Translational and post-translational regulation of mouse cation transport regulator homolog 1.

Yuki Nomura,Kentaro Oh-Hashi,Yoko Hirata,Kazutoshi Kiuchi

doi:10.1038/srep28016

Abstract

Cation transport regulator homolog 1 (Chac1) is an endoplasmic reticulum (ER) stress inducible gene that has a function as a γ-glutamyl cyclotransferase involved in the degradation of glutathione. To characterize the translation and stability of Chac1, we found that the Kozak-like sequence present in the 5′ untranslated region (5′UTR) of the Chac1 mRNA was responsible for Chac1 translation. In addition, the short form (ΔChac1), which translated from the second ATG codon, was generated in the absence of the 5′UTR. The proteasome pathway predominantly participated in the stability of the Chac1 protein; however, its expression was remarkably up-regulated by co-transfection with ubiquitin genes. Using an immunoprecipitation assay, we revealed that ubiquitin molecule was directly conjugated to Chac1, and that mutated Chac1 with all lysine residues replaced by arginine was also ubiquitinated. Finally, we showed that WT Chac1 but not ΔChac1 reduced the intracellular level of glutathione. Taken together, our results suggest that the Chac1 protein expression is regulated in translational and post-translational fashion due to the Kozak-like sequence in the 5′UTR and the ubiquitin-mediated pathways. The bidirectional roles of ubiquitination in regulating Chac1 stabilization might give us a new insight into understanding the homeostasis of glutathione under pathophysiological conditions.

Highlights

Lysine residues[12,13,14,15]
HEK293 cells expressing Chac1-Myc were treated with MG132, lysosomal acidification inhibitors (Bafilomycin A1 (Baf) or Concanamycin A (CMA)), or the calpain I inhibitor N-Acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN)
The autophagic marker LC3 conjugated to PE (PE-conjugated LC3-II)[39], was dramatically up-regulated by treatment with bafilomycin A1 (Baf) and CMA, whereas the effect of MG132 was to a lesser extent (Fig. 1e)

Summary

Introduction

Lysine residues[12,13,14,15]. the hydroxyl groups of serine and threonine residues and the thiol group of cysteine residues are found to be potential sites of ubiquitination[16,17,18,19]. Activating transcription factor 4 (ATF4) plays a key role in regulating the expression of Chac[1] by directly binding to the ATF4-consensus sequence in the 5′flanking region of the Chac[1] gene[28,29,30]. In regard to functional capability, it has been reported that Chac[1] functions as a γ-glutamyl cyclotransferase that degrades glutathione by its catalytically active residue at E116 in the mouse and E115 in the human protein[30,34,35]. Chac[1] plays an important role in regulating neurogenesis by Notch deglycination at the E1669 residue[36,37]. We investigated expression mechanisms of the mouse Chac[1] protein by focusing on its 5′UTR region as well as ubiquitination and proteasome-mediated degradation

Methods

Results

Conclusion