Method for screening substance relating to endoplasmic reticulum stress participating in onset of diabetes
View Patent ↗A system for evaluating activation of the pathway mediated by ATF6. More specifically, a method for screening an endoplasmic reticulum stressor, as well as a method for screening a substance for suppressing an endoplasmic reticulum stress induced by the activation of the pathway mediated by ATF6, and a method for screening an antidiabetic drug candidate. A method for real-time evaluation of activation of the pathway mediated by ATF6 in viable cells. The method uses a polynucleotide encoding a nonfluorescent peptide domain derived from a fluorescent protein and an ATF6 protein domain.
1. A polynucleotide encoding an amino acid sequence having:
(1) a nonfluorescent peptide domain 1 of a fluorescent protein constituted of a nonfluorescent peptide domain 1 and a nonfluorescent peptide domain 2, characterized in that when nonfluorescent peptide domain 1 and nonfluorescent peptide domain 2 are associated, fluorescent activity is gained; and
(2) an ATF6 protein domain,
wherein the polynucleotide has a region encoding (1) at the 5′ end, a region encoding (2) at the 3′ end, and a region encoding a PEST sequences between the region encoding (1) and the region encoding (2),
wherein the polynucleotide does not encode the fluorescent protein, and
wherein the fluorescent protein is GFP, GFP mutants, or a GFP-like protein family member.
2. The polynucleotide according to claim 1 , wherein the polynucleotide encodes an amino acid sequence in which the nonfluorescent peptide domain 1 of said fluorescent protein and the PEST sequences are connected via a spacer having more than 6 amino acid residues.
3. A transgenic vector comprising the polynucleotide according to claim 1 .
4. A transformed cultured cell comprising a transgenic vector according to claim 3 .
5. The transformed cultured cell according to claim 4 , further comprising a polynucleotide encoding an amino acid sequence having:
(3) the nonfluorescent peptide domain 2 of said fluorescent protein; and
(4) a nuclear localization signal peptide domain,
in a state where a protein having the amino acid sequence having (3) and (4) can be expressed.
6. The transformed cultured cell according to claim 5 , wherein the transformed cultured cell is a pancreatic β-cell.