Altieri, Filomena (2014) Does gastrokine 1 play a major role in gastric tissue? [Tesi di dottorato]

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Gastrokine1 (GKN1) is a stomach-specific protein important for maintaining the physiological function of the gastric mucosa. GKN1 is downregulated in gastric tumor tissues and derived cell lines so it has recently emerged as a potential biomarker for gastric cancer. The protein is characterized by the presence of a BRICHOS domain consisting of about 100 amino acids found in several unrelated proteins associated with major human diseases like BRI2, related to familial British and Danish dementia; Chondromodulin-I (ChM-I), linked to chondrosarcoma; Lung Surfactant Protein C (SP-C), associated with respiratory distress syndrome. Literature data show that recombinant BRICHOS domains from BRI2 and SP-C precursor (proSP-C) prevent fibrils formation of amyloid-beta peptide (Aβ) that is the major component of extracellular amyloid deposits in Alzheimer's disease. Aβ derives from the partial hydrolysis of the amyloid precursor protein (APP) catalyzed by β-and γ-secretase. The hydrolysis produces amyloid peptides of 40 (about 90%) or 42 (about 10%) amino acid residues. Main target of this thesis is to investigate the interaction between recombinant human GKN1 (rhGKN1) and Aβ(1-40) to explore whether also GKN1 was endowed by a chaperone activity toward amyloidogenic peptides. To this aim, Aβ was incubated in presence or absence of rhGKN1 and chicken cystatin, as negative control, at 10:1 molar ratios. Samples were then analyzed by SDS-PAGE. The aggregation of Aβ was also evaluated using Thioflavine T binding assay. Mass spectrometry and size-exclusion chromatography analysis were performed to characterize the interaction. The data obtained showed that rhGKN1 prevented amyloid aggregation and fibrils formation by inhibiting Aβ(1-40) polymerization. Surface plasmon resonance analysis of rhGKN1/Aβ interaction led to calculate a dissociation constant (KD) of 34 μM. These preliminary data strongly indicated that rhGKN1 possess anti-amyloid activity thus, it might play a role of molecular chaperone directed against unfolded segments with an ability to recognize amyloidogenic polypeptides and prevent their aggregation. Regarding the mechanism by which GKN1 gene is inactivated in gastric cancer, this remains still unknown; so the second part of my work is focused to clarify the causes of GKN1 gene silencing and to determine if epigenetic mechanisms, such as histonic modifications, could also contribute to its down-regulation. To this aim, chromatin immunoprecipitation assays (ChIP) for H3K9triMe and its specific HMT SUV39H1 were performed on different samples from biopsies of normal and tumor human gastric tissue. The results showed that GKN1 down-regulation in gastric cancer tissues is associated with high levels of H3K9triMe and with the recruitment of SUV39H1 on GKN1 promoter, suggesting the presence of an epigenetic transcriptional complex that negatively regulates GKN1 expression in gastric tumor.

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