in the pathogenesis of chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Mice with deficient protein L-isoaspartate (D-aspartate) O-methyl transferase 1 (PCMT1) expression reveal acceleration of aging and result in the increased proportion of D-aspartate (D-Asp) residues and dysfunction in proteins. Furthermore, mitochondrial morphology and functions are associated with COPD and IPF pathogenesis. The purpose of the current study was to investigate the role of PCMT1 on mitochondrial morphology using A549 cells. Materials and Methods: We investigated PCMT1, prohibitin1 (PHB1), mitochondrial membrane proteins expression, mitochondrial morphology, and the proportion of D-Asp residues in PHB1 in A549 cells with (PCMT1-KD) and without the context of decreased PCMT1 expression (PCMT1-Cont) using electron microscopy, fluorescence staining, Western blot analysis, and the ATP content per cells. To investigate the effects of the PCMT1-KD cells, we developed double-transfected cell lines containing either the cytosolic or the endoplasmic isoform of PCMT1. Results: We found a significantly higher proportion of D-Asp residues in PHB1 in PCMT1-KD cells than that in PCMT1-Cont cells. The PCMT1-KD cells without cigarette smoke extract exposure were characterized by a significantly increased proportion of the D-Asp residues in PHB1, damaged mitochondrial ultrastructure, and a tendency toward the fission direction of the mitochondrial dynamics followed by a significant decrease in the cellular ATP content. Conclusions: The increased proportion of the D-Asp residues may contribute to COPD pathogenesis, via irreversible protein conformational changes, followed by mitochondrial dysfunction.

KEYWORDS:

• d-aspartic acid residue
• isomerization
• protein damage

Acknowledgment

We thank Mrs. Miyuki Niisato, Iwate Medical University for technical support of cell preparation.

Declaration of interest

The authors declare that they have no competing interests. The authors alone are responsible for the content and writing of the article.

Funding

This project was supported by Grants in-Aid from the Ministry of Education, Culture, Sports Science, and Technology of Japan (26461194 and 22590842) (K.Y.) and (20590925) (M.O.).

Notes on contributors

M. Ogasawara designed this study, and performed generation of mutant cell lines and biochemical analyses. M. Otani, M. Takano, and K. Sano provided critical advices from the point of basic research. M. Shudo and T. Kiyoi performed the TEM studies. Y. Inaba and N. Kunugita collected the cigarette smoke. S. Nirasawa and S. Takahashi produced the recombinant paenidase I protein. K. Kameda was responsible for cell sorting. Y. Tanaka was responsible for GST-PHB1 protein synthesis by cell-free system protein synthesis system. K. Maeyama, M. Yamashita, and K. Yamauchi provided critical advice from the point of clinical relevance.