Pro-Vazopressin Mutantlarında Amiloid Benzeri Agregat Oluşumlarının ve Otozomal Dominant Nörohipofizal Diabetes İnsipidus ile Bağlantısının Araştırılması

Abstract

Protein misfolding or unfolding leads to the formation of aggregate structures as a result of their accumulation within or outside the cell, which is known to cause many diseases. Pathogenic protein aggregates accumulate not only in the extracellular space but also in cellular compartments such as the cytoplasm and nucleus, enabling the classification of associated diseases under the amyloidosis disease group. Autosomal dominant neurohypophyseal diabetes insipidus (ADNDI) is a rare disease caused by mutations in the arginine vasopressin-neurophysin II (AVP-NPII) gene. These mutations lead to the accumulation of precursor proteins in the endoplasmic reticulum (ER) lumen and the formation of disulfide-linked oligomers. This accumulation results in progressive degeneration of vasopressinergic neurons, allowing ADNDI to be classified as a neurodegenerative disease associated with the formation of fibrillar protein aggregates. While ADNDI forms fibrillar amyloid-like aggregates similar to amyloidosis, it differs from amyloid diseases in that these aggregates accumulate in the ER lumen rather than in the cytosol or extracellular space. In this thesis, we investigated the misfolding of mutant precursor proteins resulting from the G45C, 207_209delGGC, G88V, C98X, C104F, E108D-1, E108D-2, and R122H mutations identified by our group in ADNDI patients, as well as the resulting amyloid-like aggregate structures of disulfide-bonded oligomers. The maturation processes of mutant proteins, ER-associated degradation pathways, differences in the cytosolic fraction, and their potential retrotranslocation into the cytosol were evaluated. Additionally, differences in the formation of disulfide complexes were identified, intracellular localization was determined using immunofluorescence techniques, and physiological states were analyzed through electron microscopy. Furthermore, mutant proteins were expressed in E. coli to analyze their fibril-forming potential. Experimental results revealed that mutant precursor proteins formed disulfide-linked homo-oligomer structures. Immunofluorescence and electron microscopy analyses showed that aggregate structures accumulated within the ER. Moreover, bacterial expression studies demonstrated that purified mutant precursor proteins spontaneously formed fibrillar structures. These findings support the classification of ADNDI as a neurodegenerative disease associated with fibrillar protein aggregation.