A split-luciferase-based trimer formation assay as a high-throughput screening platform for therapeutics in Alport syndrome

[Speaker] Mary Ann Suico:1
[Co-author] Kohei Omachi:1,2, Misato Kamura:1,2, Keisuke Teramoto:1,2, Haruka Kojima:1, Tsubasa Yokota:1, Shota Kaseda:1,2, Jun Kuwazuru:1, Tsuyoshi Shuto:1, Hirofumi Kai:1,2
1:Department of Molecular Medicine, Kumamoto University, Japan, 2:Program for Leading Graduate School, HIGO (Health Life Science: Interdisciplinary and Glocal Oriented) Program, Kumamoto University, Japan

Alport syndrome (AS) is a hereditary glomerular disease caused by mutation in type IV collagen alpha (COL4A) 3, 4, 5 chains, which disrupts trimerization, leading to glomerular basement membrane degeneration. The most common mutations are those in COL4A5, which comprise more than 80% of AS-associated mutations. Correcting the trimerization of COL4A3/4/5 is a feasible therapeutic approach, but is hindered by lack of information on the regulation of intracellular COL4A5 chain and the absence of high-throughput screening (HTS) platforms to assess COL4A3/4/5 trimer formation.
We investigated the intracellular stability and regulation of the COL4A5 by immunoblotting of lysates from HEK293T cells transfected with plasmids of COL4A5 and typical endoplasmic reticulum (ER) chaperones. To check the trimerization of COL4A3/4/5, we used the split nanoluciferase binary technology (Nanoluc BiT) system in which a subunit (Large BiT (LgBiT) or Small BiT (SmBiT)) is fused to a COL4A monomer. When the split NanoLuc-tagged proteins interact, LgBiT and SmBiT complementation system produces luminescence. This quantifiable luminescence reflects COL4A3/4/5 trimerization of wild-type (WT) and clinically-associated mutant COL4A5.
Despite harboring a mutation, intracellular COL4A5 mutant monomers have the same stability and are regulated similarly as WT COL4A5. This makes the intracellular trafficking pathway of mutant COL4A5 a complicated target for therapy. These data also suggested that trimer formation may be a critical defect in mutant COL4A5. Therefore, we focused on the trimerization ability of mutant COL4A5. The COL4A3/4/5 trimer assay that we developed satisfied the acceptance criteria for HTS, reflected established features of collagen regulation and enabled the characterization of intracellular- and secretion-dependent defects of mutant COL4A5. Notably, using this assay system together with si-RNA-based and chemical screening targeting the ER, we revealed that single targeting of ER chaperones did not rescue the trimer formation defect of mutant COL4A5, but several chemical chaperones had the potential to promote COL4A3/4/5 trimer formation.
This split luciferase-based trimer formation assay is a functional HTS platform that realizes the feasibility of targeting COL4A3/4/5 trimers to treat Alport syndrome.
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