TRIM13 regulates ubiquitination and turnover of NEMO to suppress TNF induced NF-κB activation
Graphical abstract
Introduction
The NF-κB family of transcription factor is activated by various physiological and pathological stimuli. It had been implicated in several cellular processes like cell proliferation, death, inflammation and innate immunity by regulating the expression of several inflammatory cytokine [1]. The dysregulation of NF-κB leads to several pathological conditions like cancer, neurodegeneration, metabolic and aging related diseases [2], [3]. It is now well understood that different cytokines like TNF, IL-1β, IL-8 activate NF-κB pathway leading to distinct outcomes like proliferation, death or inflammation. Similarly, different intracellular stresses like proteotoxic, oxidative, genotoxic and endoplasmic reticulum stress activate NF-κB, ultimately deciding the fate of cells [4], [5], [6]. The regulatory mechanisms of NF-κB activation by different physiological stimuli leading to unique cellular response is of immense importance however it is still not well understood.
The ubiquitination of different proteins involved in NF-κB pathway is an additional level of regulation in different physiological and pathological conditions [7], [8]. In quiescent cells, NF-κB inhibiting molecule, IκBα retains the NF-κB1/RelA dimer in the cytoplasm [2]. In stimulated cells, the phosphorylation of IκBα, by the IκBα kinase (IKK) complex, primes it for K-48-linked poly-ubiquitination and subsequent degradation by the proteasome [9]. The free NF-κB dimer, translocates to the nucleus and binds to specific sequences (kappa B elements) in promoter regions of responsible genes [3]. The subunits of IKK complex also undergo different types of ubiquitination. The K-63 and linear poly-ubiquitination of NF-κB essential modulator (NEMO) subunit is known to regulate the assembly of IKK complex and its kinase activity [10], [11]. The mono-ubiquitination of IKKβ involved in its autophagic degradation [12]. The process of ubiquitination also plays essential role in the initiation of NF-κB pathway after the binding of cytokines to their cognate receptor [13]. The binding of TNF to TNF-R leads to the recruitment of specific ubiquitin ligases at the different time point leading to distinct pattern of ubiquitination and discrete outcome. TRAF proteins (RING E3 Ligase) are recruited to TNF-R1, which may self-ubiquitinate or target other proteins to regulate NF-κB pathways [13]. Similarly, emerging evidences suggest that ubiquitination acts at different levels of NF-κB pathway and may either inhibit or activate it [9].
The process of ubiquitination involves sequential action of three enzymes: E1, E2 and E3, for transferring the ubiquitin to the target protein [14]. The terminal enzyme E3, transfers Ub from E2 to a lysine residue on a substrate protein, resulting in an iso-peptide bond formation between the substrate lysine and the C-terminus glycine of Ub [15]. E3 ligases provide specificity to the pathway as they recognize the substrate, interact with definite E2 and determine the topology of ubiquitination [15], [16]. Several ubiquitin ligases have been identified that critically determine the pattern of ubiquitination leading to either degradation of proteins through UPS or regulation of their activity [17], [18].
TRIM family proteins (more than 70 proteins) are members of RING type ubiquitin E3 ligases and characterized by the presence of N-terminal RING, B-Box, Coiled Coil (CC) domain and variable C-terminal domain [19]. The role of TRIM family proteins is emerging in several cellular processes like innate immune response, cell survival, miRNA biogenesis and stem cell maintenance [20], [21], [22]. TRIM13 is an ER anchored ubiquitin E3 ligase, involved in degradation of ERAD substrates via proteasome and autophagy pathway [23], [24]. TNF is one of the pleotropic cytokine that regulates NF-κB and caspase-8 in distinct cellular conditions leading to either cell survival or cell death [25]. We previously reported that TRIM13 regulates caspase-8 ubiquitination and induces cell death during ER stress [26], however role of TRIM13 in regulation of TNF induced NF-κB and its physiological relevance had not yet been explored. Here we described the role of TRIM13 in regulation of TNF induced NF-κB pathway. TRIM13 modulates the IKK complex by regulating ubiquitination and turnover of NEMO in the presence of TNF and decreases the clonogenic ability of the cells.
Section snippets
Cell culture and reagents
The human cell lines HEK293, HeLa, A549 and MCF7 cells were grown using procedures as described previously [27]. Full length TRIM13, GST-TRIM13, Flag-TRIM13, deleted constructs of TRIM13 (TRIM13-ΔRING, GST-TRIM13-ΔCC and GST-TRIM13-ΔTM) and TRIM13-shRNA has been described previously [24]. HA-NEMO was from Dr. Gilles Courtois (INSERM, Paris, France). The shRNA for ATG5, Beclin1 and control have been described earlier [26]. The primary antibodies used were: Anti-HA-peroxidase (Roche, Germany),
TRIM13 suppresses TNF induced NF-κB
TNF is one of the important regulators of inflammation and cell death [28]. RING E3 Ligases mediated ubiquitination plays important role in regulation of TNF induced NF-kB activation. The role of TRIM13, RING E3 Ligase, in regulation of TNF induced NF-κB activation is not known. To elucidate the role of TRIM13 in regulation of NF-κB pathway, HEK293 cells were transfected with TRIM13 and vector, treated with TNF and monitored for NF-κB activation. The NF-κB activity significantly increased in
Discussion
TNF is one of the pleotropic cytokine that activates NF-κB leading to secretion of many trophic factors involved in different physiological and pathological conditions. The regulation of TNF induced NF-κB pathway generating different outcomes in different conditions is one of the major focus of recent studies. The post-translational modification of target proteins by E3 ubiquitin ligases plays an important role in regulation of NF-κB pathway. Here we provided several evidences that TRIM13, RING
Conclusion
RING E3 ligases play an essential role in regulation of NF-κB pathway at different steps. The evidences in the current study shows that ER anchored RING E3 ligase TRIM13, negatively regulates NF-κB activation. TRIM13 acts at the IKK complex by regulating NEMO ubiquitination and turnover of NEMO. TRIM13 mediated suppression of NF-κB regulate clonogenic ability of the cancer cell, hence may act as potential tumor suppressor.
Conflicts of interest
The authors declare there is no conflict of interest.
Acknowledgment
The current research work was financially supported by the Department of Biotechnology, Government of India (grant number BT/PR13924/BRB/10/794/2010 to Rajesh Singh). This work constitutes the Ph.D. thesis of Dhanendra Tomar. Authors acknowledge the research fellowships from Council of Scientific and Industrial Research (CSIR), Government of India to Dhanendra Tomar.
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