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| Author(s): |
Julio Cesar Pissuti Damalio
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Carlos. |
| Institution: | Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT) |
| Defense date: | 2011-10-26 |
| Examining board members: |
Ana Paula Ulian de Araujo;
Pietro Ciancaglini;
Márcia Regina Cominetti;
Glória Regina Franco;
Rosangela Itri
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| Advisor: | Ana Paula Ulian de Araujo |
| Abstract | |
Septins are members of a conserved group of GTP-binding and filament-forming proteins. They are involved in a variety of cellular processes, such as microtubule regulation, vesicle trafficking, the formation of scaffolding platforms and actin dynamics. Human Septin 2 (SEPT2) has an N-terminal polybasic region responsible for lipid binding, a GTPase domain, and a C-terminal domain. SEPT2 is essential for cytokinesis and it is found in many tissues, mainly in the brain. Together with SEPT1 and SEPT4, it is accumulated in deposits known as neurofibrillary tangles in Alzheimers disease, which is evidence that SEPT2 may be involved in this process. In this study, the human SEPT2, and its domains, were cloned, expressed in E.coli and purified by affinity and size-exclusion chromatographies. The proteins form homodimers in vitro, suggesting that the GTPase domain is enough to promote the oligomerization. Thermal unfolding revealed the formation of aggregates under physiological conditions, which have the ability to bind a specific amyloid dye, Thioflavin-T, suggesting them to be an amyloidal fiber. Besides, in silico prediction programs, TANGO and WALTZ, corroborate that SEPT2 contain regions with high probability of aggregation and amyloidogenic formation, respectively. Moreover, we observed 20-50 nm thick filamentous structures by electron microscopy of negatively stained. Using Langmuir monolayers at the cell membrane lipid packing, SEPT2 and SEPT2NG bound to the phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Results from in situ PM-IRRAS experiments indicated that the secondary structure of SEPT2 is preserved upon interacting with PtdIns(4,5)P2, but not when interacting with DPPC - which is not specific for SEPT2 - at the air/water interface suggesting an association with the plasma membrane and a role in septin regulation. Furthermore, we also identified protein partners of SEPT2, from both human leukocyte and brain fetal cDNA libraries, using the yeast two-hybrid system. SEPT2 was shown to interact with: septins 6 and 4; a serine-protease and a MAP inhibitory protein; an ubiquitin-conjugating enzyme; and proteins related to cellular division. Thus, taken together this study contributed for the knowledgment of the stability and the aggregation kinetic of the SEPT2, leading to a better understanding of this protein and their role in neurodegenerative disorders. (AU) | |
| FAPESP's process: | 06/53355-7 - Biochemical and structural studies of human SEPT2 septin: determining factors of the aggregates formation |
| Grantee: | Julio Cesar Pissuti Damalio |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |