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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 CYLD mediated inhibition of NF-κB activation.HeLa cells were infected with adenoviruses expressing shRNA against optineurin or with control adenoviruses. After 48 hrs of infection cells were transfected with NF-κB-Luc with or without CYLD (100 ng). The cells were treated with TNFα for 4 h after 22 h of transfection. Luciferase activities relative to untreated control are shown (n = 4). Western blot shows the expression of HA-CYLD in control and optineurin knockdown cells. RIP ubiquitination is enhanced in optineurin knockdown cells. HeLa cells were infected with Ad-shOPTN or with control (Cont-AdV) adenoviruses. After 72 h of infection cell lysates were subjected to immunoprecipitation with RIP antibody or control antibody. The immunoprecipitates were analyzed by western blotting. ns, non-specific. * indicates ubiquitinated RIP.
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pone-0017477-g005: Optineurin is required for CYLD mediated inhibition of NF-κB activation.HeLa cells were infected with adenoviruses expressing shRNA against optineurin or with control adenoviruses. After 48 hrs of infection cells were transfected with NF-κB-Luc with or without CYLD (100 ng). The cells were treated with TNFα for 4 h after 22 h of transfection. Luciferase activities relative to untreated control are shown (n = 4). Western blot shows the expression of HA-CYLD in control and optineurin knockdown cells. RIP ubiquitination is enhanced in optineurin knockdown cells. HeLa cells were infected with Ad-shOPTN or with control (Cont-AdV) adenoviruses. After 72 h of infection cell lysates were subjected to immunoprecipitation with RIP antibody or control antibody. The immunoprecipitates were analyzed by western blotting. ns, non-specific. * indicates ubiquitinated RIP.

Mentions: We next examined the requirement of optineurin in CYLD mediated abrogation of TNFα-induced NF-κB activation. HeLa cells were infected with adenoviruses expressing shRNA against optineurin or with control adenoviruses. After 48 h of infection, these cells were transfected with CYLD along with NF-κB luciferase reporter and treated with TNFα for 4 h after 22 h of transfection. As reported earlier, optineurin knockdown enhanced both basal as well as TNFα-induced NF-κB activity [16], [18]. Overexpression of CYLD resulted in strong inhibition of both basal and TNFα-induced NF-κB activity in control cells (Figure 5A). However, upon knockdown of optineurin, overexpression of CYLD resulted in only a marginal inhibition of basal and TNFα-induced NF-κB activity (Figure 5A). These results show that optineurin is essential for CYLD mediated inhibition of basal and TNFα- induced NF-κB activity.


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 CYLD mediated inhibition of NF-κB activation.HeLa cells were infected with adenoviruses expressing shRNA against optineurin or with control adenoviruses. After 48 hrs of infection cells were transfected with NF-κB-Luc with or without CYLD (100 ng). The cells were treated with TNFα for 4 h after 22 h of transfection. Luciferase activities relative to untreated control are shown (n = 4). Western blot shows the expression of HA-CYLD in control and optineurin knockdown cells. RIP ubiquitination is enhanced in optineurin knockdown cells. HeLa cells were infected with Ad-shOPTN or with control (Cont-AdV) adenoviruses. After 72 h of infection cell lysates were subjected to immunoprecipitation with RIP antibody or control antibody. The immunoprecipitates were analyzed by western blotting. ns, non-specific. * indicates ubiquitinated RIP.
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Related In: Results  -  Collection

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pone-0017477-g005: Optineurin is required for CYLD mediated inhibition of NF-κB activation.HeLa cells were infected with adenoviruses expressing shRNA against optineurin or with control adenoviruses. After 48 hrs of infection cells were transfected with NF-κB-Luc with or without CYLD (100 ng). The cells were treated with TNFα for 4 h after 22 h of transfection. Luciferase activities relative to untreated control are shown (n = 4). Western blot shows the expression of HA-CYLD in control and optineurin knockdown cells. RIP ubiquitination is enhanced in optineurin knockdown cells. HeLa cells were infected with Ad-shOPTN or with control (Cont-AdV) adenoviruses. After 72 h of infection cell lysates were subjected to immunoprecipitation with RIP antibody or control antibody. The immunoprecipitates were analyzed by western blotting. ns, non-specific. * indicates ubiquitinated RIP.
Mentions: We next examined the requirement of optineurin in CYLD mediated abrogation of TNFα-induced NF-κB activation. HeLa cells were infected with adenoviruses expressing shRNA against optineurin or with control adenoviruses. After 48 h of infection, these cells were transfected with CYLD along with NF-κB luciferase reporter and treated with TNFα for 4 h after 22 h of transfection. As reported earlier, optineurin knockdown enhanced both basal as well as TNFα-induced NF-κB activity [16], [18]. Overexpression of CYLD resulted in strong inhibition of both basal and TNFα-induced NF-κB activity in control cells (Figure 5A). However, upon knockdown of optineurin, overexpression of CYLD resulted in only a marginal inhibition of basal and TNFα-induced NF-κB activity (Figure 5A). These results show that optineurin is essential for CYLD mediated inhibition of basal and TNFα- induced NF-κB activity.

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