A glaucomaassociated mutant of optineurin shows reduced binding to CYLD. This mutant, unlike wild type optineurin, does not inhibit Z-VAD-FMK 187389-52-2 TNFa-induced NF-kB activation. Optineurin is essential both for inhibition of TNFa-induced NF-kB activation by CYLD and its association with RIP. In addition we show that optineurin is required for CYLD mediated deubiquitnation of RIP. Our results thus show that the interaction of optineurin with CYLD is important for the regulation of TNFa-induced NF-kB activity. Ubiquitin binding proteins, with their diverse range of UBDs, have emerged as key regulators of NF-kB signalling. Optineurin was shown to inhibit TNFa-induced NF-kB activation by competing with NEMO for the binding of ubiquitinated RIP. However, our results suggest that the regulation of NF-kB activation by optineurin is more complex and binding of ubiquitinated RIP to UBD of optineurin is only one of the steps in this complex regulation. Although ubiquitinated RIP is known to be a substrate of CYLD, the mechanism by which CYLD is recruited to RIP to deubiquitinate it, is not clear. The results presented in this manuscript suggest that optineurin mediates interaction of deubiquitinase CYLD with polyubiquitinated RIP and this interaction is essential for deubiquitination of RIP by CYLD. Thus an important function of optineurin in the regulation of NF-kB signalling is to act as an adaptor protein bringing CYLD and its substrate RIP together to facilitate deubiquitination of ubiquitinated RIP by CYLD. CYLD targets multiple players of the NF-kB signalling pathway. How CYLD recognises and is targeted to a specific substrate like RIP is not well understood. CYLD interacts directly with some of its substrates like TRAF2 and NEMO. Emerging evidence suggests that CYLD might indirectly associate with some of its substrates through intermediary adaptor proteins. It has been shown that p62/sequestosome1 binds to TRAF6 through its UBD and recruits CYLD to TRAF6 to regulate its ubiquitination. An adaptor protein should be able to specifically bind to substrates polyubiquitinated with Lys63-linked ubiquitin chains since CYLD specifically deconjugates Lys63-linked polyubiqitin chains. In addition, such an adaptor protein should also interact with CYLD. Since optineurin interacts with Lys63-linked polyubiquitin chains through its UBD and also with CYLD, it is ideally suited to act as an adaptor for CYLD to recognise its substrates. The ABIN proteins with their homologous UBD similarly act as adaptors for the recruitment of A20 to its targets like NEMO. TNFa stimulus triggers assembly of Lys63-linked polyubiquitin chains on RIP which initially binds to NEMO to activate IKK complex. Optineurin binds to polyubiquitinated RIP through its UBD and recruits CYLD; this may prevent association of NEMO with RIP. Close proximity of binding sites of both CYLD and RIP on optineurin provides support for the adaptor function of optineurin in facilitating interaction of RIP and CYLD.