Autocatalytic Processing of HtrA2/Omi Is Essential for Induction of Caspase-dependent Cell Death through Antagonizing XIAP

Young-Mo Seong,Ju-Youn Choi,Hyo-Jin Park,Ki-Joong Kim,Sang-Gun Ahn,Geun-Hye Seong,In-Kyung Kim,Seongman Kang,Hyangshuk Rhim

doi:10.1074/jbc.m401408200

Abstract

A mature form of nuclear-encoded mitochondrial serine protease HtrA2/Omi is pivotal in regulating apoptotic cell death; however, the underlying mechanism of the processing event of HtrA2/Omi and its relevant biological function remain to be clarified. Here, we describe that HtrA2/Omi is autocatalytically processed to the 36-kDa protein fragment, which is required for the cytochrome c-dependent caspase activation along with neutralizing XIAP-mediated inhibition of caspases through interaction with XIAP, eventually promoting apoptotic cell death. We have shown that the autocatalytic processing of HtrA2/Omi occurs via an intermolecular event, demonstrated by incubating an in vitro translated HtrA2/Omi (S306A) mutant with the enzymatically active glutathione S-transferase-HtrA2/Omi protein. Using N-terminal amino acid sequencing and mutational analysis, we identified that the autocatalytic cleavage site is the carboxyl side of alanine 133 of HtrA2/Omi, resulting in exposure of an inhibitor of apoptosis protein binding motif in its N terminus. Our study provides evidence that the autocatalytic processing of HtrA2/Omi is crucial for regulating HtrA2/Omi-mediated apoptotic cell death.

Highlights

A mature form of nuclear-encoded mitochondrial serine protease HtrA2/Omi is pivotal in regulating apoptotic cell death; the underlying mechanism of the processing event of HtrA2/Omi and its relevant biological function remain to be clarified
HtrA2 Is Autocatalytically Processed in an Intermolecular Manner—To investigate whether a mature form of HtrA2 observed in mammalian is generated by autocatalytic processing, we synthesized the proteolytically inactive mutant form of HtrA2 in a cell-free transcription and translation system (Fig. 1)
We found that ⌬129 is a suitable form for biochemical studies of its enzyme activity and proteolytic processing, since the ⌬129 protein expressed in E. coli is as soluble as other truncated portions of HtrA2 and contains additional amino acid residues preceding the putative cleavage site, 130MVLAAVPSPP (Fig. 1A) [36]

Summary

The abbreviations used are

HtrA, high temperature requirement A; TM, transmembrane; DTT, dithiothreitol; GST, glutathione S-transferase; IAP, inhibitor of apoptosis protein; IBM, IAP binding motif; Smac, second mitochondria-derived activator of caspase; XIAP, X chromosome-linked IAP; IB, immunoblot; ICAD, anti-inhibitor of the caspaseactivated DNase; GFP, green fluorescent protein. Mature HtrA2 is involved in regulating apoptotic induction along with neutralizing XIAP-mediated inhibition of caspases through interaction with XIAP in the cytosol in response to the apoptotic stimuli [15, 31,32,33,34]. These previous studies indicate that the processing of the HtrA2 precursor to the mature form is indispensable for apoptotic regulation. We demonstrate that the mature HtrA2 generated by the autocatalytic processing can promote caspase activation by inhibiting the XIAP activity, inducing apoptotic cell death

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION