SFAS

Amyloids are fibrillar protein aggregates involved in the etiology of a series of neurodegenerative pathologies such as Alzheimer’s and Parkinson’s disease whose medical and social impacts are ever increasing. Recently it was found that these same protein structures can also fulfill biological functions in a variety of organisms. In particular, it has also been reported, by us and others, that amyloid motifs function as signaling motifs in immune-related activation cascades both in mammals and in filamentous fungi. This biological function relies on the ability of amyloids to propagate their conformational state and thus to become infectious entities (prions). These motifs ensure the activation of effector proteins with roles in host defense and immune programmed cell death in response to activation of immune receptors. Activation occurs through templating of an amyloid fold which is transmitted from the receptor protein to the effector protein. This original mode of prion-based signal transduction is widespread and has been conserved over an extended evolutionary time frame. The detailed understanding of this prion-based signaling but also the comparison of pathological and functional amyloids requires high-resolution structural characterization of these amyloid motifs which at present remains extremely limited. Solid-state NMR constitutes the method of choice for this approach. The aim of the present project is to establish the receptor-effector transmission mechanisms governing the activity of three novel functional amyloid systems that we have recently identified in fungi. For each of these systems (HELLF, HELLP and SESB), the methods for producing 13C/15N labelled proteins in mg quantities required for solid-state NMR characterization have been set up. Preliminary solid-state NMR data indicate homogeneous, highly ordered samples and importantly exhibiting very low structural polymorphism, paving the way for 3D structure determination of the three fibrillar assemblies. In the same way, in vivo methods allowing measuring the activity of these motifs in cell death induction and prion propagation have been established. Our central hypothesis, based on previous observations is that, although the three receptor-effector amyloid systems are all involved in immune-related programmed cell death, amyloid-based signal transduction is structurally diverse for these three entities. Our goal is compare the structure-function relationship for the three motifs by solving the 3D structure of these amyloid assemblies by solid-state NMR and by exploring by site directed mutagenesis approaches the specific role of given residues and key structural elements in the folding and activity of these motifs. The integration of the structure-function studies obtained during this project for the three amyloid systems will provide the molecular and structural determinants of receptor-effector amyloid transmission. The outcome will highlight what are the common determinants during the amyloid activation and transmission, and will provide the first molecular and structural basis on the amyloid diversity in immune programmed cell death. The project will yield a global picture of the structural diversity underlying this mechanism of signal transduction and the phenomenon of amyloid templating. We will gain access to the different structural solutions that have been devised by evolution to fulfil this task of regulated induction of immune-related cell death. The project will provide three novel prion amyloid structures. The completion of the project will constitute a significant step in the understanding of the role of functional amyloids and by comparison in our understanding of the basis of amyloid toxicity in disease.