TRIM32 regulates mitochondrial mediated ROS levels and sensitizes the oxidative stress induced cell death

Highlights

• TRIM32 sensitizes cells to oxidative stress induced cell death.

• Level of TRIM32 decreases during oxidative stress.

• E3 ligases activity of TRIM32 modulates K63-link ubiquitination.

• TRIM32 regulates XIAP levels.

• TRIM32 modulates super complex assembly and ROS levels during the oxidative stress.

Abstract

Emerging evidence suggests that ubiquitin mediated post translational modification is a critical regulatory process involved in diverse cellular pathways including cell death. During ubiquitination, E3 ligases recognize target proteins and determine the topology of ubiquitin chains. Recruitment of E3 ligases to targets proteins under stress conditions including oxidative stress and their implication in cell death have not been systemically explored. In the present study, we characterized the role of TRIM32 as an E3 ligase in regulation of oxidative stress induced cell death. TRIM32 is ubiquitously expressed in cell lines of different origin and form cytoplasmic speckle like structures that transiently interact with mitochondria under oxidative stress conditions. The ectopic expression of TRIM32 sensitizes cell death induced by oxidative stress whereas TRIM32 knockdown shows a protective effect. The turnover of TRIM32 is enhanced during oxidative stress and its expression induces ROS generation, loss of mitochondrial transmembrane potential and decrease in complex-I activity. The pro-apoptotic effect was rescued by pan-caspase inhibitor or antioxidant treatment. E3 ligase activity of TRIM32 is essential for oxidative stress induced apoptotic cell death. Furthermore, TRIM32 decreases X-linked inhibitor of apoptosis (XIAP) level and overexpression of XIAP rescued cells from TRIM32 mediated oxidative stress and cell death. Overall, the results of this study provide the first evidence supporting the role of TRIM32 in regulating oxidative stress induced cell death, which has implications in numerous pathological conditions including cancer and neurodegeneration.

Introduction

Mitochondria are dynamic organelles and are implicated in various cellular functions including metabolism, cell death, inflammation, and immunity apart from its role in bioenergetics to maintain cellular homeostasis [[1], [2], [3]]. The equilibrium of the healthy mitochondrial network is maintained in the cells through the dynamic process of fusion and fission. The stressed or damaged mitochondria are labeled with ubiquitin (Ub) and selectively degraded through the process known as mitophagy in order to remove them from the healthy network [4]. Any defect in mitophagy or in the fusion and fission process leads to accumulation of defective mitochondria, resulting in the production of excessive ROS and initiates cell death [5]. This phenomenon has been observed in many pathological conditions including neurodegeneration and autoimmune diseases [6,7].

Ub is versatile molecule that can form different types of Ub chains of different topology on the target proteins through seven conserved lysine residues [8,9]. The presence of atypical Ub chains on the target proteins regulate their stability and impart unique functional outcomes. The reported evidence suggests that ubiquitination during oxidative stress is initiated on the mitochondria which regulates mitophagy and cell death [10]. It was observed that K63 linked ubiquitination is initiated through Bre1 ubiquitin ligase during oxidative stress that determines cell survival [11]. The process of ubiquitination is achieved by the sequential action of three enzymes: E1 (Ub activating enzyme), E2 (Ub conjugating enzyme), E3 (Ub ligases). The E3 ligases are terminal proteins in ubiquitination process and provide pathway specificity as they recognize the substrate and initiate the transfer of Ub. Interestingly, the human genome contains more than 1000 E3 Ligases and their functions are not well understood. Moreover, the role of specific E3 Ligases, their recruitment to mitochondria and regulation of cell death pathways during oxidative environment have not been well studied [12].

There are three major families of E3 Ligases of which the RING (really interesting new gene) family constitutes the most members [13,14]. Tripartite motif (TRIM) family proteins are a subfamily of RING E3 Ligases and characterized by the presence of conserved RBCC domain structures, which include the RING finger, one or two B-box motifs, a coiled-coil region and a variable c-terminal domain. There are more than 76 TRIM proteins and their role is associated with the regulation of the innate immune response during viral infections [15,16]. Recent reports suggest that TRIMs play multiple roles beyond immune responses including cell survival, stem cell maintenance, miRNA biogenesis, embryogenesis, and transcriptional regulation [17,18]. However, the role of TRIMs in pathophysiological processes has only recently begun to emerge and remains uncharacterized.

We have systematically investigated the role of TRIMs in the regulation of cell death and mitochondrial function [13,14,19]. In this study, we report that TRIM32 turnover increases during oxidative stress, translocates to the mitochondria, induces mitochondrial aggregation, and sensitizes the cells to H₂O₂ induced death. In addition, we found that TRIM32 regulates the turnover of XIAP which is important for the regulation of apoptotic cell death under oxidative stress conditions.