ESPN 50th Annual Meeting

ESPN 2017


 
Targeting the type IV collagen misfolding in hereditary nephritis Alport syndrome
KOHEI OMACHI 1 KEISUKE TERAMOTO 1 MISATO KAMURA 1 HARUKA KOJIMA 1 MARY ANN SUICO 1 TSUYOSHI SHUTO 1 HIROFUMI KAI 1

1- KUMAMOTO UNIVERSITY
 
Introduction:

Alport syndrome (AS) is a hereditary kidney disease that is caused by a mutation in Type IV collagen (COL4A) α3, α4 or α5 gene. Genetic re-expression of α3 gene rescued the phenotype of α3 knockout AS mouse model. Furthermore, the expression level of α345 proteins correlated with the severity of nephritis in clinical study. These results indicated that targeting the causal misfolded protein is a promising therapeutic strategy for AS. However, progress in this research area is hindered by the insufficient knowledge about the regulation of COL4A3/4/5 proteins and the absence of cellular model and tools for AS.    

Material and methods:

Split nanoLuc-fusion α3/ α5 and α4 were transfected into 293T cells, and luminescence was assessed in the cell lysate for intracellular trimer and in culture media for secreted trimer. The trimerization of wild type (WT) and some mutant α5 chains were also analyzed in the same way.

Results:

 Luciferase-based assay showed that WT α345 trimer but not monomer and dimer is detected with high sensitivity. Domain deletion mutant α5 (delCOL, delNC1) had significantly reduced trimerization, and some α5 mutants (G227S, G325R, G624D, G869R, G1030S, G1107R, G1244D, C1567R, R1569Q, L1649R, R1683Q) had decreased trimerization compared with WT α5. Moreover, our preliminary data suggested that some chemical chaperones could rescue trimer formation of mutant α5.

Conclusions:

The α345 trimer assay system that we developed is applicable for HTS, and could be a powerful tool to elucidate the regulation of causal protein in AS. Although further detailed studies are necessary, the results using chemical compounds suggested that it is possible to correct the trimerization of mutant α5, and targeting α345 in AS becomes more realistic.