Ca 074

Cell lines (~10⁷) were treated for 24, 48, and 72 h at 37 °C in a complete medium containing 10 μM inhibitor of E64-d (Sigma-Aldrich, St. Louis, MO, USA), CA074 (Merck, Rockville, MD, USA), leupeptin (Sigma Aldrich, St. Louis, MO, USA), and pepstatin A (ThermoFischer, Waltham, MA, USA). Dimethylsulphoxide solvent (final concentration 0.1%) was used as a control.

Commercially available inhibitors of cathepsin B (Ca074 and Ca074-OMe), cathepsin L (L, L1, LII, LIII and LIV), cathepsin K (KI, KII and KIII) and cathepsin S were purchased from Merck Millipore (Cork, Ireland; Supplementary Figure S2). The broad-spectrum papain peptidase inhibitor Z-Phe-Ala-FMK was purchased from Enzo Life Sciences (Exeter, UK). These were used to determine and compare the inhibition constant (K_i) against the recombinant FhCBs and FhCL3. All the assays were carried out in 0.1 M C-P buffer with 1 mM DTT and 0.01% Brij^® L23, at the pH corresponding to the optimum for each enzyme in a total 200 µL reaction volume. The peptidases (140 nM) were pre-incubated with the inhibitor (1000-15 nM) at room temperature, for 30 min, before starting the reaction by the addition of the substrate that was optimal for each enzyme (FhCB1, Z-Phe-Arg-AMC; FhCB2 and FhCB3, Z-Val-Ile-Arg-AMC; FhCL3, Z-Gly-Pro-Arg-AMC). Substrate concentrations were equal to K_M (shown in Table 1) and the apparent inhibition constant (K_i^app) values were calculated with GraphPad Prism 5, using the Morrison equation for tight binding inhibition (Equation (3)). For competitive inhibitors, such as those used in this study, K_i^app was fitted to a second equation (Equation (4)) from which K_i can be determined [30 ].
$$\frac{v_i}{v_o}=1-\frac{\left(\left[\mathrm{E}\right]\right)+\left[\mathrm{I}\right]+K_{\mathrm{i}}^{\mathrm{app}})-\sqrt{{(\left[\mathrm{E}\right]+\left[\mathrm{I}\right]+K_{\mathrm{i}}^{\mathrm{app}})}^2-4\left[\mathrm{E}\right]\left[\mathrm{I}\right] }}{2\left[\mathrm{E}\right]}$$
$$K_{\mathrm{i}}^{\mathrm{app}}=K_{\mathrm{i}}\left(1+\frac{\left[\mathrm{S}\right]}{K_{\mathrm{M}}}\right)$$

Human proximal tubular epithelial cells (HK-2) were obtained from American Type Cell Culture (ATCC). Cells were cultured in DMFM/F-12 (Gibco, USA) containing 10% fetal bovine serum in a cell incubator with 5% CO₂ maintained at 37°C. HK-2 cells were treated with different concentrations of LPS for 24 h or 48 h. CTSB inhibitor CA074 was purchased from MERCK (134448-10-5). HK-2 cells were treated with 5 μM CA074.

The stimuli used to activate cells were LPS, Escherichia coli Serotype R515 (Enzo Life Sciences) and alhydrogel (Brenntag Biosector). Inhibitory molecules used in this research included: MCC950 (Cayman Chemical), caspase1-Z-YVAD-FMK (Bachem), caspase8-Z-IETD-FMK (Bachem), CA-074 (Sigma-Aldrich), and cathepsinB-CA-074-Me (Sigma-Aldrich). Antibodies used for western blotting included: polyclonal anti bovine IL-1β (Bio-Rad), polyclonal (N-15-R) antimouse ASC (Santa Cruz Biotechnology sc-22514-R), and monoclonal (AC-74) β-actin (Sigma-Aldrich). ELISA kits used to detect bovine IL-1β and human IL-1β were sourced from ThermoScientific and R&D Systems, respectively. The FLICA™ Assay Kit (FAM-YVAD-FMK) for caspase-1 detection was acquired from ImmunoChemistry Technologies.

Cell viability was evaluated using the MTT (methylthiazolyldiphenyl‐tetrazolium bromide, Sigma) assay. Briefly, 3 × 10⁴ neurons were plated in 96‐well plates coated with poly‐L‐lysine (0.1 mg/ml). At DIV12, serum‐free 48 h‐conditioned media from ASMko or wt BMDMs was added to neuronal media at a 1:1 ratio in the presence or absence of the non‐cell permeable CathB inhibitor Ca074 (50 μM, Sigma‐Aldrich). After 24 h, 1 mM MTT was added to the wells and incubated for 3 h. Then, the supernatant was discarded, and 50 μl DMSO was added to the plates. The colour intensity was measured at 570 nm using a microplate reader (FLUOstar Optima, BMG Labtech). Cells treated with DMSO were used as a control.

The cysteine protease inhibitor E64d (100 mM, Peptanova, Sandhausen, Germany), the H⁺-ATPase inhibitor Bafilomycin A1 (20 μM, Sigma Aldrich, Munich, Germany) the cathepsin B inhibitors CA-074 Me (25 mM, Peptanova), and CA-074 (25 mM, Sigma Aldrich, Munich, Germany) were dissolved in dimethyl sulphoxide (DMSO, Carl Roth, Karlsruhe, Germany) and stored at −20 °C. For the analysis of the released Aβ, the expression of APP, of BACE1, and of CatB, a complete medium change with serum-free medium was performed prior to treatment with drugs or with DMSO alone, yielding maximum final concentrations of 0.2% v/v DMSO. The cells were treated over 48 h. The conditioned media were subsequently centrifuged at 500 g for 5 min and stored at −20°C. Cells were washed with phosphate buffered saline (PBS, Biochrom) for 5 min at room temperature (RT) and lysed in detergent buffer [50 mM HEPES, 0.037 w/v Complete Mini Protease Inhibitor Cocktail (Thermo Fisher Scientific/Roche), 150 mM NaCl, 1% v/v Non-idet P-40, 0.5% w/v sodium deoxycholate, and 0.1% w/v sodium dodecylsulfate (SDS)] or in CytoBuster™ Protein Extraction Reagent (Merck Millipore) for 10 min at 4°C. The lysed cells were centrifuged (13,000 g, 5 min, 4°C), and supernatants were stored at −70°C.

To measure protease activity of worm lysates, planarians were ground with mortar and pestle for thirty seconds and incubated with lysis buffer (100mM Tris pH 7.5, 200mM NaCl, 1% NP-40, 0.1% SDS, 1XTBS) for one hour on ice with occasional vortexing. Samples were spun in a microcentrifuge at maximum speed, 4°C for twenty minutes. The supernatant was saved and activity was measured by adding assay mix (5mMDTT, 50mM sodium citrate, pH5.5) containing 50μM fluorescent peptide, either Z-FR-AMC or Z-RR-AMC (BACHEM). Fluorescence was measured (excitation 360nm, absorbance 460nm) using a FlexStation fluorometer (Molecular Devices) and SoftMax Pro 4.8 software. Both kinetic and endpoint assays were used. Chemical inhibitors of cathepsin B, K11777 (UCSF, CDIPD) and CA-074 (Sigma), were added at 50μM concentration two hours prior to addition of fluorescent peptides for select experiments. Protease activity was also measured with activity-based probes DCG-04 and BMV109, both gifts from Matthew Bogyo [31 (link), 32 (link)]. Probes were added to 1μM final volume in samples containing 5mM DTT, pH 5.5 for 1 hour at 37°C before imaging Cy5 levels via Typhoon Trio (GE Healthcare Life Sciences).