Abstract: TH-PO072

Altered Hydrolysis Function of Ubiquitin C-Terminal Hydrolase L1 (UCH-L1) by Oxidative Modification Affects Protein Homeostasis in Podocytes

Session Information

Category: Glomerular

  • 1002 Glomerular: Basic/Experimental Pathology

Authors

  • Reichelt, Julia, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany
  • Reinicke, Anna, University Clinic Eppendorf Hamburg, Hamburg, Germany
  • Fehlert, Julia M., University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Sachs, Marlies, UKE , Hamburg, Germany
  • Zahner, Gunther, University hospital hamburg, Hamburg, Germany
  • Meyer-Schwesinger, Catherine, University of Hamburg, Hamburg, Germany
Background

The ubiquitin proteasome system (UPS) represents the major system for protein degradation and is important for maintenance of protein homeostasis. Ubiquitin C-terminal hydrolase L1 (UCH-L1) regulates the pool of monoubiquitin required for tagging target proteins. The hydrolase-deficient mutant UCH-L1I93M is highly associated with the development of neurodegenerative disease in humans and structurally resembles oxidative-modified UCH-L1. During podocyte injury UCH-L1 is de novo expressed resulting in proteasomal impairment and accumulation of polyubiquitinated proteins by unknown mechanisms. Aim of this project is to investigate the toxic gain of function of UCH-L1.

Methods

In order to dissect UCH-L1 hydrolysis versus UCH-L1 hydrolase-independent mechanisms transgenic mice with podocyte-specific overexpression of UCH-L1 wildtype or an enzymatic-deficient form UCH-L1I93M were generated. The effects of altered degradation were investigated in cultured podocytes and in 5-7 week old naïve mice morphologically, clinically and biochemically. By use of PAS and immunofluorescent stainings for podocyte-specific proteins and by measurement of proteinuria the glomerular integrity was characterized. To analyze the biochemical properties of UCH-L1 (such as expression levels or proteolytic capacity) quantitative real-time PCR, Western blot, activity assays and immunohistochemical stainings were executed.

Results

In cultured podocytes UCH-L1 expression was induced following oxidative stress. UCH-L1I93M induced accumulations of polyubiquitinated as well as oxidative modified proteins in cultured podocytes and isolated glomeruli of transgenic mice. This conglomeration resulted from impaired proteasomal activity despite an enhanced proteasomal capacity. UCH-L1WT mice on the other hand were characterized by increased proteasomal activity and a dedifferentiated phenotype. This was indicated by decreasing levels of podocyte-specific proteins nephrin and α-actinin-4 influenced by declining amount of WT-1 which seems to be regulated by the proteasome.

Conclusion

De novo expression of UCH-L1 in podocyte injury could mediate two effects: first, dedifferentiation of the podocyte and second, accumulation of polyubiquitinated proteins conditioned by defective enzymatic function.