Ciano, Michela (2017) Tackling the Most Frequent Wilson Disease-Causing ATP7B Mutation: Mechanism of Rescue by αB-Crystallin and its Derived Peptide. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Lingua: English
Title: Tackling the Most Frequent Wilson Disease-Causing ATP7B Mutation: Mechanism of Rescue by αB-Crystallin and its Derived Peptide
Creators:
CreatorsEmail
Ciano, Michelamichelaciano@libero.it
Date: 11 December 2017
Number of Pages: 82
Institution: Università degli Studi di Napoli Federico II
Department: dep14
Dottorato: phd054
Ciclo di dottorato: 30
Coordinatore del Corso di dottorato:
nomeemail
Avvedimento, Vittorio Enricovittorioenrico.avvedimento@unina.it
Tutor:
nomeemail
Bonatti, StefanoUNSPECIFIED
Date: 11 December 2017
Number of Pages: 82
Uncontrolled Keywords: Wilson disease; ATP7B; alphaB-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 23:42
Last Modified: 19 Mar 2019 11:51
URI: http://www.fedoa.unina.it/id/eprint/12221

Abstract

Mutations in copper (Cu) transporter ATP7B are the primary cause of Wilson Disease (WD). WD is an autosomal recessive disorder characterized by hepatic cirrhosis and neuronal degeneration due to a severe impairment in biliary Cu excretion. The defective gene in WD encodes ATP7B, a Cu-trasporting protein primarily expressed in the liver. Generally, ATP7B senses and responds to intracellular Cu levels via trafficking from the trans-Golgi network (TGN) toward post late-endosome (LE)/lysosomal compartments and plasma membrane (PM) to eliminate excessive Cu from the cell via bile canaliculi. The most frequent ATP7B disease-associated mutation (H1069Q) results in aberrant protein products that are mis-targeted from the Golgi complex to the endoplasmic reticulum (ER). However, ATP7B-H1069Q mantains a residual Cu-translocating activity believed to be sufficient to rescue Cu homeostasis when the protein is targeted to TGN (Kaler SG. et al., 2011). My research project expands previous studies conducted in my laboratory showing that the cytosolic holdase alphaB-Crystallin (CryAB or Hsp-B5) rescues the localization and trafficking response of ATP7B-H1069Q to Cu overload (D’Agostino M. et al., 2013). In brief, the project was divided into two lines of research performed in collaboration with another Ph.D student Simona Allocca. In the first line of research, we studied the effects of phosphorylation on CryAB full length structure and function and how phosphorylation can influence CryAB chaperone activity on ATP7B mutant. Our results show that mimicking phosphorylation of serine 19 and 45, either singularly or in combination reduces CryAB chaperone function. By contrast, phosphorylation of serine 59 has no such effect and counteracts the inhibition caused by single phosphorylation at S19 and S45. In addition, we found that all phosphomimetic substitutions lead to the formation of small oligomeric complexes containing CryAB, thereby suggesting that the reduction of the oligomerization state of CryAB does not correlate with the inhibition of its chaperone activity (Ciano M. et al., 2016). In the second line of research, we verified the rescue ability of CryAB domains on ATP7B-H1069Q. To this aim, we dissected CryAB in its main domains: N-terminal, alpha-crystallin, and C-terminal. Subsequently, we assayed them through immunofluorescence analysis in cells co-transfected with ATP7B-H1069Q. We found that the alpha-crystallin domain is able to rescue the Golgi localization of the ATP7B-H1069Q mutant. Then, since several studies have shown that small CryAB derived peptides are able to mimic the effect of the full length protein, (Sreekumar P.G. et al., 2013; Nahomi R.B. et al., 2015), we focused on peptide 73-92, derived from the crystallin-domain of CryAB (Peptide1 or Pept1) in our WD cellular model. Our results revealed that Peptide1 retains the ability to rescue the Golgi localization of ATP7B-H1069Q, thereby fully mimicking the effects of the isolated α-crystallin domain and full-length CryAB. Furthermore, we observed that Peptide1 and Control Peptide (Ctrl Pept) are taken up by COS7 and do not affect cell viability. Interestingly, we also demonstrated that Pept1, but not Ctrl Pept, is able not only to interact with ATP7B WT and H1069Q, but also to correct the localization of ATP7B-H1069Q from the ER to the TGN and to the post-TGN trafficking in response to toxic Cu. Moreover, our experiments revealed that Pept1 is also able to stabilize ATP7B-H1069Q, thereby increasing its half-life. Finally, in collaboration with Dr. Silvia Parisi, Anna Musto, Simona Allocca, and Prof. Roman Polishchuk, we generated an isogenic cell model from iPSCs obtained from skin fibroblasts of a WD patient carrying the H1069Q mutation (H1069Q/H1069Q) and a familiar control (WT/H1069Q), and differentiated them into hepatocyte-like cells HLCs. Therefore, our future objective is to exploit these cells to study the expression, localization, mobilization, and function of the mutant ATP7B-H1069Q, and to test the rescue efficiency of peptide CryAB 73-92 (Peptide1).

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