Proteolytic processing of dynamin by cytoplasmic cathepsin L is a mechanism for proteinuric kidney disease
Sanja Sever, … , Boris Nikolic, Jochen Reiser
Sanja Sever, … , Boris Nikolic, Jochen Reiser
Published August 1, 2007
Citation Information: J Clin Invest. 2007;117(8):2095-2104. https://doi.org/10.1172/JCI32022.
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Proteolytic processing of dynamin by cytoplasmic cathepsin L is a mechanism for proteinuric kidney disease

Abstract

Kidney podocytes and their foot processes maintain the ultrafiltration barrier and prevent urinary protein loss (proteinuria). Here we show that the GTPase dynamin is essential for podocyte function. During proteinuric kidney disease, induction of cytoplasmic cathepsin L leads to cleavage of dynamin at an evolutionary conserved site, resulting in reorganization of the podocyte actin cytoskeleton and proteinuria. Dynamin mutants that lack the cathepsin L site, or render the cathepsin L site inaccessible through dynamin self-assembly, are resistant to cathepsin L cleavage. When delivered into mice, these mutants restored podocyte function and resolve proteinuria. Our study identifies dynamin as a critical regulator of renal permselectivity that is specifically targeted by proteolysis under pathological conditions.

Authors

Sanja Sever, Mehmet M. Altintas, Sharif R. Nankoe, Clemens C. Möller, David Ko, Changli Wei, Joel Henderson, Elizabetta C. del Re, Lianne Hsing, Ann Erickson, Clemens D. Cohen, Matthias Kretzler, Dontscho Kerjaschki, Alexander Rudensky, Boris Nikolic, Jochen Reiser

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Figure 4

Effects of the nucleotide-bound and assembly state of dynamin on CatL cleavage in vitro and in vivo.

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Effects of the nucleotide-bound and assembly state of dynamin on CatL cl...
(A) Domain structure of dynamin, corresponding antibodies, and amino acid sequence of predicted CatL cleavage sites. Note that the ELSGGA sequence is a highly conserved motif throughout the species. PH, pleckstrin homology domain; PRD, proline-arginine rich domain; Shi, dynamin homolog in Drosophila; Vsp1, dynamin homolog in yeast. (B) Schematic depiction of dynamin GTPase cycle. In its basal state, dynamin is a homotetramer. Self-assembly into higher-order structures such as rings or spirals can be promoted by GTPϒS and activates assembly-mediated GTP hydrolysis, which in turn drives disassembly. Dynamin’s middle domain is located inside the spiral. (C) Recombinant dyn1 (20 pmol) (CON) was mixed with CatL (1 pmol) (top panel), CatB (middle panel), or Furin (bottom panel). The reactions were performed at pH 7.0 under nonassembly conditions (200 mM NaCl). Where indicated, 200 μM GTP or 1 mM GTPϒS was present. Proteolytic products were detected by monoclonal anti-dynamin antibody against the GTPase domain. (D) Silver staining of recombinant dyn1 incubated with CatL at different pHs in the presence or absence of GTPϒS. (E) Western blot analysis using GTPase antibody of podocyte extracts infected with various dynamin mutants before or after addition of 100 μg/ml LPS for 20 hours. Note the appearance of a 40-kDa fragment (arrow). When indicated, podocytes were treated with 1 μM of the selective CatL inhibitor Z-FF-FMK for the duration of the LPS treatment. (F) Western blot analysis of subcellular fractionation of podocytes expressing dynWT for 24 hours after LPS treatment. Extracts were blotted using antibodies against the GTPase domain (N-terminal), GAP domain (C-terminal), LAMP-2, and tubulin.