Naslov
Luciferase-based reporter for studying human tau protein oligomerization in living yeast cells

Autori
Zubčić, Klara ; Šimić, Goran ; Boban, Mirta

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Proceedings of the 7th Mediterranean Neuroscience Society Conference / - , 2019, 264-264

Skup
7th Mediterranean Neuroscience Society Conference

Mjesto i datum
Marakeš, Maroko, 23.06.2019. - 27.06.2019

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Alzheimer's disease, protein aggregation, tau, luciferase, Saccharomyces cerevisiae

Sažetak
Tauopathies, including Alzheimer’s disease, are neurodegenerative disorders characterized by tau protein aggregates in brain cells. Degeneration is characterized by a pathological accumulation of hyperphosphorylated tau, called neurofibrillary tangles, which show a high positive correlation with the degree of dementia. However, tau aggregation pathways leading to tau filament formation and toxicity are unclear. To better understand molecular pathways leading to tau pathology, we constructed a simple tool for studying tau oligomerization in living cells, based on the luminescent reporter NanoBiT. In short, proteins of interest are fused to luciferase subunits 11S and 114. Protein- protein interaction results in enzyme complementation, generating luminescence that can be measured in living cells. Since molecular pathways that prevent accumulation of protein aggregates in the cell are largely evolutionarily conserved, we selected a simple model, yeast Saccharomyces cerevisiae, to look for the factors that influence tau oligomerization. We constructed C-terminal fusions of human 2N4R-tau protein with the luciferase subunits 11S and 114. The DNA sequence encoding human tau protein was codon optimized for yeast and placed under the control of a weak promoter and short synthetic terminators. To be able to distinguish between the expression of tau- 11S and tau-114 within the same cell, we tagged the constructs with two different epitope tags. We will validate the reporter by comparing the luminescent signal of cells expressing constructs tau-11S and tau-114 with the cells additionally overexpressing untagged tau, and with the cells carrying constructs 11S and tau- 114. This work is supported by Croatian Science Foundation (grant IP-2014-09-9730 and DOK-01- 2018/European Social Fund).

Izvorni jezik
Engleski

POVEZANOST RADA