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Item Type: Tesi di dottorato
Lingua: English
De Paola,
Date: 30 November 2011
Number of Pages: 124
Institution: Università degli Studi di Napoli Federico II
Department: Scienze biologiche
Scuola di dottorato: Biotecnologie
Dottorato: Scienze biotecnologiche
Ciclo di dottorato: 24
Coordinatore del Corso di dottorato:
Sannia, GiovanniUNSPECIFIED
Benedetti, EttoreUNSPECIFIED
Date: 30 November 2011
Number of Pages: 124
Uncontrolled Keywords: Design helix peptide
Settori scientifico-disciplinari del MIUR: Area 03 - Scienze chimiche > CHIM/03 - Chimica generale e inorganica
Area 03 - Scienze chimiche > CHIM/08 - Chimica farmaceutica
Date Deposited: 06 Dec 2011 11:29
Last Modified: 30 Apr 2014 19:48
DOI: 10.6092/UNINA/FEDOA/8845


Malfunctions in transcriptional regulation are associated with many human diseases and there is considerable interest in biomedical and biotechnological field in developing artificial transcription factors (ATFs) that must minimally contain a DNA binding domain (DBD) and a transactivation domain (TAD). Designing artificial TADs is proven difficult because these systems interact with multiple target proteins as part of their normal function and, moreover, there are only a limited number of high-resolution structures of TADs in complex with target proteins. The p53 protein is a transcriptional activator factor with a fundamental role in cellular survival. Recent structural studies by Omichinski and co-workers gave insights into interaction between p53 TAD2 and tfb1/p62 subunit of the transcription factor TFIIH and contributed to the understanding of the way p53 protein works in transcriptional activation. On the basis of these information new molecules, able to interfere with the binding between p53 TAD2 and tfb1 of TFIIH, were designed. In our hypothesis peptide analogues of p53 TAD2 with enhanced helical propensity could yield a more potent artificial TAD. Therefore, several peptides mimicking the helical fragment 47-55 of p53 TAD2 were designed by using different molecular tools, such as N- and C-capping boxes, and synthesized. A structural analysis of all obtained peptides by CD experiments highlighted an increased helical content relative to native p53-13 peptide and one of these peptides, E-Cap, showed a higher binding affinity than other peptides in ITC experiments. Furthermore NMR experiments were performed to elucidate how E-Cap peptide interacts with tfb1. NMR titrations spectra displayed that E-Cap peptide binds along the same interface of p5325-75, forming a 9-residue α-helix when in complex with tfb1. NMR studies of E-Cap peptide in complex demonstrated the presence of both the i, i+3 and the i, i+4 interactions for Leu6 with Leu9 and Trp10 residues, respectively. The potential ability of E-Cap peptide to activate transcription in vivo was investigated in yeast cells by activation assay of lacZ with LexA fusion peptide. E-Cap peptideshowed to be a more potent transcriptional activator than positive control Gal4, p53- 13 native sequence and AH and VP2 transcriptional activators. Besides mutation studies for E-Cap and p53-13 peptides provided evidence that both i, i+3 interaction between Leu6-Leu9, and i, i+4 interaction between Leu6-Trp10 have an important role for the in vivo activity. In conclusion, a new peptide with high content of helical conformation and able to activate transcription in vitro was designed using only proteinogenic amino acids. This result will be used to develop a new interesting biotechnological system such as "programmable ATF", linking obtained TAD to new synthetic DBD. BTB/POZ domain-containing proteins are identified in diverse cellular locations and participate in different processes ranging from transcriptional repression/activation to cytoskeletal organization. In particular, KCTD are a class of protein containing BTB/POZ domains with different biological function as KCTD11 that inhibits tumor growth in medulloblastoma. POZ/BTB domain of KCTD proteins mediates interaction with Cullin, a family of proteins ubiquitin ligases that induce proteolysis of target proteins by proteasome. Recently, different studies highlighted that several members of KCTD family protein bind to Cullin3 (Cul3). The understanding of the structural features required for the interaction of the Cul3 with KCTDs is an intriguing challenge and is the basis for the future design of molecules with agonist/antagonist function for biotechnological applications. To achieve this goal the attention was focused on KCTD11 that mediates interaction with Cul3 by its POZ/BTB domain at N-terminus. Starting from the proposed model of KCTD11-Cul3 interaction, peptide molecules mimicking the helical fragment of Cul3 49-68, were designed. To evaluate the role of some aromatic residues of Cul3 in the interaction with KCTD11 two mutant peptides were developed (Cul3-KK and Cul3-Y(P)). Structural analysis by CD experiments highlighted that these peptide and Cul3-wt native sequence peptide are unfolded. ELISA assays showed that Cul3-KK and Cul3-Y(P) have a lower affinity than Cul3-wt for KCTD11. Subsequently, in order to test if the enhance of the helical propensity match with a gain in binding affinity, opportunely constrained sequences were designed and synthesized. Cul3 stapled peptide, with a hydrocarbon bridge in central position of sequence, showed indeed a higher helical structure than the native Cul3-wt peptide. Preliminary ELISA assays indicate that both Cul3-wt and Cul3 stapled peptides were able to interact with KCTD11. Future studies by ITC and NMR will allow the evaluation of the Kd and the molecular features governing the interaction with KCTD11 required for the future perspectives in biotechnology field.

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