Fig. 2: UCH-L1 is modified in experimental MN.

a Calculated electrostatic surface potential of the crystal structures of UCH-L1 wildtype (PDB: 2ETL, UCH-L1^WT), and its I93M (PDB:3IRT, UCH-L1^I93M) variant and of the predicted crystal structure of an oxidative-modified variant (UCH-L1^ox_mod) in two orientations in surface representation. Surface is colored according to the electrostatic surface potential, using a ramp from −5kT/e to +5kT/e at which the surface colors are clamped at red for a negative or blue for a positive electrostatic potential, visualized by a slider. Location of the amino acid residues Lys123 and Met1 in UCH-L1^WT and UCH-L1^I93M or methionine sulfoxide MetO1 in UCH-L1^ox_mod are indicated. While the surface of the active site entrance (arrows) in the oxidative-modified variant is less positively charged compared to the wildtype (upper panel), the dorsal side of the oxidative-modified variant shows positive surface charge which is missing in the wildtype (lower panel). b, c Doxycycline-induced overexpression of I93M-UCH-L1 or WT-UCH-L1 in murine podocytes. b K48 pUB levels were determined by immunoblot and normalized to β-actin, n = 3 (WT&mock) or n = 4 (I93M) of 2 independent experiments, mean + /-SEM, two-tailed Mann Whitney U test. c Examination of the main proteasomal chymotrypsin-like activity by measurement of Suc-LLVY-AMC peptide hydrolysis, n = 5 (WT&I93M) or n = 8 (mock) of 2 independent experiments, mean + /-SEM, **p = 0.0062, two-tailed Mann Whitney U test. d Experimental MN was initiated by i.v. injection of AP-abs or sheep IgG (Ctrl) in mice. Kidneys were collected on day 14. Glomerular enzymatic in-gel activity of UCH-L1 was measured using a ubiquitin-based activity probe (Cy5-Ub-VME), which irreversibly binds to the catalytic center of active deubiquitinating enzymes. Brain lysates of Uchl1^-/- mice were used as negative control (NC), of Uchl1^+/+ mice as positive control (PC). Levels of active UCH-L1 were quantified by measuring in-gel fluorescence of labeled probe at 35 kDa subsequently normalized to respective total UCH-L1 protein levels (normalized to β-actin of the same membrane). Values are presented as mean + /-SEM, n = 3 (Ctrl) or n = 9 (exp. MN) of 1 experiment, *p = 0.0364, two-tailed Mann Whitney U test. Source data are provided as a Source Data file.