In vitro Şartlarda Mutant AVPR2 Proteinlerinin Yarı Ömür ve Glikozilasyon Özelliklerinin Araştırılması
Özet
Diabetes insipidus (DI) is a disease characterized by excessive urine formation (polyuria) and excessive thirst (polydipsia). DI is classified into gestational DI, primary polydipsia, Nephrogenic DI (NDI), and Central DI (CDI), according to their causes. Hereditary NDI is caused by mutations in the AVPR2 gene on the X chromosome (Xq28) with a rate of 90%. A mutation in the 371 amino acid-encoded DNA of the AVPR2 protein causes the emergence of this disease, which can be observed in different symptoms. Because of these mutations, the AVPR2 protein is misfolded and directed to degradation. This changes the half-life of the protein.
The half-life of the intracellular proteome varies from a few minutes to several months. The reason for this change is that the protein degradation rate varies depending on the cellular response given in cell cycles and various environmental/physiological conditions. The increase or decrease in the rate of degradation caused by the mutation is related to the glycosylation modifications the protein undergoes.
The purpose of this thesis is to reveal the half-life times and glycosylation modifications of the G12E, H80Y, V88M, V88L, R106C, V215M, and L219P mutations occurring in the AVPR2 gene which were previously defined by our group. In the experimental studies carried out in this context, expression vectors containing the related mutant AVPR2 gene sequences were prepared by using site-directed mutagenesis method with the expression vector carrying the wild-type coding AVPR2 sequence. Deglycosylation experiments were carried out to investigate the characterization of the intracellular maturation process of mutant proteins expressed in COS-7 cells. In addition, in order to determine the half-lives of proteins, cycloheximide analysis was performed and the results of both deglycosylation experiments and cycloheximide experiments were evaluated by immunoblot analysis. Thus, the relationship between the degradation state of mutant proteins and the glycosylation modifications they undergo was revealed.
As a result, it is seen that each mutant is trapped in the ER at different levels. It is thought that the half-life times are not directly related to the functionality of the protein and the signal response it generates, and have meaning when evaluated together with glycosylation modifications. Comparisons between mutants and wild type for half-life values were statistically significant for each mutant. According to the results obtained, L219P mutant protein has the lowest half-life value and when evaluated with the deglycosylation results, it was seen that most of it were trapped in the ER. It has been observed that the half-life value of the R106C mutant increases and it has been suggested that this may be related to the inhibition of degradation mechanisms. Although the H80Y mutant protein has the longest half-life, when evaluated by deglycosylation analysis, it was observed that most of it remained stuck in the ER. The fact that the half-life length is close to the wild type has been associated with the resistance of the mutant protein to exit from the ER and degradation. These mutations, which occur at different points of the AVPR2 protein and with different amino acid changes, have different mechanisms of action, and this may help explain the symptoms of hereditary NDI that vary between patients.
