Riassunti dei progetti del corso di dottorato di ricerca in biologia molecolare e cellulare
Project leader: ANNA MORONI (anna.moroni@unimi.it)
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- Project leader: MARCO MUZI FALCONI ( marco.muzifalconi@unimi.it)
- Project leader: MARCO NARDINI (marco.nardini@unimi.it)]
Project leader: ANNA MORONI (anna.moroni@unimi.it)Location: Department of Biosciences, University of Milan, ItalyRESEARCH PROJECT SUMMARYThe structural mechanism of KAT1 channel regulation by the cytoplasmic domain CNBHD The KAT1 family of voltage-dependent potassium channels (AKT1, SKOR, GORK) are important regulators of nutrient uptake and osmotic responses in plant cells. KAT1 in particular plays a key role in stomata function, thus controlling carbon dioxide uptake for photosynthesis and water balance of the entire organism. The KAT1 channel activity depends on membrane voltage as well as on extra- and intracellular modulatory factors which act by shifting the voltage range of KAT1 activation. KAT1 channels have two large intracellular regions that underlie the specialized gating and regulation of this channel family (Marten and Hoshi, PNAS 94, 3448-3453,1997). Indeed, voltage-dependent gating depend on the N and C- termini in a manner that is reminiscent of mammalian KCNHD channels. Moreover, the carboxy-teminal region of KAT1 channels domain is structurally and functionally related to those of mammalian KCNH channels that contains a cyclic nucleotide- binding homology domain (CNBHD) which is connected to the pore through a C-linker domain (Haitin et al., Nature 501, 444-448, 2013). The CNBHD however does not bind cyclic nucleotide and presumably regulate channel gating in a cyclic nucleotide-independent manner. We propose in this project to solve the structure of the CNBHD of KAT1 and to dissect its functional role by a combination of X-ray crystallography and cell electrophysiology. Project leader: MARCO MUZI FALCONI (marco.muzifalconi@unimi.it)Location: Department of Biosciences, University of Milan, ItalyRESEARCH PROJECT SUMMARYHaspin kinase: a new regulator of cell polarity and cell division Haspin is an atypical serine-threonine protein kinase which is evolutionarily conserved in all eukaryotes (Higgins, 2003). The structure of human haspin has been determined, but very little is known regarding its substrates and its role in vivo. In human cells, Haspin is involved in mitotic phosphorylation of histone H3-Thr3 and in protecting centromeric cohesion during mitosis (Dai et al., 2005; Dai et al., 2006; Yamagishi et al., 2010; Kelly et al., 2010; Wang et al, 2010). Budding yeast contains two haspin-coding genes (ALK1 and ALK2) and their gene products are cell cycle regulated both at the level of protein stability and phosphorylation (Nespoli et al. 2010). Since in yeast H3-Thr is not phosphorylated this model system could be exploited to identify new functions for Haspin. We recently showed that Alk1 and Alk2 play a role in modulating cell polarization, actin distribution and mitotic spindle alignment (Panigada et al. 2013). In this project we propose to investigate the molecular mechanisms involved in this new function of Haspin. Preliminary evidence suggest a genetic interaction between Haspin and Cdc42. Moreover, Alk2 physically interacts with the Cdc42, a GTPase controlling cell polarity (Etienne-Manneville, 2004), and elevated Alk2 levels reduce phosphorylation of the Cdc42 activator Cdc24. These findings suggest that haspin may impact on cell polarity by regulating Cdc42 activity or one of its several effectors. Through the combined use of cell biology, biochemistry, molecular biology and genetics we will analyze how haspin affects Cdc42 localization, activation/inactivation, interaction with downstream effectors and how haspin modulates cell polarization, and asymmetric cell division. Dai et al., (2005) Genes Dev 19:472 Dai et al (2006) Dev Cell 11:741 Etienne-Manneville (2004) J Cell Sci 17:1291 Higgins J., (2003) Cell Mol Life Sci 60:446 Kelly et al. (2010) Science 330:235 Nespoli et al., (2006) Cell Cycle 5:1464 Panigada et al., (2013) Dev Cell 26:483 Wang et al. (2010) Science 330:231 Yamagishi et al (2010) Science 330:239
Location: Department of Biosciences, University of Milan, ItalyRESEARCH PROJECT SUMMARYStructural analysis of transcription factor/DNA complexes One of the key issues in biology is how the genetic information is transferred to biological functions. Binding of transcription factors (TFs) to discrete sequences in gene promoters and enhancers, is crucial to the process, which needs to interface with chromatin, whose fundamental unit is the nucleosome, formed by core histones wrapped by 146 bp of DNA. Binding of TFs entails the recruitment of histone modifying and chromatin remodeling machines, thus helping to define the chromatin status (“euchromatin” vs “heterochromatin”). TFs fall in essentially two categories: (i) “pioneer” TFs, with intrinsic chromatin association capacity; (ii) "activating" TFs, binding to a favorable chromatin landscape pre-set by pioneers. In this context, the present PhD project focus on NF-Y, a histone-like TF that binds and activates the CCAAT box [1], and on MYC, a proto-oncogene that binds the E-box (5’-CACGTG-3’), whose altered expression transforms cells [2]. NF-Y and MYC are deemed to be paradigms of pioneer and activating factors, respectively, and indeed they were shown to interact directly. Furthermore, the availability of the 3D structures for both NF-Y and MYC [3, 4] makes both TFs potential targets for development of anti-cancer drugs. Because of their direct binding, the strong correlation between the NF-Y and MYC loci in vivo, and the fact that regulation of MYC and CCAAT genes is crucial to oncogenic transformation, understanding of their interplay at the structural level will further increase druggable surfaces. The present PhD project will be carried out in the Nardini’s (structural biology) lab as a continuation of an ongoing research project that led recently to the successful determination of the X-ray structure of the NF-Y in complex with its target DNA [3]. The project will pursue analyses of NF-Y in complex with DNA containing multiple CCAAT boxes, and the NF-Y/MYC/MAX-DNA complex, both by X-ray crystallography and solution scattering methods (Small/Wide Angle X-ray Scattering, SAXS/WAXS). The SAXS/WAXS experiments, being performed on solution samples, are always practicable, provided that the sample is sufficiently pure and monodisperse. For the X-ray crystallography approach, the Nardini lab experience in growing protein-DNA complex crystals will be crucial for the achievement of this step: several E-box and CCAAT-containing fragments will be designed and tested in an approach that proved successful for the NF-Y/CCAAT complex [3]. The 3D structure of the NF-Y/DNA complex, will also allow to search rationally for potential inhibitors. As a part of the PhD project, inhibitor/NF-Y docking simulations will be carried out to screen virtual chemical libraries of low molecular weight compounds, searching for inhibitors of the NF-Y quaternary assembly and of its DNA-binding capacity. X-ray crystallography will be then applied to characterize the structure of the complexes between NF-Y and the best inhibitors. A similar approach will be eventually applied to interfere with the interactions between NF-Y and MYC/MAX. Potential inhibitors selected through the in silico approaches will be cross-validated in vitro through Thermal shift and electrophoretic mobility shift assay (EMSA) experiments, and in cells by ChIPs. [1] Dolfini D, Gatta R, Mantovani R. Crit Rev Biochem Mol Biol. (2012) 47: 29-49. [2] Prendergast GC, Lawe D, and Ziff EB. Cell (1991) 65: 395-407. [3] Nardini M, Gnesutta N, Donati G, Gatta R et al. Cell (2013) 152: 132-143. [4] Nair SK, Burley SK. Cell (2003) 112: 193-205.
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