Cimmaruta, Chiara (2018) ANALYSIS OF A RARE GENETIC DISEASE DUE TO PROTEIN INSTABILITY: STUDY AND CHARACTERIZATION OF CONGENITAL DISORDER OF GLYCOSYLATION TYPE IA. [Tesi di dottorato]
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| Item Type: | Tesi di dottorato |
|---|---|
| Resource language: | Italiano |
| Title: | ANALYSIS OF A RARE GENETIC DISEASE DUE TO PROTEIN INSTABILITY: STUDY AND CHARACTERIZATION OF CONGENITAL DISORDER OF GLYCOSYLATION TYPE IA |
| Creators: | Creators Email Cimmaruta, Chiara chiara.cimmaruta@unina.it |
| Date: | December 2018 |
| Number of Pages: | 66 |
| Institution: | Università degli Studi di Napoli Federico II |
| Department: | Biologia |
| Dottorato: | Biologia |
| Ciclo di dottorato: | 31 |
| Coordinatore del Corso di dottorato: | nome email Cozzolino, Salvatore salvatore.cozzolino@unina.it |
| Tutor: | nome email Cubellis, Maria Vittoria UNSPECIFIED |
| Date: | December 2018 |
| Number of Pages: | 66 |
| Keywords: | CDG-IA; fosfomannomutasi2 |
| Settori scientifico-disciplinari del MIUR: | Area 05 - Scienze biologiche > BIO/10 - Biochimica |
| Date Deposited: | 03 Jan 2019 14:20 |
| Last Modified: | 30 Jun 2020 08:51 |
| URI: | http://www.fedoa.unina.it/id/eprint/12499 |
Collection description
Congenital disorder of glycosylation type Ia (CDG-Ia ) is an autosomal recessive disease caused by mutations in PMM2 gene encoding phosphomannomutase 2. In humans two PMM enzymes exist, PMM1 and PMM2; both of them catalyze the interconversion of mannose-6-phosphate into mannose-1-phosphate but only PMM1 can also hydrolyze bis-phosphate hexoses after stimulation with inosine monophosphate (IMP). Although no mutation in PMM1 gene has been associated with the disease, the role of the paralogous enzyme in CDG-Ia should be clarified. PMM1 and PMM2 were analyzed by conventional enzymatic assays as well as by novel techniques such as 31P-NMR and thermal shift assay. In order to evaluate the role of IMP in regulating mannose-1-phosphate production and ultimately protein glycosylation, it was performed the characterization of a triple mutant of PMM1 that retains mutase and phosphatase activity, but not sensitive to IMP. For the purpose of developing a therapeutic approach for CDG-Ia, for which there is no cure at the moment, it was generated a new cell model in order to identify biomarkers for cell-based screening, such as glycosylated proteins. A cancer cell line HepG2 was edited by Crispr/Cas9 system to introduce the hypomorphic PMM2 mutation, F119L, that has been observed in CDG-Ia patients in homozygosity. Hypo-glycosylated forms of α-1-anti-trypsin (AAT) was detected by Western blot in HepG2-F119L similarly to the serum from CDG-Ia patients. Furthermore, a difference in the amount of a secreted protein, alpha-fetoprotein (AFP), which is N-glycosylated, was found in HepG2-F119L secretome compared to the control cells. These results suggest that HepG2-F119L represent a good cell model for CDG-Ia and a useful tool for drug screening since they are defective in N-glycosylation of serum proteins that are found altered in patients. Further studies will be performed to propose AFP as a suitable biomarker.
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