Dokument: Biochemical and biophysical characterization of the centrosomal protein TACC3
| Titel: | Biochemical and biophysical characterization of the centrosomal protein TACC3 | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=23455 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20130703-092548-8 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | M.Sc. Thakur, Harish Chandra [Autor] | |||||||
| Dateien: |
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| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | The centrosomal protein TACC3 belongs to the TACC (Transforming Acidic Coiled Coil) family of mitotic spindle-associated proteins, which display a highly conserved C-terminal coiled coil (TACC) and protein interaction domain and a rather functionally uncharacterized N-terminus. TACC3 is critical in particular for embryonic development and stem cell proliferation, a phenotype that became evident upon TACC3 gene inactivation in the mouse.
TACC3 is highly expressed in a cell cycle-dependent manner during the G2/M phase where its centrosomal localization depends on its phosphorylation mediated by the mitotic kinase Aurora-A. Functionally, TACC3 forms a complex with the microtubule polymerase chTOG (colonic and hepatic Tumor Overexpressed Gene) thereby regulating spindle microtubule stability and dynamics and hence correct chromosomal segregation. It was proposed that the TACC domain of the X. laevis TACC3 homologue is being “masked” by an unknown molecular mechanism and that phosphorylation by Aurora-A kinase may “unmask” the C-terminus possibly required for binding to XMAP215, the chTOG homologue of X. laevis. However, despite this, the modus and directionality of this protein interaction that is mediated through the TACC domain of TACC3 and the C-terminus of chTOG/XMAP215 were largely uncharacterized at the molecular level at the beginning of this work. Therefore, GST-fused murine TACC3 deletion mutants and single fragments as well as the C-terminal part of murine chTOG were overexpressed in E. coli, purified, and thereafter subjected to an in-depth analysis regarding their molecular interaction, Aurora-A mediated phosphorylation, and lastly subcellular localization employing various biochemical, biophysical, and cell imaging-based methods, the latter following transient transfection of GFP-fused constructs. In particular, the following major questions were addressed: (I) Does the N-terminus of TACC3 mask the C-terminal TACC domain via an intramolecular interaction? Which domain of TACC3 is involved in such an intramolecular interaction? (II) How does the TACC domain interact with the C-terminus of chTOG? Which part of the TACC domain is involved in this intermolecular interaction? Does the interaction of chTOG with the TACC domain interfere with an intramolecular interaction and thus TACC domain masking? (III) Is Aurora-A-mediated phosphorylation of TACC3 dependent from its intramolecular (“masked”) or intermolecular (chTOG-bound) interaction state? (IV) What structural insight can be gained from the biophysical analysis of TACC3? Subjecting murine TACC3 to an in-depth in silico comparison within the vertebrate and mammalian TACC family revealed that TACC3 contains (i) a conserved N-terminal domain of approximately 100 amino acids, (ii) a central serine/prolin-rich repeat region, and (iii) a TACC domain consisting of two previously not described coiled coil modules, CC1 and CC2, separated by a breaking peptide region. CC2 displays thereby a clearly higher conservation than CC1. Based on these findings and addressing the first point, it is shown in protein-protein interaction studies using complementary methods (pulldown, gel filtration analysis, ITC) that the central repeat region undergoes a specific intramolecular interaction with the CC2 module of the TACC domain of TACC3 thereby likely mediating the previously proposed masking function. In contrast, the CC1 module within the TACC domain binds selectivily to the C-terminus of chTOG and thereby precludes the intramolecular interaction between CC2 and the central repeat region. With this, intramolecular “masking” of the TACC domain may regulate its stability prior to chTOG binding. Interestingly, in in vitro kinase assays, TACC3 was efficiently phosphorylated by Aurora-A independent of its intra- or intermolecular interaction status thereby suggesting that, in contrast to its critical function in centrosomal recruitment of TACC3 or the TACC3-chTOG complex, Aurora-A does not play a decisive role in the “unmasking” action of the TACC domain prior to chTOG binding. Thus, these findings overall establish a novel role of the TACC domain of TACC3 where CC1 and CC2 as two functionally diverse modules which direct and determine TACC3-chTOG protein complex formation probably prior to its Aurora-A regulated centrosomal localization. Purified murine TACC3 was characterized at a biophysical and structural level. Employing various methods, including gel filtration, multi angle light scattering (MALS), analytical ultracentrifugation (AUC), and electron microscopy (EM) clearly revealed an elongated, fiber-like oligomeric structure of full length TACC3 with the coiled coil-containing TACC domain displaying a parallel -helical orientation throughout the CC1 and CC2 modules. Interestingly, the oligomeric/multimeric nature of TACC3 could be confirmed by transient transfection studies of TACC3-GFP fusion proteins in eukaryotic cells. The occurrence of punctuate-like structures in interphase cells was thereby dependent on the presence of the TACC domain and clearly reduced when either one of the CC modules was deleted. In first crystallization attempts crystals for the CC1 module could be successfully generated which are now being optimized and subjected to molecular structure determination. The clinical relevance of human TACC3 alterations becomes increasingly evident. Various alterations in TACC3 gene expression have been linked to cancer development and several TACC3 point mutations have been identified in primary human tumors. Recently, a Fibroblast Growth Factor Receptor 3-TACC3 gene fusion with transforming activity has been reported in glioblastoma patients. Thus, future work will focus on the molecular elucidation of the mechanistic impact of cancer associated structural TACC3 alterations on TACC3-chTOG interaction and function and ultimately chromosomal segregation and stability. In particular, blocking the molecular CC1-chTOG binding interface may represent a candidate anti-neoplastic approach against TACC3 and chTOG overexpressed in tumors. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät | |||||||
| Dokument erstellt am: | 03.07.2013 | |||||||
| Dateien geändert am: | 03.07.2013 | |||||||
| Promotionsantrag am: | 05.12.2012 | |||||||
| Datum der Promotion: | 19.12.2012 |
