Allocca, Simona (2017) New Therapeutic Perspectives for the Most Frequent ATP7B Mutation in Wilson Disease: Development of Pharmacologically Active Peptides and Generation of a Novel WD Isogenic Cell Model. [Tesi di dottorato]


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Item Type: Tesi di dottorato
Lingua: English
Title: New Therapeutic Perspectives for the Most Frequent ATP7B Mutation in Wilson Disease: Development of Pharmacologically Active Peptides and Generation of a Novel WD Isogenic Cell Model
Date: 11 December 2017
Number of Pages: 76
Institution: Università degli Studi di Napoli Federico II
Department: dep14
Dottorato: phd054
Ciclo di dottorato: 30
Coordinatore del Corso di dottorato:
Avvedimento, Vittorio
Bonatti, StefanoUNSPECIFIED
Date: 11 December 2017
Number of Pages: 76
Uncontrolled Keywords: Wilson disease; ATP7B; αB-crystallin
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/11 - Biologia molecolare
Area 05 - Scienze biologiche > BIO/12 - Biochimica clinica e biologia molecolare clinica
Area 05 - Scienze biologiche > BIO/13 - Biologia applicata
Date Deposited: 27 Dec 2017 23:41
Last Modified: 19 Mar 2019 11:47


Background Wilson Disease (WD) is an autosomal recessive inherited disorder, which if left untreated can be lethal. Its development is due to abnormal copper (Cu) metabolism. In particular, reduced copper excretion causes an excessive deposition of the ion in many organs such as the liver, central nervous system, cornea, kidney, and cardiac muscle, thereby damaging the physiological functions of the affected organs. Mutations in the P-type adenosine triphosphatase (ATP7B), the gene encoding the copper transporting P-type ATPase, are responsible for hepatic copper accumulation. Cu overload in the liver produces toxic effects via modulating several molecular pathways. The recessive ATP7B-H1069Q mutation alone is the most frequent cause of WD among Caucasians (~60%). Physiologically, ATP7B transfers copper across the membrane into the lumen of the trans-Golgi network (TGN). In the Golgi, the metal is incorporated into the serum protein ceruloplasmin (CP). In the presence of excessive cytosolic Cu, the metal is localized by ATP7B to post-Golgi vesicles and plasma membrane (PM), where it is removed via bile canaliculi. Overexpression studies in heterologous cell systems show that H1069Q mutation results in aberrant protein products that are strongly mis-targeted from the TGN towards the endoplasmic reticulum (ER). Despite that, this mutant still exhibits residual Cu translocating activity, which might be sufficient enough to ward off the disease if the protein is correctly localized to the TGN. In this context, our previous work has demonstrated that the cytosolic holdase αB-crystallin (CRYAB or Hsp-B5) rescues the proper folding, localization and response to Cu overload of ATP7B-H1069Q in Hep-G2 and COS-7 cells. Rationale Evidence that residual Cu transport activity is retained by ATP7B-H1069Q and that correcting agents can indeed localize this mutant to the Golgi complex has spurred our great interest in investigating the potentially therapeutic implications of these findings. My PhD thesis describes the properties of a CRYAB-derived mini-chaperone in heterologous or hepatoma-derived cell lines overexpressing ATP7B-H1069Q and the generation of an isogenic cell model based on induced pluripotent stem cells (iPSCs) created from skin fibroblasts of a homozygous WD patient and his mother, used as control, followed by differentiation into hepatocyte-like cells (HLCs). Main results We found that the CRYAB peptide enters the cytosol and only a small fraction is associated with lysosome structures. The peptide interacts with ATP7B-H1069Q, rescuing its Golgi localization and trafficking response to increased Cu level. Other important results, obtained by virtue of our new WD cell model are the following: 1) the expression level of the mutant protein in HLCs was only 30% compared to the control, although ATP7B mRNA levels were similar; 2) a significant amount of ATP7B-H1069Q (about 35%) reached the Golgi complex and was able to redistribute to the endo/lysosomal compartment in the presence of Cu excess; 3) most of the newly synthesized mutant underwent rapid ER-associated degradation. Conclusion Biochemical optimization of the CRYAB peptide could provide helpful insights into its mechanism of action and its potential use for WD treatment. Meanwhile, the new isogenic WD cell model is a powerful system to validate in vitro results, obtained in heterologous or hepatoma-derived cell lines. In this context, ATP7B-H1069Q dysfunction could be tackled by treatments that reduce ER-associated degradation, thereby increasing the number of molecules reaching the TGN.

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