Tetramethylpyrazine blocks TFAM degradation and up-regulates mitochondrial DNA copy number by interacting with TFAM.

Linhua Lan,Bin Lu,Ying Zheng,Lili Niu,Yongzhang Liu,Yihua Zhang,Carolyn K Suzuki,Fuhong Chen,Miaomiao Guo,Ya Zhang,Lei Xia,Yong Ai,Dawei Huang

doi:10.1042/bsr20170319

Abstract

The natural small molecule compound: 2,3,5,6-tetramethylpyrazine (TMP), is a major component of the Chinese medicine Chuanxiong, which has wide clinical applications in dilating blood vessels, inhibiting platelet aggregation and treating thrombosis. Recent work suggests that TMP is also an antitumour agent. Despite its chemotherapeutic potential, the mechanism(s) underlying TMP action are unknown. Herein, we demonstrate that TMP binds to mitochondrial transcription factor A (TFAM) and blocks its degradation by the mitochondrial Lon protease. TFAM is a key regulator of mtDNA replication, transcription and transmission. Our previous work showed that when TFAM is not bound to DNA, it is rapidly degraded by the ATP-dependent Lon protease, which is essential for mitochondrial proteostasis. In cultured cells, TMP specifically blocks Lon-mediated degradation of TFAM, leading to TFAM accumulation and subsequent up-regulation of mtDNA content in cells with substantially low levels of mtDNA. In vitro protease assays show that TMP does not directly inhibit mitochondrial Lon, rather interacts with TFAM and blocks degradation. Pull-down assays show that biotinylated TMP interacts with TFAM. These findings suggest a novel mechanism whereby TMP stabilizes TFAM and confers resistance to Lon-mediated degradation, thereby promoting mtDNA up-regulation in cells with low mtDNA content.

Highlights

Protein quality control in mitochondria is essential for proper mitochondrial protein homoeostasis, which maintains mitochondrial function and prevents the proteotoxic stress leading to cellular pathology
We proceeded to analyse the stability of wild-type TFAM (TFAMwt) and TFAMHMG1/2 in HeLa ρ+ and EC-1 cells under the condition of TMP treatment to gain insight into the effect of TMP on Lon-mediated degradation of mitochondrial transcription factor A (TFAM) in cells
Both TFAMHMG1/2 and TFAMwt carry a hexahistidine, which distinguish overexpressed from the endogenous TFAM

Summary

Introduction

Protein quality control in mitochondria is essential for proper mitochondrial protein homoeostasis ( termed as mitochondrial proteostasis), which maintains mitochondrial function and prevents the proteotoxic stress leading to cellular pathology. Down-regulation of Lon impairs mitochondrial function, leads to cell apoptosis and decreases cellular bioenergetics [7,8,9,10]. Lon up-regulation has been shown to be tightly correlated with tumorigenesis [8,9,11,12,13,14,15] Taking together, abnormal expression of c 2017 The Author(s).

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