Maturation of Mitochondrially Targeted Prx V Involves a Second Cleavage by Mitochondrial Intermediate Peptidase That Is Sensitive to Inhibition by H2O2.

Juhyun Sim,Jiyoung Park,Sue Goo Rhee,Hyun Ae Woo

doi:10.3390/antiox10030346

Abstract

Prx V mRNA contains two in-frame AUG codons, producing a long (L-Prx V) and short form of Prx V (S-Prx V), and mouse L-Prx V is expressed as a precursor protein containing a 49-amino acid N-terminal mitochondria targeting sequence. Here, we show that the N-terminal 41-residue sequence of L-Prx V is cleaved by mitochondrial processing peptidase (MPP) in the mitochondrial matrix to produce an intermediate Prx V (I-Prx V) with a destabilizing phenylalanine at its N-terminus, and further, that the next 8-residue sequence is cleaved by mitochondrial intermediate peptidase (MIP) to convert I-Prx V to a stabilized mature form that is identical to S-Prx V. Further, we show that when mitochondrial H2O2 levels are increased in HeLa cells using rotenone, in several mouse tissues by deleting Prx III, and in the adrenal gland by deleting Srx or by exposing mice to immobilized stress, I-Prx V accumulates transiently and mature S-Prx V levels decrease in mitochondria over time. These findings support the view that MIP is inhibited by H2O2, resulting in the accumulation and subsequent degradation of I-Prx V, identifying a role for redox mediated regulation of Prx V proteolytic maturation and expression in mitochondria.

Highlights

Immunoblot analysis of mouse heart lysates with antibodies to Prx V revealed a major band of 17 kDa and a minor band of slightly larger size, which we designated as intermediate Prx V (I-Prx V); the latter was not observed in liver lysates (Figure 1A, left panel)
In order to identify the minor band, a fraction enriched with both the major and minor Prx V bands was obtained by subjecting mouse heart lysates to several HPLC (High-performance liquid chromatography) column chromatography steps as described in the Materials and Methods section
We show that the maturation of mouse L-Prx V involves the two-step cleavage by mitochondrial processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP); that the intermediate I-Prx V has a destabilizing Phe at its N-terminus; and that removal of an octapeptide from the N-terminus of I-Prx V by MIP converts unstable I-Prx V into stable mature short form of Prx V (S-Prx V) with a stabilizing N-terminalAla

Summary

Introduction

The peroxidase reaction depends on a reactive cysteine residue in their active sites that is conserved among all Prxs [2] [3]. This catalytic Cys is referred to as the peroxidatic Cys (CP ) to reflect its sensitivity to oxidation by peroxides [4]. On the basis of the location or absence of the CR residue, Prxs are classified into typical 2-Cys, atypical 2-Cys, and 1-Cys Prx subfamilies [3]. Mammalian cells express six isoforms of Prx: four 2-Cys Prx isoforms (Prx I to IV), one atypical 2-Cys

Results

Discussion

Conclusion