טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentHaskin Joseph
SubjectCharacterization of AF-6 as a Novel Parkinson's
Disease Related Protein
DepartmentDepartment of Medicine
Supervisor Professor Simone Engelender
Full Thesis textFull thesis text - English Version


Abstract

Parkinson's disease (PD) is caused by the death of dopaminergic neurons and the presence of inclusion bodies, termed Lewy bodies (LB), in surviving dopaminergic neurons. Recent advances have been made by the identification of several genes implicated in familial forms of PD.

            The first protein which was identified as related to PD is a-synuclein. Mutations in a-synuclein are responsible for autosomal dominant forms of PD. Another protein associated with PD is synphilin-1, which is an a-synuclein-interacting protein. We identified a synphilin-1 isoform, synphilin-1A, which is neurotoxic, aggregation prone and accumulates in LBs, suggesting that it may play a role in the formation of LB.

            We now identified a protein, called AF-6, that interacts with synphilin-1A. The AF-6 gene has been previously shown to translocate in certain types of leukemias. We obtained data showing that AF-6 interacts with synphilin-1A in vitro and in vivo. This interaction is mediated through the last four amino acids of the synphilin-1A and the PDZ domain of AF-6. AF-6 accumulates in the presence of increasing levels of synphilin-1A and AF-6 co-aggregates with synphilin-1A inclusions in transfected cells, suggesting that AF-6 could participate in the formation of Lewy bodies and in PD.

            We also found that AF-6 interacts in vitro and in vivo with SIAH. SIAH was shown to interact with and to ubiquitinate both a-synuclein and synphilin-1. We now found that SIAH can also ubiquitinate and promote the degradation of AF-6. SIAH interacts with both the N-terminus and C-terminus of AF-6. At the C-terminus, the interaction with SIAH is modulated by the PDZ domain, where possibly that the occupancy of AF-6 PDZ domain by synphilin-1A, or additional interactors, may facilitate the association of SIAH to AF-6's C-terminus.

            Finally, we identified parkin as an additional AF-6 interacting protein. The interaction between AF-6 and parkin occurs in vitro and in vivo, and is mediated by the association of parkin’s last three amino acids with the PDZ domain of AF-6. Parkin is an E3 ubiquitin-ligase that is mutated in families with autosomal recessive PD. Parkin ubiquitinates and promotes the degradation of AF-6. On the other hand, increasing amounts of AF-6 inhibited parkin's ligase activity, suggesting that AF-6 exerts a mechanism of feedback inhibition to prevent its own degradation. The interaction and degradation of AF-6 by different E3 ubiquitin-ligases related to PD suggest that the homeostasis of AF-6 might be important to the pathogenesis of the disease.