Data di Pubblicazione:
2010
Citazione:
THE PHD FINGER OF SP140: A STRUCTURAL AND FUNCTIONAL STUDY / C. Zucchelli ; tutor : Giovanna Musco ; Marcello Maria Duranti; coordinatore del dottorato: Francesco Bonomi. Universita' degli Studi di Milano, 2010 Dec 09. 23. ciclo, Anno Accademico 2010.
Abstract:
Sp140 is an IFNg-inducible leukocyte-specific member of the Sp100 family proteins. Along with the PML tumor suppressor, Sp100 family proteins are major components of PML-nuclear bodies (PML-NBs), they interact with DNA and various regulatory factors and may be involved in chromatin-dependent transcriptional activation or repression. Sp140 is expressed in all human mature B cells and plasma cell lines, as well as some T cells, suggesting an important role in cellular functions that are peculiar to these cells. In mammalian cells Sp140 behaves as a transcription co-activator for a reporter gene (Bloch et al, 2000). Despite the involvement in B-cell Chronic Lymphocytic Leukaemia (CLL, OMIM151400) (Di Bernardo et al, 2008) and HIV-1 replication (Guldner et al, 1992), until now Sp140 physiological and pathological role is unknown and unexplored, both at cellular and molecular level.
The predicted Sp140 amino acid sequence, 867 residues in length, indicates a modular structure similar to other Sp100 family members (Bloch et al, 1996). The N-terminus HSR domain (amino acids 36-157) is responsible both for PML-NBs targeting and homo/hetero-dimerization. The region between residues 306-404 is strongly negatively charged, while the central portion contains a putative bipartite nuclear localization signal, a SAND domain (residues 588-661), a PHD finger (residues 692-734) and a bromodomain (residues 756-859). In this thesis we investigated Sp140 PHD finger both at functional and structural level.
Structural determination was performed in solution by means of NMR spectroscopy. Analysis of 1H-15N HSQC spectra of Sp140 15N PHD finger wild type and 15N PHD finger P45A mutant revealed peptidyl-prolyl cis trans isomerization of the T44-P45 peptide bond and a ratio between cis and trans conformers of 1:2. As the NetPhos 2.0 server predicts phosphorylation of the T44 residue by the p38 mitogen-activated protein kinase (p38 MAPK), we suppose that the phosphorylated T44-P45 bond might be a site of regulation of the domain structure through the activity of PIN-1, an enzyme that efficiently and specifically bind to and isomerizes the phosphorylated S/T-P motifs in proteins (Wulf et al, 2005).
Using triple resonance NMR experiments we assigned 95% of backbone resonances, 96% of side chain 1H resonances and 73% of side chain non-1H resonances (side chain resonances of OH, SH, CO, NH and NH2 groups were not assigned) of the Sp140 PHD finger trans conformer. The tautomeric state of the two histidines was determined, analyzing a 1H-15N long range correlation HSQC spectrum. The 3J(HNHa) coupling constants and the corresponding phi dihedral angles were calculated by analysis of the 3D HNHA spectrum and were then compared to those predicted by TALOS+ software. We manually assigned more than 850 NOE cross-peaks of the 2D and 3D NOESY spectra. NOEs restraints and 12 dihedral angles were employed for structure calculation of the Sp140 trans conformer by means of ARIA 2.1.3 software. The best NMR ensemble achieved up to now showed a compact globular fold with two short a-helices (stretches 12-EVCR-15 and 50-FCRM-53) as the sole elements of secondary structure. Analysis of the electrostatic surface potential revealed the strong negative charged character of the Sp140 PHD finger trans conformer, with a positive patch only on one side of the domain.
Heteronuclear NOE experiments revealed that loop 2, containing the T44-P45 bond which undergoes cis trans isomerization is flexible from E40 to N47. Also the N-terminal tail of the domain is flexible up to residue L8. Flexibility caused a limited number of available NOE restraints in these two regions, especially in loop 2, and consequently an overall high backbone RMSD of the NMR ensemble. In order to increase the n
Tipologia IRIS:
Tesi di dottorato
Keywords:
Sp140 ; PHD finger ; SUMOylation
Elenco autori:
C. Zucchelli
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