Identification and analysis of dominant negative mutants of RAIDD and PIDD

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Abstract

Caspases are cysteine proteases that are essential during the initiation and execution of apoptosis and inflammation. The formation of large oligomeric protein complexes is critical to the activation of caspases in apoptotic and inflammatory signaling pathways. These oligomeric protein complexes function as a platform to recruit caspases, which leads to caspase activation via a proximity-induced mechanism. One well-known oligomeric caspase-activating complex is the PIDDosome for caspase-2 activation, which is composed of 3 protein components, PIDD, RAIDD and Caspase-2. Despite the significant role that caspase-2 activated by PIDDosome plays during genotoxic stress-induced apoptosis, the oligomerization mechanism and the method by which the caspase-activating process is mediated by the formation of PIDDosome is currently not well understood. Here, we show that the assembly mechanism of the core of PIDDosome is time-dependent and salt concentration-dependent. In addition, we demonstrate that point mutations on RAIDD (R147E) and on PIDD (Y814A) exert a dominant negative effect on the formation of the PIDDosome, and that this effect cannot be applied after the PIDDosome has been formed.

Introduction

Apoptosis is a form of programmed cell death in multicellular organisms that is critical for tissue homeostasis, embryonic development, and immune cell regulation [1], [2], [3], [4]. However, a malfunction of apoptosis can cause serious human diseases such as cancer and autoimmunity [5], [6], [7], [8].

This important process is mediated by the sequential activation of caspases, which are cysteine proteases that cleave proteins specifically after aspartic acid residues [9], [10]. Caspases can be classified into two groups according to their sequence of activation, initiator caspases such as caspases-2, 8, 9 and 10, and effector caspases such as caspases-3 and 7 [11], [12]. Initiator caspases, which have N-terminal pro-domains for the formation of caspase-activating complexes, are activated by a proximity-induced mechanism. Conversely, effector caspases are activated by initiator caspases. Oligomeric initiator caspase-activating complexes function as a platform to recruit caspases, providing proximity for self-activation [9], [11], [13], [14], [15]. Well-known initiator caspase-activating complexes include 1) DISC (Death Inducing Signaling Complex), which activates Caspases-8 and 10 [16], [17], 2) Apoptosome, which activates Caspase-9 [18], [19], 3) Inflammasome, which activates Caspase-1 [20], [21], [22], and 4) PIDDosome, which activates Caspase-2 [23], [24], [25], [26].

PIDD [27], [28], RAIDD [29], [30] and caspase-2 [26] are three protein components that form the most recently identified caspase-activating complex, PIDDosome. PIDD contains 910 residues with seven leucine rich repeats (LRRs), two ZU-5 domains and a C-terminal Death Domain (DD). PIDD is essential for cell death, which it facilitates by activating caspase-2. In addition, PIDD is also critical for cell survival due to its interaction with RIP1, a kinase that has been implicated in the activation of NF-κB [31], [32]. The results of several studies indicate that PIDD may be a molecular switch that controls the balance between life and death upon genotoxic stress [32]. RAIDD is an adapter protein containing Caspase Recruiting Domain (CARD) at the N-terminus and a DD domain at the C-terminus. Caspase-2 is the second caspase to be identified and is the most evolutionarily conserved caspase cross the species of animal [33], [34], [35]. Despite this, due to the lack of a distinct phenotype in knock-out mice there has been little interest in this caspase. However, identification of the PIDDosome as a caspase-2 activating complex, as well as additional studies that have shown that caspase-2 acts upstream of the mitochondrial pathway in response to genotoxic stress to induce caspase-2 mediated apoptosis, has renewed interest in this field [34]. Caspase-2 possesses the CARD domain at its N-terminus.

The assembly of PIDDosome is totally dependent on the protein–protein interacting module known as the death-domain superfamily, which is comprised of 4 subfamilies, Death Domain (DD), Death Effector Domain (DED), Caspase-Recruitment Domain (CARD), and Pyrin Domain (PYD) [4], [36]. PIDDosome is assembled via a DD:DD interaction between RAIDD and PIDD and a CARD:CARD interaction between RAIDD and caspase-2 [26]. The PIDD DD:RAIDD DD complex forms the core oligomeric platform in the PIDDosome, while the RAIDD CARD:caspase-2 CARD interaction is responsible for caspase-2 recruitment.

The molecular structure of the core portion of the PIDDosome formed by RAIDD DD and PIDD DD was recently elucidated [37]. Although the description of the structure of PIDDosome provided vast information enabling a better understanding of its oligomerization mechanism, there is still little biochemical information regarding its assembly mechanism.

In this biochemical study, we showed that the formation of the core of PIDDosome is dependent on time and salt concentration. We also found that two mutations generated during structural-based mutagenesis studies, R147E on RAIDD DD and Y814A on PIDD DD, are dominant negative mutations that inhibit complex formation in the presence of wild-types. Interestingly, those dominant negative effects are abolished after the PIDDosome core portions are pre-formed.

The discovery of dominant negative mutants R147E on RAIDD and Y814A on PIDD in this study that might inhibit genotoxic stress-induced apoptosis could open a new potential application for the therapeutic approach to reduce the side-effects of cancer treatment and to treat human diseases that are caused by excessive apoptosis such as degenerative diseases.

Section snippets

Protein expression and purification

PCR was conducted to amplify the death domain of PIDD and RAIDD from the cDNA of full-length human PIDD (isoform3) and RAIDD, respectively. The plasmid vector, pET 26b (Novagen), was used to add a hexahistidine tag to the carboxy-terminus of PIDD DD, RAIDD DD and full-length RAIDD for affinity purification. The PCR products of PIDD DD were then digested with the NdeI and NotI (NEB) restriction enzymes, while the RAIDD DD and RAIDD PCR products were digested with NdeI and XhoI. The digested PCR

PIDDosome core part assembly is time-dependent

Previous studies have shown that time is required for the PIDDosome dependent apoptosis [26], [37]. Therefore, to determine if the formation of the core of PIDDosome is time-dependent, we biochemically assayed the formation of the complex after various lengths of time. Previous studies have shown that the core of PIDDosome, which is composed of 7 RAIDD DD and 5 PIDD DD, was fully assembled after 1 h of incubation at room temperature [37]. In current study, the increasing amount of the PIDDosome

Discussion

In this study, we biochemically characterized the assembly mechanism by which PIDDosome is formed. The formation of the core portion of PIDDosome, which is composed of 7 RAIDD DD and 5 PIDD DD, was found to be dependent on time and the salt concentration. Consistent with the results of previous studies that suggested that approximately 1 h of incubation at 37 °C or room temperature is necessary for the assembly of active PIDDosome, the results of the present study showed that the PIDDosome core

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the ministry of Education, Science and Technology (2009-0065643).

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