Iacobucci, Ilaria (2021) In vitro and ex vivo investigation of functional protein complexes for the elucidation of biological processes. [Tesi di dottorato]


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Item Type: Tesi di dottorato
Resource language: English
Title: In vitro and ex vivo investigation of functional protein complexes for the elucidation of biological processes
Iacobucci, Ilariailaria.iacobucci@unina.it
Date: 15 April 2021
Number of Pages: 255
Institution: Università degli Studi di Napoli Federico II
Department: Scienze Chimiche
Dottorato: Scienze chimiche
Ciclo di dottorato: 33
Coordinatore del Corso di dottorato:
Lombardi, Angelinaalombard@unina.it
Date: 15 April 2021
Number of Pages: 255
Keywords: Protein complexes; Proteomics, Biomolecular mass spectrometry.
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Date Deposited: 26 Apr 2021 16:30
Last Modified: 07 Jun 2023 10:48
URI: http://www.fedoa.unina.it/id/eprint/13855

Collection description

Functional Proteomics aims to the identification of in vivo protein-protein interaction (PPI) in order to piece together protein complexes, and therefore, cell pathways involved in biological processes of interest. Over the years, proteomic approaches used for protein-protein interaction investigation have relied on classical biochemical protocols adapted to a global overview of protein-protein interactions within so-called "interactomics" investigation. In particular, their coupling with advanced mass spectrometry instruments and innovative analytical methods led to make great strides in the PPIs investigation in proteomics. The structural investigation of proteins and protein complexes, as well as the identification of protein-ligand interacting regions, is also a powerful tool to understand the biological roles of protein-protein complexes and develop new and more efficient drugs in the therapy treatment of diseases. In these perspectives, the present PhD project has been addressed to the investigation of molecular mechanisms at the basis of human diseases, host-virus interaction by functional and structural proteomics approaches. Furthermore, the binding mechanisms of metal-based complexes with protein/peptides have been investigated. In chapter two, ex vivo proteomic strategies have been discussed to investigate the molecular mechanisms of Fabry disease, Huntington's disease, and in vitro mass spectrometry-based techniques have been employed to investigate the interaction between Osteopontin (OPN) and ICOS ligand (ICOSL) responsible for tumor metastatization. The role that a specific protein plays in cellular processes is clarified by the identification of its molecular partners. Indeed, the association of an individual protein, whose 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 isolating protein complexes involving the target protein (bait) from a cell lysate by immunoprecipitation. Proteins so purified are fractionated by SDS-PAGE, in situ digested with trypsin, and identified by nanoLC-MS/MS methodologies integrated with a protein database search. This strategy has been applied to investigate the functional role of α-galactosidase in Fabry disease and ADAM10 in Huntington's disease. Fabry disease is a genetic disorder caused by a mutation in the GLA gene encoding for α-galactosidase (α-GAL) enzyme. α-GAL is a lysosomal hydrolase that degrades some substrates such as Globotriaosylceramide (Gb3). The enzyme deficiency causes an accumulation of Gb3, triggering organ dysfunctions. The investigation of the intracellular pathways involved in the route from the endoplasmic reticulum (ER) to lysosome of the wild-type enzyme and the two recombinant enzymes used in the enzyme replacement therapy (ERT) may elucidate the internalization process of the drugs compared to the physiological traffic of α-GAL. The proteomic investigation of the internalization process of both enzymes revealed that it occurs through endocytosis mediated by caveolae and clathrin vesicles. Huntington's disease (HD) is a neurodegenerative disorder caused by Huntingtin (HTT) gene mutation. Disintegrin and metalloproteinase domain-containing protein 10 was found to accumulate in HD patient brains. The dysregulation of ADAM10 protein levels has been associated with reduced neurotransmission and cognitive decline in HD mice models. The role of ADAM10 in wild-type and HD mice models has been studied with the functional proteomics approach described above to clarify its role in HD dysregulated processes. Proteomics results revealed, for the first time, that ADAM10 is involved in presynaptic functions, specifically in the regulation of synaptic vesicles (SVs) dynamics at the Active Zone. In HD density of SVs is reduced for dysregulation of the ADAM10/Piccolo complex. Previous reports demonstrated the migration of cells high expressing ICOSL in the presence of OPN. In this study, we demonstrated ICOSL as a novel receptor for OPN. The interaction was structurally investigated with cross-link and limited proteolysis approaches coupled to mass spectrometry to understand the functional role. These strategies allowed the definition of a binding site upstream of the RGD motif and the thrombin cleavage site. Furthermore, cross-linking experiments indicate that the region downstream the RGD domain (including K170, K172, and K173) becomes exposed upon ICOSL binding, which in turn indicates that OPN changes its conformation in the complex with ICOSL. In chapter three, the host-virus interaction processes have been investigated for the SARS-CoV-2 and the Sulfolobus spindle shape 1 (SSV1) viruses. The molecular mechanisms of SARS-CoV-2 viral infection have been focused on two fields of application: 1) the interaction between Angiotensin-Converting Enzyme 2 (ACE2), the SARS-CoV-2 main target, and potential virus inhibitors, 2) the study of additional SARS-CoV-2 targets on colon and renal cell surfaces. SARS-CoV-2 is a novel coronavirus discovered because of several pneumonia cases in the Hubei region in China at the end of 2019, but it has rapidly spread worldwide, causing a global pandemic. Besides vaccines, the development of novel and efficient therapies is an urgent issue to be addressed. Long-chain inorganic polyphosphates (PolyPs) demonstrated to have antiviral activities against HIV-1 infection. To assess the potential antiviral molecular mechanism of PolyPs, we studied their interaction with ACE2 receptor by Size Exclusion Chromatography (SEC) and limited proteolysis-mass spectrometry approaches. SEC data suggested that PolyP120 and Spike S1 have different binding sites on ACE2 and limited proteolysis experiments confirmed the docking calculation for the prediction of a binding pocket. The investigation of additional SARS-CoV-2 targets on human cells provided the employment of a pull-down experiment using the S1 subunit of the SARS-CoV-2 S protein to purify its putative binding proteins on the human cell membrane. NanoLC-MS/MS technique was used for protein identification according to a "shotgun" proteomic approach. Sulfolobus spindle shape 1 (SSV1) virus is an archaeal virus well known as extremophile. SSV1 infects Sulfolobus solfataricus bacteria. The viral transcription factor F55 has been found to regulate the viral life cycle through the crosstalk between the host and the virus, but the molecular mechanism at the basis of this process is still unknown. Therefore, an electromobility shift assay was employed to isolate the DNA-bound F55 host protein partners, then identified by a bottom-up proteomic technique. Functional experiments allowed us to propose a model explaining the effect of the F55 interaction with the identified host interactor RadA on the T6 promoter. In chapter four, the binding features of platinum complexes and potential anti-aggregation metallodrugs with β-lactoglobulin protein and the beta-amyloid peptide (Aβ), respectively, have been probed by the native-MS technique. The latter is based on a particular approach in which biological analytes are ionized and gas-phase transferred through electrospray ionization (ESI) in a non-denaturing solvent and setting the source parameters as much soft as possible to achieve a good compromise between ionization and complex stability. Native ESI-MS can provide powerful information on protein and protein-ligand complexes binding and stoichiometry. β-Lactoglobulin is a major globular milk whey carrier with potential applications as an oral drug delivery system thanks to its biochemical and biophysical features. Cisplatin and oxaliplatin anticancer agents have been tested to define their binding aspects with β-Lactoglobulin by the native ESI-MS technique. Metallocomplexes have been proposed as potential drugs in amyloidogenic neurodegenerative diseases such as Alzheimer's disease (AD). Indeed, they have some anti-aggregation properties towards fibrils generated by the amyloidogenic peptide Aβ. The amyloid inhibitory activity of the metal-based drugs can be exploited through different mechanisms: 1) coordination chemistry, 2) oxidative, 3) proteolytic reactions for peptide modifications. A native ESI-MS approach aimed to investigate the mechanism of action of several Pt-, Pd-, and Au-based complexes in the aggregation modulation of the C-term of the Aβ peptide spanning from residues 21 to 40 (Aβ21-40).


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