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| Author(s): |
Joci Neuby Alves Macêdo
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: | 2010-09-24 |
| Examining board members: |
Richard Charles Garratt;
Maria Celia Bertolini;
Glória Regina Franco;
Jorg Kobarg;
Ricardo De Marco
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| Advisor: | Ana Paula Ulian de Araujo |
| Abstract | |
Septins are proteins that belong to the superfamily of GTPases, which were initially identified in Saccharomyces cerevisiae, and then in higher eukaryotes, except plants. These proteins are involved in a variety of cellular processes such as chromosome segregation, cell polarity, membrane dynamics, vesicle trafficking, exocytosis, apoptosis, among others. Mutations or changes in the expression of septins have been associated with various cancers and neurological diseases. Aiming to provide functional information about these proteins, the human septins 1, 5 and 7 were used as baits in yeast two-hybrid assay in order to identify their protein partners. After screening cDNA libraries from human leukocytes and fetal brain, the protein partners predominantly found were septins from others groups. The septin-septin interactions involved the GTP binding domain. Others non-septins interactors have also been identified in the libraries and were functionally related to endocytosis, the regulation of the GTPase activity, intracellular trafficking, sumoylation, maintenance of metaphase plate and centrosome maturation. Some of these functions are new and were related to the septins for the first time. This work also focused on the biophysical characterization of the septins 3 and 5 (SEPT3 and SEPT5). Many different protocols were developed aiming to obtain homogeneous samples of SEPT5, but were not successful. On the other hand, recombinant SEPT3, without the amino-terminal domain (SEPT3GC), was produced in a homogeneous form in E. coli. SEPT3GC is monomeric in solution as confirmed by size exclusion chromatography and small-angle X-ray scattering (SAXS). Also, SEPT3GC has shown to be active and able to hydrolyze GTP in vitro. The SEPT3GC affinity by GTPγS and GDP were evaluated by isothermal titration calorimetry (ITC). The KD for GTPγS was about 5,43 μM and it was observed to be Mg2+ dependent. The binding to GDP was not detectable. SEPT3GC aggregates induced by temperature were able to bind the thioflavin-T fluorescent probe, suggesting an amyloid nature for such structures. (AU) | |
| FAPESP's process: | 06/57573-9 - Biochemical and structural studies of human SEPT3 and SEPT5 septins and identification of protein partners |
| Grantee: | Joci Neuby Alves Macedo |
| Support Opportunities: | Scholarships in Brazil - Doctorate |