Limits...
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.

Show MeSH

Related in: MedlinePlus

H486R optineurin is defective in interaction with CYLD.Yeast strain PJ69-4A was co-transformed with wild-type optineurin or its mutants and CYLD. Transformants were grown on selection media lacking adenine to assay interaction. GST-optineurin, GST-H486R or GST alone bound to glutathione agarose beads were incubated with lysates of HeLa cells transfected with HA-CYLD. The bound proteins were eluted and immunoblotted with anti-HA antibodies. Western blot was done with optineurin antibody (lower panel) to confirm the identity of the GST fusion proteins. HeLa cells were co-transfected with HA-CYLD and Myc-OPTN or Myc-H486R. Lysates were immunoprecipitated with anti-HA antibody and subjected to western blotting. WCL, whole cell lysate.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3049778&req=5

pone-0017477-g002: H486R optineurin is defective in interaction with CYLD.Yeast strain PJ69-4A was co-transformed with wild-type optineurin or its mutants and CYLD. Transformants were grown on selection media lacking adenine to assay interaction. GST-optineurin, GST-H486R or GST alone bound to glutathione agarose beads were incubated with lysates of HeLa cells transfected with HA-CYLD. The bound proteins were eluted and immunoblotted with anti-HA antibodies. Western blot was done with optineurin antibody (lower panel) to confirm the identity of the GST fusion proteins. HeLa cells were co-transfected with HA-CYLD and Myc-OPTN or Myc-H486R. Lysates were immunoprecipitated with anti-HA antibody and subjected to western blotting. WCL, whole cell lysate.

Mentions: We tested the hypothesis that some of the glaucoma-associated mutants of optineurin may be altered in their interaction with CYLD. Wild-type optineurin or its glaucoma-associated mutants (E50K, H486R and R545Q) were co-transformed with CYLD in yeast. One of the glaucoma-associated mutants, H486R, failed to interact with CYLD (Figure 2A). In vitro binding assays using GST-optineurin and GST-H486R with cell lysates expressing HA-CYLD were carried out. As compared with wild type optineurin, the H486R mutant showed 5-fold less binding with HA-CYLD (Figure 2B). These observations were further validated by co-imunoprecipitation. The H486R mutant showed less interaction with HA-CYLD than wild type optineurin (Figure 2C). Optineurin is present in the cell as a high molecular weight homo-hexameric complex perhaps in association with other proteins which may influence its interaction with CYLD [33]. Overall our binding experiments suggest that the H486R mutant is altered in its interaction with CYLD.


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

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

H486R optineurin is defective in interaction with CYLD.Yeast strain PJ69-4A was co-transformed with wild-type optineurin or its mutants and CYLD. Transformants were grown on selection media lacking adenine to assay interaction. GST-optineurin, GST-H486R or GST alone bound to glutathione agarose beads were incubated with lysates of HeLa cells transfected with HA-CYLD. The bound proteins were eluted and immunoblotted with anti-HA antibodies. Western blot was done with optineurin antibody (lower panel) to confirm the identity of the GST fusion proteins. HeLa cells were co-transfected with HA-CYLD and Myc-OPTN or Myc-H486R. Lysates were immunoprecipitated with anti-HA antibody and subjected to western blotting. WCL, whole cell lysate.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017477-g002: H486R optineurin is defective in interaction with CYLD.Yeast strain PJ69-4A was co-transformed with wild-type optineurin or its mutants and CYLD. Transformants were grown on selection media lacking adenine to assay interaction. GST-optineurin, GST-H486R or GST alone bound to glutathione agarose beads were incubated with lysates of HeLa cells transfected with HA-CYLD. The bound proteins were eluted and immunoblotted with anti-HA antibodies. Western blot was done with optineurin antibody (lower panel) to confirm the identity of the GST fusion proteins. HeLa cells were co-transfected with HA-CYLD and Myc-OPTN or Myc-H486R. Lysates were immunoprecipitated with anti-HA antibody and subjected to western blotting. WCL, whole cell lysate.
Mentions: We tested the hypothesis that some of the glaucoma-associated mutants of optineurin may be altered in their interaction with CYLD. Wild-type optineurin or its glaucoma-associated mutants (E50K, H486R and R545Q) were co-transformed with CYLD in yeast. One of the glaucoma-associated mutants, H486R, failed to interact with CYLD (Figure 2A). In vitro binding assays using GST-optineurin and GST-H486R with cell lysates expressing HA-CYLD were carried out. As compared with wild type optineurin, the H486R mutant showed 5-fold less binding with HA-CYLD (Figure 2B). These observations were further validated by co-imunoprecipitation. The H486R mutant showed less interaction with HA-CYLD than wild type optineurin (Figure 2C). Optineurin is present in the cell as a high molecular weight homo-hexameric complex perhaps in association with other proteins which may influence its interaction with CYLD [33]. Overall our binding experiments suggest that the H486R mutant is altered in its interaction with CYLD.

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