PhD Programme in Cellellular, Molecular and industrial Biology (Project n.1: Physiology and Cellular Biology)

Proteomics and protein function
1. MolecularBasis of Myoclonous Epilepsy 1 (EPM1). Cystatin B is an antiprotease that inhibits proteins of the cathepsin family. Mutations of the cystatin B gene are found in patients affected by EPM1. Cystatin B is a ubiquitous protein but the damage caused by EPM1 occurs only the central nervous system, particularly the cerebellum. In fact, the cellular damage caused by knocking out of the cystatin B gene is the apoptosis of the majority of the cerebellar granule cells. The molecular basis of this pathology is not clear. Through the yeast Two- Hybrid system we have identified seven proteins that interact with cystatin B in a multiprotein complex, none of which is a protease. The interaction is found also in the adult rat cerebellum and during development. By studying Cystatin B expression in rat cerebellum, it has been shown that it is mainly present in two cell types: Purkinje cells and Radial Glial cells. We are now analyzing the role of these proteins in relation to cystatin B function and to the EPM1 disease. Using mass spectrometry we plan to identify every polypeptide within the Cystatin B multiprotein complex.
2. Functional analysis of the transcription factor SOX10 The transcription factor SOX10 is an essential protein for glial cell development in both central and peripheral nervous system in mammals. In humans, heterozygous mutations of SOX10 are linked to Waardenburg-Hirschsprung syndrome (WS4). This is a pathology of the neural crests which gives rise to intestinal aganglionosis, pigmentation, and hearing abnormalities. In order to identify SOX10 interacting proteins we used the Yeast Two-Hybrid system. Several clones were identified and are still the object of research. The identification of the mechanisms of action of SOX10 is extremely important in order to understand the molecular basis of the differentiation of glial cells in the mammalian nervous system. This knowledge could also help to explain the involvement of SOX10 mutations in the development of Waardenburg-Hirschsprung syndrome. 3. Role of Tollip in the pathway of IL-1 signal transduction Tollip is a protein which mediates the interaction between the protein kinase IRAK-1 and the IL-1 receptor. According to the proposed model, in the absence of IL-1 stimulation, Tollip interacts and inhibits IRAK-1 kinase activity. Binding with the cytokine activates the receptor complex, enabling Tollip to interact with both of the subunits that take part in the complex, namely IL-1R1 and the accessory protein. This results in the interaction of IRAK-1 with the receptor. Despite having been cloned as an IL-1 receptor- interacting protein, Tollip’s role in the IL-1 signal transduction pathway is still unclear. By studying the function of Tollip through the yeast Two-Hybrid system, we have identified several proteins, which are now in the process of being characterized. Using mass spectrometry we plan to identify all the cellular proteins which interact with Tollip after IL-1 induction.

Post doctoral fellowships on specific research projects

-

Proteomics and protein function

Research Group

Marialuisa Melli (Full professor)

Davide Ambrosetti (Research Associate)

Valentina Sancisi (PhD student)

Elena Cipollini (PhD student)