Medulloblastoma tumor suppressor factor RENKCTD11: structure and function based design and development of new compounds of theraupetical interest.
Pirone, Luciano (2009) Medulloblastoma tumor suppressor factor RENKCTD11: structure and function based design and development of new compounds of theraupetical interest. [Tesi di dottorato] (Inedito)
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Hedgehog (Hh) signaling is suggested to be a major oncogenic pathway in medulloblastoma, which arises from aberrant development of cerebellar granule progenitors. Hh signaling is regulated by ubiquitin ligases E3, that process the downstream transcription factors Gli via dual Cullin-based ubiquitin-dependent pathways. Interestingly, Cullin3 has been shown to require proteins containing BTB (Broad Complex, Tramtrack and Bric a Brac) domain to target substrates. Recently a human BTB protein has been identified, RENKCTD11, which displays allelic deletion as well as significantly, reduced expression in medulloblastoma. Data in vitro have suggested that RENKCTD11 binds Cullin3 and indirectly promotes the degradation of Gli1 thus inhibiting the transactivation of the Hedgehog target genes. Although the available literature data clearly show the role of RENKCTD11 as tumour suppressor, its biochemical properties remain to be defined. In this context we have undertaken a structural and functional study on RENKCTD11 protein. Human genomic region coding for RENKCTD11 has been amplified by PCR, cloned and over-expressed in Escherichia coli in soluble form, as a fusion product with Maltose Binding Protein (MBP). The protein has been purified to homogeneity by only one purification step utilizing the presence of His-tag. Size exclusion chromatography allowed to structurally characterize RENKCTD11 as an homotetramer in agreement with in silico analysis. The TEV-digested protein sample was extremely unstable. The alignment of RENKCTD11 sequence with sequence databases shows that a region of almost one hundred residues presents a significant degree of similarity with the POZ/BTB domain of potassium (kv4) channel. Bioinformatic analyses have allowed to identify the exact extension of POZ/BTB domain of RENKCTD11. It has been cloned, expressed as a fusion product with Thioredoxin-A (TrxA), purified in soluble form as a tetramer and characterized by mass spectroscopy, circular dichroism and light scattering. The homogeneous protein has been used for crystallization tests. Site-directed mutagenesis has been used to validate the ionic interactions responsible of the POZ/BTB domain tetrameric form. The mutant D69A/R74A/N78A/R81A resulted strongly unstable. These data confirmed the key role of these residues in the stabilization of the tetrameric form of the protein. The experimental evidence that POZ/BTB of RENKCTD11 forms a stable tetramer in solution prompted us to check the possibility to generate by homology modelling a RENKCTD11 POZ/BTB-Cul3 complex with a 4:4 stoichiometry. A peptide corresponding to residues 49-68 (NH2-NSGLSFEELYRNAYTMVLHK-COOH) of Cul3 has been individuated as responsible of interaction with the domain POZ/BTB of RENKCTD11. The peptide binds POZ/BTB of RENKCTD11 with an apparent affinity constant of 0.8 μM. Then, we tried to investigate whether the same region of Cul3 was responsible for interaction with POZ/BTB domains of other KCTD proteins. It was chosen as the starting point KCTD5, a cytosolic protein whose crystal structure has been recently deposited. The POZ/BTB domain of KCTD5 with five aminoacids added at N-terminus (region 40-151) was expressed in E. coli and obtained in large amount. The results obtained by ELISA show that this POZ/BTB domain binds the helix 49-68 of Cul3 with an affinity similar to that of the BTB/POZ of RENKCTD11. Finally we present a mechanism of function of RENKCTD11 applicable to other members of KCTD family that, interacting with the ubiquitin-ligase-E3, regulate some of the most important cellular metabolic pathways by their POZ/BTB domains.
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