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Galeno, Lauretta (2014) Structural features of the intracellular domains of the cystic fibrosis transmembrane conductance regulator. [Tesi di dottorato]

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Abstract (inglese)

Cystic fibrosis (CF) is the most common human genetic disease, occurring prevalently in the Caucasian population at a rate of 1 to 2500 newborns. It is an autosomal recessive disease caused by mutations in the cystic fibrosis trans-membrane conductance regulator (CFTR) gene, which encodes a chloride channel expressed mainly in epithelial cells, but which is also involved in the bicarbonate–chloride exchange. The most common CF symptoms include progressive lung disease and chronic problems of the digestive apparatus (Riordan et al., 1989), whose degree of severity depends on other genetic and/or environmental factors. CF pathogenesis is characterised by the build-up of thick, sticky mucus in multiple mucin-producing organs, such as lungs, sinuses, intestine, pancreas and reproductive organs. For this reason, CF is also denominated mucoviscidosis, implying that mucins - polymeric, gel-forming O-linked glycoproteins responsible for the viscoelastic properties of the mucus - play a critical role in the disease (Kreda et al., 2012).
The aim of the present Ph. D. work was to investigate the structural features of two CFTR domains: the nucleotide binding domains (NBDs) - responsible for the gating mechanisms of the channel, and which have been proposed to serve as drug targets - and the regulatory domain (RD), directly involved in the activation of the channel. Knowledge of these aspects could likely improve understanding of the aberrant functionality of defective CFTR, and also help designing therapeutic strategies to either correct the defective protein in situ, and/or to potentiate its physiologic channel activity.
The present thesis refers essentially to the four published papers containing most of the results obtained during the 3 year-doctorate course. The first one reports on some biochemical and structural features of NBDs, which were investigated using biochemical assays and measures of small angle x ray scattering (SAXS), while the second paper dealt with the interaction of NBDs with a potentiator (2-pyrimidin-7,8-benzoflavone, PBF) of CFTR activity. Instead, the third and the fourth papers considered RD under non-phosphorylated and phosphorylated conditions, and the influence of phosphorylation on the conformation of the domain as followed by circular dichroism (CD) and SAXS experiments.
Briefly, the obtained results allowed us to draw the following principal conclusions.
NBDs When in an equimolar mixture and in the presence of ATP, NBDs form a dimer, whose conformation can be significantly changed by PBF. In addition, data could be exploited to reconstruct the ab-initio model of NBDs both as dimer (with or without PBF) and as isolated monomers.
RD In this case, obtained results on biochemical, structural and thermodynamic RD aspects allowed us to reconstructing a low-resolution, 3-D model of the native and phosphorylated protein, and to underline how phosphorylation induces the conformational change of the domain and the decreasing of RD stability.

Abstract (italiano)

La fibrosi cistica (FC) è la malattia genetica umana più comune, che si verifica prevalentemente nella popolazione caucasica con incidenza pari a 1 su 2500 neonati. È una malattia autosomica recessiva causata da mutazioni nel gene che codifica per il canale CFTR, canale del cloro espresso prevalentemente in cellule epiteliali, ma che è anche coinvolto nello scambio bicarbonato-cloruro. I sintomi FC più comuni includono la malattia polmonare progressiva e problemi cronici dell'apparato digerente (Riordan et al., 1989), il cui grado di gravità dipende da altri fattori genetici e / o ambientali. La patogenesi di FC è caratterizzata dalla formazione di muco denso e appiccicoso in diversi organi che producono mucina, come i polmoni, seni paranasali, intestino, pancreas e organi riproduttivi. Per questo motivo, FC anche denominata mucoviscidosi, poichè mucine - polimeriche, glicoproteine O-linked gelificanti responsabili delle proprietà visco-elastiche del muco, giocano un ruolo critico nella malattia (Kreda et al, 2012.).
Lo scopo del presente lavoro di Ph. D è stato quello di indagare le caratteristiche strutturali dei due domini di CFTR: i domini leganti nucletidi (NBDS) - responsabili dei meccanismi di gating del canale, e che sono stati individuati essere siti bersaglio di farmaci utilizzati nella terapia di FC - e il dominio regolatorio (RD), direttamente coinvolto nell'attivazione del canale. La conoscenza di questi aspetti potrebbe probabilmente migliorare la comprensione delle funzionalità di CFTR difettosa, e anche aiutare la progettazione di strategie terapeutiche per correggere la proteina difettosa in situ, e / o potenziando l'attività fisiologica del canale.
La presente tesi si riferisce essenzialmente ai quattro articoli pubblicati ed allegati che presentano la maggior parte dei risultati ottenuti durante il corso di 3 anni di dottorato. Il primo articolo tratta le caratteristiche biochimiche e strutturali degli NBD (1 e 2), che sono state studiate attraverso saggi biochimici e misure di Small Angle X-ray Scattering (SAXS), mentre il secondo articolo è relativo alla pubblicazione dei dati riguardanti lo studio dell’interazione tra gli NBD (1 e 2) e un potenziatore (2-pirimidin-7,8-Benzoflavone, PBF) dell’attività di CFTR. Invece, il terzo e il quarto articolo riportano i dati relativi allo studio di RD in condizioni non-fosforilate e fosforilate, e l'influenza della fosforilazione sulla conformazione del dominio, misurata tramite esperimenti di dicroismo circolare (CD) e esperimenti di SAXS.
In sintesi, i risultati ottenuti ci hanno permesso di trarre le seguenti conclusioni principali.
NBD1 e NBD2 se presenti in una miscela equi-molare formano un dimero che in presenza di ATP risulta avere una conformazione più compatta; tale conformazione può essere significativamente cambiato da PBF. Inoltre, i dati possono essere sfruttati per ricostruire il modello ab-initio di NBD1 e NBD2, sia come dimero (con o senza PBF) sia come monomeri isolati.
I risultati ottenuti sulle caratteristiche biochimiche, strutturali e termodinamiche di RD, ci ha permesso di ricostruire un modello 3-D a bassa risoluzione, della proteina nativa e fosforilata, e sottolineare come la fosforilazione induca un cambiamento conformazionale del dominio e la diminuzione di stabilità di RD.

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Tipo di EPrint:Tesi di dottorato
Relatore:Sorgato, Maria Catia
Correlatore:Moran, Oscar
Dottorato (corsi e scuole):Ciclo 26 > Scuole 26 > BIOSCIENZE E BIOTECNOLOGIE > BIOCHIMICA E BIOFISICA
Data di deposito della tesi:28 Luglio 2014
Anno di Pubblicazione:28 Luglio 2014
Informazioni aggiuntive:alla tesi sono allegati 4 articoli integranti il lavoro stesso
Parole chiave (italiano / inglese):fibrosi cistica, CFTR, mutazioni, purificazione di proteine, saxs
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/10 Biochimica
Struttura di riferimento:Dipartimenti > Dipartimento di Biologia
Codice ID:6999
Depositato il:29 Lug 2015 10:07
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