Proteolytic processing of dynamin by cytoplasmic cathepsin L is a mechanism for proteinuric kidney disease
J. Clin. Invest. Sanja Sever, et al. 117:2095 doi:10.1172/JCI32022 [Go to this article.]

Figure 4
Effects of the nucleotide-bound and assembly state of dynamin on CatL cleavage in vitro and in vivo. (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 GTP_ϒS 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 GTP_ϒS 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 GTP_ϒS. (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 dyn^WT 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.