Active-site-directed chemical tools for profiling mitochondrial Lon protease

Abstract

Lon and ClpXP are the only soluble ATP-dependent proteases within the mammalian mitochondria matrix, which function in protein quality control by selectively degrading misfolded, misassembled, or damaged proteins. Chemical tools to study these proteases in biological samples have not been identified, thereby hindering a clear understanding of their respective functions in normal and disease states. In this study, we applied a proteolytic site-directed approach to identify a peptide reporter substrate and a peptide inhibitor that are selective for Lon but not ClpXP. These chemical tools permit quantitative measurements that distinguish Lon-mediated proteolysis from that of ClpXP in biochemical assays with purified proteases, as well as in intact mitochondria and mitochondrial lysates. This chemical biology approach provides needed tools to further our understanding of mitochondrial ATP-dependent proteolysis and contributes to the future development of diagnostic and pharmacological agents for treating diseases associated with defects in mitochondrial protein quality.

Figure 1. Structures of peptide-based substrates and inhibitor used in these experiments

FRETN 89–98 contains an anthranilamide fluorescent donor at the carboxy terminus and a nitrotyrosine quencher at the amino terminus. Upon cleavage of the peptide at Cys-Ser, the donor and quencher separate and an increase in fluorescence emission can be monitored over time. FRETN 89–98Abu contains a substitution of Abu for Cys at the cleavage site. Inhibitor DBN93 was designed from a product of FRETN 89–98 peptide hydrolysis by Lon with a boronic acid moiety on the carboxy end to interact with the active site of the protease.

Figure 2. Peptide-based substrates are used to monitor activity of human Lon and human ClpXP

(a) FRETN 89–98 is cleaved by both human Lon (black) and human ClpXP (dark gray) in the presence of ATP with different efficacies. The time course for hLon cleavage of FRETN 89–98 in the absence of ATP is shown in light gray. (b) FRETN 89–98Abu is a specific substrate for human Lon (black) and is not cleaved by human ClpXP (gray).

Figure 3. DBN93 inhibits human Lon by a two-step mechanism

(a) Representative time course of peptide cleavage by human Lon in the presence of ATP and DBN93. Two rates of cleavage, v_i and v_ss, and a rate constant of interconversion between the two, k_inter, can be quantified from the experimental time course. (b) Reactions containing 150 nM human Lon were preincubated with 1 mM FRETN 89–98 prior to the addition of 1 mM ATP. After 90 s, varying amounts of DBN93 (0–2 µM) were added and peptide hydrolysis was monitored over 45 min. All experiments were done in at least duplicate and the v_i and v_ss values determined by fitting the time courses as done previously for bacterial Lon (23). The averaged v_i (■), v_ss (▲) in the presence of inhibitor normalized to 1 with the average velocity in the absence of inhibitor were plotted against corresponding inhibitor concentrations. The solid lines represent the best fit of the data, as described in Materials and Methods. (c) Experimental time courses were fit to both one-step and two-step inhibition mechanisms using the global non-linear fitting program, DynaFit (Biokin, Ltd.). Black lines represent the averaged experimental time courses at 150 nM hLon, 1 mM ATP, 1 mM peptide and varying concentrations of DBN93. Gray lines represent the best fit of the data to the two-step time-dependent mechanism which was most consistent with the experimental data. The kinetic parameters yielded from this fit are summarized and compared to those previously determined for bacterial Lon (23) in Table 1.

Figure 4. DBN93 inhibits degradation of proteins by human Lon, but not by ClpXP, as resolved by SDS-PAGE

Reactions containing 10 µM λN (a) or StAR (b) protein, 1 µM human Lon and 5 mM ATP were analyzed by SDS-PAGE in the absence and presence of 10 µM DBN93 inhibitor. The band beneath StAR corresponds to an unidentified product. Casein (10 µM) digests with 1 µM hLon (c) or 2 µM ClpX and 2 µM ClpP (d) with 5 mM ATP in the absence and presence of 10 µM DBN93 were analyzed by SDS-PAGE.

Figure 5. DBN93 inhibits 20S, but not ClpXP peptidase activity

(a) Cleavage of FRETN 89–98 by ClpXP was monitored in the absence (black) and presence of 1 µM (light gray) or 4 µM (dark gray) DBN93. (b) In the presence of 500 µM ATP, 200 nM human 20S proteasome cleaves 100 µM FRETN 89–98 peptide (black). The addition of 2 µM DBN93 inhibited the cleavage (gray). This illustrates the need to isolate mitochondria from cytosol so Lon can be preferentially inhibited by DBN93.

Figure 6. Monitoring ATP-dependent peptide cleavage of FRETN 89–98 by isolated mitochondria

(a) Western blots of mitochondria isolated from HeLa cells visualized with antibodies against known amounts of human Lon, ClpX, or ClpP as indicated to approximate amount of enzyme in the isolated mitochondria. (b) Cleavage of 100 µM FRETN 89–98 by 5 µg of the purified mitochondrial matrix protein mixture was monitored in the absence (black) and presence (dark gray) of 1 mM ATP. The addition of 10 µM DBN93 inhibited ATP-dependent peptidase activity (light gray) to intrinsic levels. (c) In the presence of ATP (black), there is an increase in the rate of peptide cleavage by mitochondria containing Lon over that in the absence of ATP (dark gray). In mitochondria immunodepleted of Lon (hatch marks), there is no increase in peptide cleavage in the presence of ATP. Upon addition of 10 µM DBN93 (light gray), ATP-dependent peptide cleavage was brought back down to the background rate in the mitochondria containing Lon and no decrease in peptide cleavage was seen in the immunodepleted mitochondria. Error bars indicate the standard error from least three trials.

Figure 7. DBN93 inhibits degradation of StAR in rat mitochondria

³⁵S-StAR precursor protein was imported into rat mitochondria for either 30 min, or 30 min with a 1 or 2 hr chase, with or without 10 µM DBN93 Lon inhibitor. The mean relative intensities of 3 independent experiments are shown. SEM (n=3); p-values were calculated by Student’s t-test as compared to control. * p < 0.05, ** p <0.01, *** p > 0.05.