Del Giudice, Rita (2013) Cellular models to study ApoA-I related amyloidosis: mechanism of action and search for specific targets. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Lingua: English
Title: Cellular models to study ApoA-I related amyloidosis: mechanism of action and search for specific targets
Creators:
CreatorsEmail
Del Giudice, Ritarita.delgiudice@unina.it
Date: 29 March 2013
Number of Pages: 108
Institution: Università degli Studi di Napoli Federico II
Department: Scienze Chimiche
Scuola di dottorato: Biotecnologie
Dottorato: Scienze biotecnologiche
Ciclo di dottorato: 25
Coordinatore del Corso di dottorato:
nomeemail
Sannia, Giovannigiovanni.sannia@unina.it
Tutor:
nomeemail
Piccoli, Renatapiccoli@unina.it
Date: 29 March 2013
Number of Pages: 108
Uncontrolled Keywords: protein aggregation Apolipoprotein A-I Angiogenin
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Aree tematiche (7° programma Quadro): SALUTE e TUTELA DEL CONSUMATORE > Biotecnologie, strumenti e tecnologie generiche per la salute umana
BIOTECNOLOGIE, PRODOTTI ALIMENTARI E AGRICOLTURA > Scienze della vita, biotecnologia e biochimica per prodotti e processi non-alimentari sostenibili
Date Deposited: 03 Apr 2013 14:33
Last Modified: 04 Jul 2014 11:31
URI: http://www.fedoa.unina.it/id/eprint/9218

Abstract

An increasing number of human diseases is linked to protein misfolding and aggregation in amyloid fibrils. In spite of the extensive research during the last decade, much remains to be learnt on the basis of the molecular mechanism responsible for cell damage. Among the amyloidogenic proteins, 19 mutated versions of apolipoprotein A-I (ApoA-I) have been associated to amyloid diseases. Fibrils are mainly constituted by N-terminal fragments of ApoA-I, about 90-100 residue long. The 93-residue polypeptide, [1-93]ApoA-I, is the main constituent of extracellular cardiac amyloid fibrils. My research activity was aimed at inspecting the molecular mechanisms of the pathology. Therefore, we analyzed binding and intracellular pathway of recombinant [1-93]ApoA-I in cardiac target cells, we identified potential partners and studied the effects of endogenous amyloidogenic variants of ApoA-I on cell physiology. We demonstrated that the fibrillogenic polypeptide recognizes specific binding sites on target cell membranes and partially co-localizes with ABCA1 transporter. Following binding, the polypeptide is internalized mostly by chlatrin-mediated endocytosis and by lipid rafts, whereas a macropinocytosis involvement is excluded. Upon internalization, no retro-endocytosis is observed, while the polypeptide is massively degraded by proteasomal and lysosomal machineries. The rapid degradation of the polypeptide, together with the finding that fibrils obtained in vitro have no access to the intracellular compartment, are consistent with the absence of cytotoxic effects on cardiac cells. The identification of the molecular partners of a pathogenic protein is a central issue in the comprehension of the molecular bases of the disease. GST pull-down experiments, and protein identification by mass spectrometry, were performed on cardiomyoblasts membrane extracts. This experimental approach provided a list of about 100 potential interactors of the fibrillogenic polypeptide and, among these, the β-chain of ectopic ATP synthase and nicastrin were selected to be analyzed in detail. By co-immunoprecipitation and fluorescent microscopy experiments both proteins were found to interact with the fibrillogenic polypeptide. Interestingly, the former protein is an ApoA-I receptor, the latter has a role in the production of amyloid β peptide, responsible for Alzheimer’s disease. Since patients are heterozygous for the mutated ApoA-I gene, the isolation of amyloidogenic variants from sera is impracticable. Thus, we set up a suitable cellular model, consisting in stably transfected CHO-K1 cells, to express ApoA-I amyloidogenic variant L174S. The recombinant protein, efficiently secreted in the culture medium, was isolated following a one-step purification procedure and found to be associated to fatty acids, for which a role in trafficking and secretion may be hypothesized. Finally, we analyzed the effects of an amyloidogenic ApoA-I variant on cell physiology. We obtained hepatic cells stably over-expressing amyloidogenic ApoA-I variant L75P and found that the protein is mostly retained within the cells, rather than secreted, probably because of its partial unfolding. Moreover, reduced cell viability and decreased angiogenin (ANG) levels were detected. When cells were exposed to stress conditions (e.g. serum starvation), cell viability was more severely affected than cells expressing the wild-type protein. Furthermore, ANG levels and its subcellular localization were altered. ANG is known to play a role in cell recovery from stress and, more recently, it has been associated to neurodegenerative diseases, such as Parkinson’s, Alzheimer’s and Amyotrophic Lateral Sclerosis. Our data suggest a possible role of ANG in ApoA-I related amyloidosis. According to these observations, the addition of exogenous ANG to our cell model was able to restore cell viability. The analysis of a set of ANG loss-of-function mutants allowed us to demonstrate that ANG needs to be internalized into the cells to elicit its protective action against stress.

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