Investigating the molecular mechanisms underlying ks-vFLIP and endogenous activation of the NF-kB pathway.

The main aim of our research is to understand at the atomic level how two essential cellular pathways (called the NF-?B canonical and non-canonical pathways) are activated by a viral protein linked to Kaposi Sarcoma, the main form of AIDS related cancer and a host protein of similar structure (implicated in immune cell development and non-viral cancer). For stimulation of the canonical pathway, both proteins have been found to interact with a component of an assembly of proteins known as the IKK complex that is thought to act as a molecular switch. Our studies will focus on how the host protein recognises this component (since this has already been done for the viral protein in a previous study) and how the molecular switch toggles between on and off states. To achieve this, we will use a technique known as protein crystallography that will enable us visualise the host protein bound to the IKK component in 3 dimensions and also the IKK component in isolation allowing comparison. This should ultimately aid in the design of novel anti-cancer drugs that specifically inhibit the transition. We aim to perform similar studies with our target proteins and components of the non-canonical pathway.

The canonical and non-canonical (or alternative) NF-kB pathways are key to cellular survival and are pivotal to the life cycle of several lymphoproliferative viruses. Following viral infection or the breakdown of the regulatory processes, both are rendered constitutively active culminating in the upregulated expression of anti-apoptotic and pro-survival genes. This has been associated with a number of malignancies that include cancer, lymphoproliferative disorders and auto-immune diseases. Understanding how these NF-kB pathways are activated by both endogenous and viral proteins at the molecular level is thus crucial for the development of novel therapeutics given that many of these disorders lack effective treatments. We therefore seek to establish the molecular basis underlying activation of the canonical and non-canonical NF-kB pathways by the Kaposi Sarcoma human herpes virus protein ks-vFLIP and to establish whether its cellular homologue p22-cFLIP utilises similar mechanisms. For stimulation of the canonical pathway, both ks-vFLIP and p22-cFLIP have been shown to interact with IKKgamma, an essential component of the IKK kinase. Having recently determined the crystal structure of ks-vFLIP bound to IKKgamma we wish to ascertain whether p22-cFLIP uses a similar mode of recognition through structural studies. It has been suggested that activation of the canonical pathway is dependent on a conformational transition within the IKKgamma molecule enabling it to act as a molecular switch. Using a biophysical approach, we aim to investigate the dynamics of this transition. By contrast to the canonical pathway, ks-vFLIP activation of the non-canonical pathway is achieved via a physical interaction with the transcriptional activator p100. We will analyse this interaction using a combination of structural biology and biochemical techniques and also establish whether p22-cFLIP can similarly contact p100 and thus activate the non-canonical NF-kB pathway that is so far unknown. We will also investigate downstream events to determine whether IKKgamma functions in the ks-vFLIP (and potentially p22-cFLIP) activation of the non-canonical pathway that is also unresolved.