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Optineurin is required for CYLD-dependent inhibition of TNFα-induced NF-κB activation.

Nagabhushana A, Bansal M, Swarup G - PLoS ONE (2011)

Bottom Line: It competes with NEMO (NF-κB essential modulator) for binding to ubiquitinated RIP (receptor interacting protein) to prevent NF-κB activation.Unlike wild-type optineurin, the H486R mutant did not inhibit tumour necrosis factor α (TNFα)-induced NF-κB activation.The level of ubiquitinated RIP was increased in optineurin knockdown cells.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Hyderabad, India.

ABSTRACT
The nuclear factor kappa B (NF-κB) regulates genes that function in diverse cellular processes like inflammation, immunity and cell survival. The activation of NF-κB is tightly controlled and the deubiquitinase CYLD has emerged as a key negative regulator of NF-κB signalling. Optineurin, mutated in certain glaucomas and amyotrophic lateral sclerosis, is also a negative regulator of NF-κB activation. It competes with NEMO (NF-κB essential modulator) for binding to ubiquitinated RIP (receptor interacting protein) to prevent NF-κB activation. Recently we identified CYLD as optineurin-interacting protein. Here we have analysed the functional significance of interaction of optineurin with CYLD. Our results show that a glaucoma-associated mutant of optineurin, H486R, is altered in its interaction with CYLD. Unlike wild-type optineurin, the H486R mutant did not inhibit tumour necrosis factor α (TNFα)-induced NF-κB activation. CYLD mediated inhibition of TNFα-induced NF-κB activation was abrogated by expression of the H486R mutant. Upon knockdown of optineurin, CYLD was unable to inhibit TNFα-induced NF-κB activation and showed drastically reduced interaction with ubiquitinated RIP. The level of ubiquitinated RIP was increased in optineurin knockdown cells. Deubiquitination of RIP by over-expressed CYLD was abrogated in optineurin knockdown cells. These results suggest that optineurin regulates NF-κB activation by mediating interaction of CYLD with ubiquitinated RIP thus facilitating deubiquitination of RIP.

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Optineurin is required for interaction of CYLD with RIP.HeLa cells were infected with adenoviruses expressing shRNA against optineurin or with control adenoviruses. After 48 h of infection, cells were transfected with HA-tagged mutant (H871N) CYLD, treated with TNFα for 5 min after 30 h of transfection and subjected to immunoprecipitation with HA or control antibodies. Immunoprecipitates were then subjected to western blotting. A schematic representing the regulation of TNFα-induced NF-κB signalling by optineurin. Binding of TNFα to its receptor leads to assembly of a multimolecular complex on TNF receptor in which ubiquitination of RIP takes place. Then NEMO is recruited to ubiquitinated RIP. This leads to activation of IKK (left panel). Optineurin binds to ubiquitinated RIP possibly by displacing NEMO (middle panel) as suggested by Zhu et al [16] and then recruits CYLD to the molecular complex thus facilitating deubiquitination of RIP by CYLD (right panel). In the absence of optineurin, CYLD is not recruited to ubiquitinated RIP resulting in accumulation of ubiquitinated RIP.
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pone-0017477-g007: Optineurin is required for interaction of CYLD with RIP.HeLa cells were infected with adenoviruses expressing shRNA against optineurin or with control adenoviruses. After 48 h of infection, cells were transfected with HA-tagged mutant (H871N) CYLD, treated with TNFα for 5 min after 30 h of transfection and subjected to immunoprecipitation with HA or control antibodies. Immunoprecipitates were then subjected to western blotting. A schematic representing the regulation of TNFα-induced NF-κB signalling by optineurin. Binding of TNFα to its receptor leads to assembly of a multimolecular complex on TNF receptor in which ubiquitination of RIP takes place. Then NEMO is recruited to ubiquitinated RIP. This leads to activation of IKK (left panel). Optineurin binds to ubiquitinated RIP possibly by displacing NEMO (middle panel) as suggested by Zhu et al [16] and then recruits CYLD to the molecular complex thus facilitating deubiquitination of RIP by CYLD (right panel). In the absence of optineurin, CYLD is not recruited to ubiquitinated RIP resulting in accumulation of ubiquitinated RIP.

Mentions: Since CYLD failed to deubiquitinate RIP in the absence of optineurin, it is likely that optineurin acts as an adaptor to facilitate interaction of CYLD with RIP. To test this possibility, endogenous optineurin in HeLa cells was knocked down using shRNA and then these cells were transfected with a catalytically inactive mutant (H871N) of CYLD. After 30 hrs of transfection, these cells were treated with TNFα for 5 min and CYLD was immunoprecipitated. Ubiquitinated RIP and CYLD could co-immunoprecipitate along with endogenous optineurin in control cells indicating that CYLD, RIP and optineurin exist in a single complex. Upon knockdown of optineurin the ability of CYLD to associate with ubiquitinated RIP was drastically reduced (Figure 7A). The catalytically inactive mutant of CYLD was used for this experiment because we got very little or no ubiquitinated RIP in the pulldown with wild type CYLD. These results show that optineurin is essential for interaction of CYLD with ubiquitinated RIP. In conclusion, our observations strongly suggest that optineurin mediates targeting of CYLD to its substrate (i.e ubiquitinated RIP) which facilitates its deubiquitination.


Optineurin is required for CYLD-dependent inhibition of TNFα-induced NF-κB activation.

Nagabhushana A, Bansal M, Swarup G - PLoS ONE (2011)

Optineurin is required for interaction of CYLD with RIP.HeLa cells were infected with adenoviruses expressing shRNA against optineurin or with control adenoviruses. After 48 h of infection, cells were transfected with HA-tagged mutant (H871N) CYLD, treated with TNFα for 5 min after 30 h of transfection and subjected to immunoprecipitation with HA or control antibodies. Immunoprecipitates were then subjected to western blotting. A schematic representing the regulation of TNFα-induced NF-κB signalling by optineurin. Binding of TNFα to its receptor leads to assembly of a multimolecular complex on TNF receptor in which ubiquitination of RIP takes place. Then NEMO is recruited to ubiquitinated RIP. This leads to activation of IKK (left panel). Optineurin binds to ubiquitinated RIP possibly by displacing NEMO (middle panel) as suggested by Zhu et al [16] and then recruits CYLD to the molecular complex thus facilitating deubiquitination of RIP by CYLD (right panel). In the absence of optineurin, CYLD is not recruited to ubiquitinated RIP resulting in accumulation of ubiquitinated RIP.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3049778&req=5

pone-0017477-g007: Optineurin is required for interaction of CYLD with RIP.HeLa cells were infected with adenoviruses expressing shRNA against optineurin or with control adenoviruses. After 48 h of infection, cells were transfected with HA-tagged mutant (H871N) CYLD, treated with TNFα for 5 min after 30 h of transfection and subjected to immunoprecipitation with HA or control antibodies. Immunoprecipitates were then subjected to western blotting. A schematic representing the regulation of TNFα-induced NF-κB signalling by optineurin. Binding of TNFα to its receptor leads to assembly of a multimolecular complex on TNF receptor in which ubiquitination of RIP takes place. Then NEMO is recruited to ubiquitinated RIP. This leads to activation of IKK (left panel). Optineurin binds to ubiquitinated RIP possibly by displacing NEMO (middle panel) as suggested by Zhu et al [16] and then recruits CYLD to the molecular complex thus facilitating deubiquitination of RIP by CYLD (right panel). In the absence of optineurin, CYLD is not recruited to ubiquitinated RIP resulting in accumulation of ubiquitinated RIP.
Mentions: Since CYLD failed to deubiquitinate RIP in the absence of optineurin, it is likely that optineurin acts as an adaptor to facilitate interaction of CYLD with RIP. To test this possibility, endogenous optineurin in HeLa cells was knocked down using shRNA and then these cells were transfected with a catalytically inactive mutant (H871N) of CYLD. After 30 hrs of transfection, these cells were treated with TNFα for 5 min and CYLD was immunoprecipitated. Ubiquitinated RIP and CYLD could co-immunoprecipitate along with endogenous optineurin in control cells indicating that CYLD, RIP and optineurin exist in a single complex. Upon knockdown of optineurin the ability of CYLD to associate with ubiquitinated RIP was drastically reduced (Figure 7A). The catalytically inactive mutant of CYLD was used for this experiment because we got very little or no ubiquitinated RIP in the pulldown with wild type CYLD. These results show that optineurin is essential for interaction of CYLD with ubiquitinated RIP. In conclusion, our observations strongly suggest that optineurin mediates targeting of CYLD to its substrate (i.e ubiquitinated RIP) which facilitates its deubiquitination.

Bottom Line: It competes with NEMO (NF-κB essential modulator) for binding to ubiquitinated RIP (receptor interacting protein) to prevent NF-κB activation.Unlike wild-type optineurin, the H486R mutant did not inhibit tumour necrosis factor α (TNFα)-induced NF-κB activation.The level of ubiquitinated RIP was increased in optineurin knockdown cells.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Hyderabad, India.

ABSTRACT
The nuclear factor kappa B (NF-κB) regulates genes that function in diverse cellular processes like inflammation, immunity and cell survival. The activation of NF-κB is tightly controlled and the deubiquitinase CYLD has emerged as a key negative regulator of NF-κB signalling. Optineurin, mutated in certain glaucomas and amyotrophic lateral sclerosis, is also a negative regulator of NF-κB activation. It competes with NEMO (NF-κB essential modulator) for binding to ubiquitinated RIP (receptor interacting protein) to prevent NF-κB activation. Recently we identified CYLD as optineurin-interacting protein. Here we have analysed the functional significance of interaction of optineurin with CYLD. Our results show that a glaucoma-associated mutant of optineurin, H486R, is altered in its interaction with CYLD. Unlike wild-type optineurin, the H486R mutant did not inhibit tumour necrosis factor α (TNFα)-induced NF-κB activation. CYLD mediated inhibition of TNFα-induced NF-κB activation was abrogated by expression of the H486R mutant. Upon knockdown of optineurin, CYLD was unable to inhibit TNFα-induced NF-κB activation and showed drastically reduced interaction with ubiquitinated RIP. The level of ubiquitinated RIP was increased in optineurin knockdown cells. Deubiquitination of RIP by over-expressed CYLD was abrogated in optineurin knockdown cells. These results suggest that optineurin regulates NF-κB activation by mediating interaction of CYLD with ubiquitinated RIP thus facilitating deubiquitination of RIP.

Show MeSH
Related in: MedlinePlus