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Kidney Week

Abstract: TH-PO717

SRNS-Associated MYO1E Mutations Have Differential Effects on Myo1e Activity and Stability

Session Information

Category: Genetic Diseases of the Kidney

  • 1002 Genetic Diseases of the Kidney: Non-Cystic

Authors

  • Liu, Pei-Ju, SUNY Upstate Medical University, Syracuse, New York, United States
  • Perez, Diana, SUNY Upstate Medical University, Syracuse, New York, United States
  • Bi Karchin, Jing, SUNY Upstate Medical University, Syracuse, New York, United States
  • Pellenz, Christopher D., SUNY Upstate Medical University, Syracuse, New York, United States
  • Chase, Sharon E., SUNY Upstate Medical University, Syracuse, New York, United States
  • Presti, Maria, SUNY Upstate Medical University, Syracuse, New York, United States
  • Plante, Eric L., SUNY Upstate Medical University, Syracuse, New York, United States
  • Martin, Claire E., University of Guelph, Guelph, Ontario, Canada
  • Lovric, Svjetlana, Hannover Medical School, Hannover, Germany
  • Hildebrandt, Friedhelm, Boston Children's Hospital, Boston, Massachusetts, United States
  • Krendel, Mira, SUNY Upstate Medical University, Syracuse, New York, United States
Background

Mutations in the MYO1E gene, encoding myosin 1e (Myo1e), are associated with steroid resistant nephrotic syndrome (SRNS). We set out to characterize novel MYO1E mutations identified in SRNS patients (Sadowski et al., JASN, 2015, 26(6): 1279-89).

Methods

Using adenoviral transduction of EGFP-Myo1e constructs into cultured podocytes, we compared steady-state expression levels, protein turnover rates, and localization of the wild type (wt) Myo1e and several Myo1e mutants, including mutants with point mutations in the motor domain and a frame shift mutant d3094-7 lacking the C-terminal SH3 domain.

Results

We found that two mutants, the T119I motor domain mutant and the d3094-7 frame shift mutant, had low expression levels and high turnover rates and did not localize to cell-cell junctions, where Myo1e is thought to aid in the assembly of the slit diaphragm complexes. In an attempt to restore Myo1e protein stability, we treated cells expressing these mutants with a proteasome inhibitor and observed accumulation of the mutant proteins. The restoration of protein expression in cells expressing the d3094-7 mutant resulted in a partial recovery of the junctional localization and dynamics of this protein. In contrast, an increase in the protein level of the T119I mutant achieved using proteasome inhibition was accompanied by formation of random, seemingly insoluble, aggregates (as determined using fluorescence recovery after photobleaching (FRAP)). Further investigating the properties of those Myo1e mutants that did not exhibit decreased protein stability, we found that the dynamics of one of the motor domain mutants at the junctions was decreased compared to the wt Myo1e. Using FRAP, we found that the mobile fraction of this mutant was lower than that of the wt Myo1e while the half time of fluorescence recovery was higher.

Conclusion

Overall, our findings indicate that while some SRNS-associated mutations affect Myo1e stability and folding, others may have more specific effects on motor domain activity. Furthermore, proteasome inhibition needs to be further examined as a potential therapeutic approach for alleviating the effects of those mutations that affect Myo1e stability without disrupting its motor function.

Funding

  • NIDDK Support