logo SBA

ETD

Digital archive of theses discussed at the University of Pisa

 

Thesis etd-01102022-120355


Thesis type
Tesi di laurea magistrale LM5
Author
MOSTI, LORENZO
URN
etd-01102022-120355
Thesis title
Protein-Protein Interactions in Diseases - Expression, Purification, Crystallization, and Interaction of a 14-3-3 Protein
Department
FARMACIA
Course of study
CHIMICA E TECNOLOGIA FARMACEUTICHE
Supervisors
relatore Prof.ssa Rapposelli, Simona
relatore Dott. Garau, Gianpiero
Keywords
  • biostructures
  • proteins
  • crystallography
  • pcr
  • purification of protein
  • recombinant protein
  • 14-3-3
  • ppi
  • x-ray crystallography
Graduation session start date
26/01/2022
Availability
Withheld
Release date
26/01/2062
Summary
Many relevant human pathologies, including cancer, neurodegenerative and infection diseases, are the result of abnormal protein–protein interactions (PPI) that alter the mechanism of molecular recognition and the affinity of binding partners under a given set of conditions. Despite the successes in clinic for different protein interaction systems involved in diseases, the results of the recent years have highlighted that most PPI are indeed difficult to tackle. The successful design of binding compounds modulating PPI requires a detailed knowledge of the involved protein-protein system at molecular level, insights also into the dynamics of the protein association events, and investigation of the structural motifs that drive the association of the proteins at the recognition interface. Among the used structural biophysical approaches, X-ray crystallography has a dominating role in solving biomolecular complex structures at atomic resolution and in rational design of PPI-modulating compounds.
To explore mapping, structure, and modulation of PPI in human, we focused on the class of 14-3-3 proteins as a model system, relevant to treating a variety of diseases, including various cancers, neurodegenerative disorders, asthma, and cystic fibrosis. The 14-3-3 proteins family is constituted by 28-33kDa acidic regulatory proteins (isoforms: β, ϵ, η, γ, τ (o θ), ζ , σ) that control key biological processes, including cell cycle, apoptosis, metabolism, and control of gene transcription. There are only a few ligands that can efficiently bind and modulate their protein complexes.
We engineered a novel expression construct formed by a 14-3-3 protein (isoform ζ ) fused directly with a binding partner peptide and optimize its expression and purification at a very high level for structural biophysics approaches. We successfully screened and discovered different crystallization conditions useful for experiments of X-ray diffraction at the synchrotron light source of ELETTRA (Trieste). Finally, we tested the interaction of the target with a new class of 14.3.3 protein modulators having promised anticancer profile, both in vitro and in vivo models. This class of molecules may be a good starting point to obtain selectivity over other 14-3-3 PPI partners by expanding the molecule through structure-guided design and gaining more contact with the desired binding partners.
File