Cullin-4B E3 ubiquitin ligase mediates Apaf-1 ubiquitination to regulate caspase-9 activity.

Eri Ohta,Masanori Itoh,Kazunori Ohta,Tomomi Sunayama,Yoya Ohno,Toshiyuki Nakagawa,Tana Tana,Miao-Xing Wang,Kiyomi Nakagawa,Masashi Ueda,Masashi Endo,So Hirata,Yoko Hida,Miki Hayakawa-Ogura,Shimo Li,Saiful Islam,Huayue Chen,Emika Nishida,Yi-Hsien Hsieh

doi:10.1371/journal.pone.0219782

Abstract

Apoptotic protease-activating factor 1 (Apaf-1) is a component of apoptosome, which regulates caspase-9 activity. In addition to apoptosis, Apaf-1 plays critical roles in the intra-S-phase checkpoint; therefore, impaired expression of Apaf-1 has been demonstrated in chemotherapy-resistant malignant melanoma and nuclear translocation of Apaf-1 has represented a favorable prognosis of patients with non-small cell lung cancer. In contrast, increased levels of Apaf-1 protein are observed in the brain in Huntington’s disease. The regulation of Apaf-1 protein is not yet fully understood. In this study, we show that etoposide triggers the interaction of Apaf-1 with Cullin-4B, resulting in enhanced Apaf-1 ubiquitination. Ubiquitinated Apaf-1, which was degraded in healthy cells, binds p62 and forms aggregates in the cytosol. This complex of ubiquitinated Apaf-1 and p62 induces caspase-9 activation following MG132 treatment of HEK293T cells that stably express bcl-xl. These results show that ubiquitinated Apaf-1 may activate caspase-9 under conditions of proteasome impairment.

Highlights

Apoptosis is an important mechanism in embryonic development and maintenance of tissue homeostasis[1]
apoptotic protease-activating factor 1 (Apaf-1) protein was rapidly degraded in HEK293T cells upon cycloheximide treatment (Fig 1B), while increased Apaf-1 protein levels were detected upon treatment with the proteasome inhibitor MG132 but not the autophagy inhibitors E64d and pepstatin A (Fig 1C)
Apaf-1 is degraded under normal conditions, treatment with etoposide stimulated the interaction between Apaf-1 and Cullin-4B, leading to Apaf-1 ubiquitination

Summary

Introduction

Apoptosis is an important mechanism in embryonic development and maintenance of tissue homeostasis[1]. Dysregulation of apoptosis is implicated in the pathogenesis of several diseases such as neurodegeneration and cancer. A central component of the apoptosis pathway is a family of cysteine proteases called caspases. Caspases are synthesized as inactive zymogens, and are activated by upstream caspases or by the formation of signaling complexes such as death-inducing signaling complex or DISC[2] and apoptosome[3]. The formation of apoptosome complex is critical for the initiation of apoptosis through caspase activation[4], and comprises homo-oligomers of apoptotic protease-activating factor 1 (Apaf-1) in mammals[3] or its homologs cell-death abnormal (CED)-4 in Caenorhabditis elegans[5] and Drosophila Apaf1-related killer or Dark [ known as hac-1 (homolog of Apaf-1 and ced-4) or Dapaf-1.

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Conclusion