Canetti, Diana (2017) Investigation of molecular mechanisms involving protein trafficking. [Tesi di dottorato]


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Tipologia del documento: Tesi di dottorato
Lingua: English
Titolo: Investigation of molecular mechanisms involving protein trafficking
Data: 9 Aprile 2017
Numero di pagine: 164
Istituzione: Università degli Studi di Napoli Federico II
Dipartimento: Scienze Chimiche
Dottorato: Scienze chimiche
Ciclo di dottorato: 29
Coordinatore del Corso di dottorato:
Monti, Maria[non definito]
Data: 9 Aprile 2017
Numero di pagine: 164
Parole chiave: Protein trafficking; Proteomics; Mass spectrometry;
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Depositato il: 03 Mag 2017 17:21
Ultima modifica: 14 Mar 2018 09:56
DOI: 10.6093/UNINA/FEDOA/11677


The cellular protein traffic and the molecular mechanisms involved in the transport of each individual protein towards a specific destination are crucial events for cell physiology. The 2013 Nobel Prize in Physiology or Medicine honoured three scientists: Dr. Rothman, Dr. Schekman and Dr. Südhof, who have solved the mystery of how the cell organizes its complex transport system. They demonstrated that mechanisms employed by a cell to accomplish vesicle transport and fusion are independent by cell type, and are the same in all eukaryotic organisms. The description of the machinery regulating vesicle traffic, the major transport system in cells, represented a shift in our understanding of how the eukaryotic cell, characterised by a complex internal compartmentalization, organizes the routing of molecules and proteins to various intracellular destinations, as well as to the outside of the cell. The protein traffic plays a very critical role for a variety of physiological processes in which vesicle fusion and molecules delivery must be strictly controlled, such as during hormones and/ or cytokines release. In absence of this wonderful and precise organization, the cell would lapse into chaos. Defective vesicle transport leads to the development of a variety of neurological and immunological disorders, as well as in chronic pathologies, such as the diabetes. Many of these disorders occur in presence of mutation at level of different genes, that often give rise to proteins still catalytically active, but not able to reach the right cell compartment where operate, leading often to the pathological accumulation of metabolite over a toxic threshold. The comprehension at the molecular level how the protein trafficking process occurs in vivo and how it is impaired in pathological conditions might lead to the identification of new targets for therapeutic treatments. In this perspective, my PhD project was focused on the investigation of molecular mechanisms involved in traffic processes of several proteins associated to genetic diseases when they are mutated. These investigations have been carried out basically by using functional proteomic approaches. The role that a specific protein plays in intra- and extra-cellular processes is clarified by the identification of its molecular partners. Indeed, the association of an individual protein, whose its function is unknown, with protein complexes involved in well definite cellular processes would be strongly suggestive of its biological function. A classical functional proteomics approach consists of isolation of protein complexes involving the target protein (bait) from a cell lysate by immunoprecipitation. Proteins so purified were then fractionated by SDS-PAGE, digested in situ with trypsin and identified by nano-LC-MS/MS methodologies integrated with protein database search. In this PhD thesis, the above strategy was employed in the investigation of molecular mechanisms impaired in two rare genetic disorders: Wilson and Pompe disease. Wilson disease, described in Chapter 2, is a disorder characterised by defective copper excretion, due to mutations in copper transporter ATP7B. In normal conditions, ATP7B binds Cu in the trans-Golgi network and moves to the plasma membrane where delivery the Cu in bile channels. Although the most frequent ATP7B mutants, such as ATP7B(H1069Q), potentially are able to bind Cu, they cannot reach the plasma membrane where the excess of Cu has to be removed. In order to evaluate which molecular pathways result altered by expression of ATP7B mutants, a comparison between the interactomes of the wild type protein and the mutant ATP7B(H1069Q) was performed by functional proteomics approach. Pompe disease, discussed in Chapter 3, is a rare disorder of glycogen metabolism caused by mutations in gene encoding GAA, essential enzyme for the degradation of glycogen to glucose in lysosomes. Although the biochemistry GAA activity and genetic basis of the associated disorder are well characterized, the routes followed by wild type GAA to reach lysosomes and impaired in presence of mutants are still unclear. This work was focused on the investigation of the intracellular pathways controlling GAA traffic by the identification of protein partners associated to the enzyme on the route from ER to lysosomes. Similarly, the uptake pathway of recombinant GAA (rhGAA), used in Enzyme Replacement Therapy (ERT), was also studied in order to define the fate of recombinant protein once got into the cell. In Chapter 4, the investigation of molecular mechanisms impaired in amyloidosis has been described. Amyloidosis diseases are associated to the formation of protein aggregates at level of different organs, often associated to the presence of genetic variants of secreted proteins. The study concerning amyloidosis has been carried out by using a double approach: the conformational characterization of some amyloidogenic ApoAI variants was investigated in vitro by a strategy based on complementary proteolysis coupled with mass spectrometry (LC-MS). This methodology allowed clarifying the effect of single point mutations on the protein folding and stability thus to make hypothesis on the structural basis of amyloidosis as protein misfolding/mistrafficking diseases. Moreover a new proteomic strategy for typing amyloid deposits was developed during the period spent abroad at Wolfson Drug Discovery Unit, Centre for Amyloidosis and Acute Phase Proteins, Division of Medicine, University College London (Royal Free Campus), London, UK. Diagnosis and treatment of systemic amyloidosis depends on the correct identification of the amyloid protein triggering the formation of aggregates. In fact, a precise identification of the amyloid fibril protein is essential for the definition of an appropriate therapy. In this field, the proteomic analysis of amyloid deposits provides a chemical characterization of fibrillar constituents, which add important details to genetic sequencing and immunohistochemistry analysis. However, there are ambiguous cases, in which more than one potentially amyloidogenic protein is found within the patient’s biopsy and an accurate diagnosis becomes challenging. The work was aimed at the development of a methodology based on decellularization of human fat biopsies, tryptic digestion and mass spectrometry analysis (LC-MS/MS) in order to eliminate background contamination and improve the specificity of amyloid typing. The use of deoxycholate detergent and the shaking of fat biopsy in tissue lyser resulted fundamental in decellularization procedure. The innovative strategy of decellularization proved to be a simple way to improve the accuracy and specificity of proteomic identification of amyloid fibril type removing most of cellular and plasma proteins background from tissue without altering properties of amyloid fibrils.

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