Investigation of Fibrillar Aggregates Formed by Pathogenic Pre-Pro-Vasopressin Mutants that Cause ADNDI

Refika Dilara Vaizoglu; Beril Erdem; Mehmet Gul; Ceren Acar; Huseyin Ozgur Ozdemirel; Emel Saglar Ozer; Hatice Mergen

doi:10.1210/clinem/dgae749

Investigation of Fibrillar Aggregates Formed by Pathogenic Pre-Pro-Vasopressin Mutants that Cause ADNDI

J Clin Endocrinol Metab. 2024 Oct 25:dgae749. doi: 10.1210/clinem/dgae749. Online ahead of print.

Authors

Refika Dilara Vaizoglu¹, Beril Erdem¹, Mehmet Gul², Ceren Acar³, Huseyin Ozgur Ozdemirel¹, Emel Saglar Ozer¹, Hatice Mergen¹

Affiliations

¹ Hacettepe University, Faculty of Science, Department of Biology, Molecular Biology Section, Ankara, Turkey.
² Inonu University, Faculty of Medicine, Basic Medical Sciences, Histology and Embryology Section, Malatya, Turkey.
³ Inonu University, Faculty of Arts and Science, Department of Molecular Biology and Genetics, Malatya, Turkey.

PMID: 39449655
DOI: 10.1210/clinem/dgae749

Abstract

Context: Aggregations of unfolded or misfolded proteins, both inside and outside cells, are implicated in numerous diseases, collectively known as amyloidosis. Particularly, Autosomal dominant neurohypophyseal diabetes insipidus (ADNDI) is a rare disease caused by mutations in the AVP-NPII gene, leading to the inability to secrete arginine vasopressin (AVP). These misfolded proteins accumulate within the endoplasmic reticulum (ER), causing cellular dysfunction.

Objective: This study aimed to investigate the formation of amyloid-like aggregates within the cell resulting from misfolded mutant precursor proteins, which induce disulfide-linked oligomers due to the G45C, 207_209delGGC, G88V, C98X, C104F, E108D-1, E108D-2 and R122H mutations identified by our group in the AVP-NPII gene of ADNDI patients.

Methods: De-glycosylation studies were performed to analyze the glycosylation patterns of mutant protein precursors. The involvement of these precursors in the ER-related degradation pathway was studied by conducting protease inhibition experiments. Disulfide-linked oligomer analysis determined the oligomerization status of the mutant precursors. Immunofluorescence and electron microscopy studies provided evidence of aggregate structures in the ER lumen. In vitro studies involving bacterial expression and fibril formation in E. coli.

Results: Our findings demonstrated that the N-glycan structure of mutant precursors remains intact within the ER. Protease inhibition experiments indicated the involvement of these precursors in the ER-related degradation pathway. Disulfide-linked oligomer analysis revealed homo-oligomer structures in mutations. Immunofluorescence and electron microscopy studies confirmed the presence of aggregate structures in the ER lumen. In vitro studies showed that mutant precursors could form fibril structures in E. coli.

Conclusion: Our study may support the idea that ADNDI belongs to the group of neurodegenerative diseases due to the formation of fibrillar amyloid aggregates in the cell.

Keywords: AVP-NPII; ADNDI; Aggregates; Amyloid; Neurodegenerative Diseases.