Masters- Geyer collaborative projects

Melbourne subproject:
Structure function studies of the Pyrin inflammasome

photo of Seth Masters
Seth Masters
Walter & Eliza Hall
Institute of Medical
Email Seth

We recently identified a new disease caused by a mutation in the innate immune receptor Pyrin, which results in a severe chronic inflammation (Masters et al, Sci Trans Med 2016). This mutation prevents phosphorylation of S242, and binding of 14-3-3. Normally, 14-3-3 is bound to inhibit the Pyrin “inflammasome”, a protein complex that produces the cytokine IL-1b. The mutation increases activation of the inflammasome, and therefore the patients have now been treated by blocking IL-1b.

In this project we will study the function of Pyrin with regard to the domains that activate the inflammasome, and how 14-3-3 acts to prevent complex formation. This will be integrated with information from patient mutations. The three dimensional structure of Pyrin together with 14-3-3 in the inactive conformation, and with the S242 mutation in an active conformation will also be generated to provide greater detail. This will involve protein biochemistry techniques for modifications and purification, followed by structural analysis. Based on these results we will perform additional functional studies to confirm the basis for Pyrin activation, with relevance for chronic inflammatory diseases, and infectious pathogens that trigger the Pyrin inflammasome.

This project is a collaborative effort between the Walter and Eliza Hall Institute and the University of Bonn, supported by the Bonn &Melbourne Research and Graduate training group (Bo&merang). The student will perform 2 years research in the laboratory of Dr Seth Masters at WEHI, and 1 year in the laboratory of Dr Matthias Geyer at the University of Bonn (Germany). The Masters laboratory studies innate immunity based on investigations of genetic changes that result in inflammatory diseases. This is mostly focused around a family of innate immune receptors that can form inflammasome complexes and trigger production of the potent inflammatory cytokine IL-1b. The Geyer lab is interested in the molecular mechanisms that govern immune receptor activation. This incorporates a variety of techniques from molecular biology and biochemistry to structural biology to analyze interaction between proteins, RNA, lipids, and ligands.

Bonn subproject:
Structural basis of NLRP1 inflammasome formation

photo of Matthias Geyer
Matthias Geyer
Institute of Innate
Email Matthias
photo of Jonas Mocking
Jonas Möcking
PhD Student
Institute of Innate
Email Jonas

As part of the innate immune system, inflammation plays a major role in defending the human body against pathogens. Cytosolic sensors known as NOD-like receptors (NLRs) recognize different stimuli caused by invading pathogens and can trigger an inflammatory response. Upon activation, these proteins assemble into oligomeric complexes and recruit the adapter protein ASC (Apoptosis-associated speck-like protein containing a CARD). Multiple ASC molecules form speck-like filaments and can bind and activate Procaspase-1. Active Caspase-1 is responsible for the cleavage of Pro-IL-1b and Pro-IL-18, ultimately resulting in inflammation and a form of cell death called pyroptosis. Within the family of NLRs there are four different subgroups, the NLRAs, NLRBs, NLRCs, and NLRPs. NLRP1 was the first member of the NLRPs to be discovered. The domain architecture of NLRP1 differs from other members of the NLRP subgroup. Apart from the typical N-terminal PYD, NLRP1 contains a C-terminal FIIND and CARD domain1. Both, the PYD and the CARD, are known to be effector domains in NLRs. However, it has been shown very recently that for NLRP1 only the CARD domain is able to trigger the activation of Caspase-1. Contrary, the PYD has an autoinhibitory effect on NLRP1 function 2. Using recombinant expression of full length human NLRP1 as well as separate domains in baculo virus infected Sf9 cells or bacterial E.coli cells we aim to generate structural data to understand the underlying mechanisms of NLRP1 activation and ASC and Caspase-1 recruitment. To this end, methods like protein crystallization and electron microscopy are applied. Additionally, biochemical assays like the malachite green assay are used to investigate the importance of ATP as a cofactor in activating NLRP1. One of the challenges that arise is the fact that the activating trigger for human NLRP1 has not been discovered yet.

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