L-leucyl-L-leucine methyl ester does not release cysteine cathepsins to the cytosol but inactivates them in transiently permeabilized lysosomes

Abstract

L-leucyl-L-leucine methyl ester (LLOMe) induces apoptosis, which is thought to be mediated by release of lysosomal cysteine cathepsins from permeabilized lysosomes into the cytosol. Here, we demonstrated in HeLa cells that apoptotic as well as sub-apoptotic concentrations of LLOMe caused rapid and complete lysosomal membrane permeabilization (LMP), as evidenced by loss of the proton gradient and release into the cytosol of internalized lysosomal markers below a relative molecular mass of 10,000. However, there was no evidence for the release of cysteine cathepsins B and L into the cytosol; rather they remained within lysosomes, where they were rapidly inactivated and degraded. LLOMe-induced adverse effects, including LMP, loss of cysteine cathepsin activity, caspase activation and cell death could be reduced by inhibition of cathepsin C, but not by inhibiting cathepsins B and L. When incubated with sub-apoptotic LLOMe concentrations, lysosomes transiently lost protons but annealed and re-acidified within hours. Full lysosomal function required new protein synthesis of cysteine cathepsins and other hydrolyses. Our data argue against the release of lysosomal enzymes into the cytosol and their proposed proteolytic signaling during LLOMe-induced apoptosis.

Lysosomal membrane permeabilization and cell death

“…Moreover, cationic nanoparticles first induce the release from the lysosome of smaller cleaved‐cathepsin D (~27 kDa), followed later by the larger cathepsin B (~37 kDa) . A recent study reported that the lysosomotropic compound l ‐leucyl‐ l ‐leucine O ‐methyl ester renders lysosomes permeable to protons prior to the release of preloaded dextrans, and that ESCRT machinery is initiated to repair the damaged membrane . These findings suggest that LMP is a progressive and dynamic process and they also point to the existence of a molecular “sieve” that dictates the specific translocation of the lysosomal contents.…”

Triggered recruitment of ESCRT machinery promotes endolysosomal repair

“…The lysosomotropic compound LLOME (L-leucyl-L-leucine O -methyl ester) is commonly used to rupture endolysosomes (3, 23, 24). LLOME rapidly permeates cellular membranes and accumulates in the lumen of acidified organelles, where it is condensed into a membranolytic polymer by the lysosomal enzyme cathepsin C (9, 23). We confirmed LLOME-induced damage in HeLa cells by noting concentration of cytoplasmic GAL3 (a galectin frequently used to monitor damage (24)) on punctate structures (Fig.…”

“…The effects of LLOME on ESCRTs were abolished by preincubating cells with the cathepsin inhibitor E64d (Fig. 1D), which prevents LLOME processing by cathepsin C and consequent membrane disruption (9). Accordingly, ESCRT-enriched structures induced by LLOME corresponded to late endosomes and lysosomes, coinciding well with LAMP1 but not with the early endosome protein EEA1 (Fig.…”

“…LLOME forms hydrophobic adducts that destabilize the lipid bilayer, causing damage that ranges from small perturbations permeable to protons and small molecules (9) to larger ruptures that allow exchange of higher molecular weight material (24). Lysophagy of ruptured endolysosomes is directed in part by an influx of galectins, which bind to injury-exposed lumenal β-galactosides (1, 2) and would thus require sufficiently large membrane disruptions to access the compartmental interior.…”

“…We thus sought a way to follow ESCRT dynamics during both the onset and resolution of endolysosomal injury. Briefly exposing cells to lysosomotropic rupturing agents induces transient membrane disruption (9, 11, 27, 36) and accordingly, cells treated with LLOME for less than a minute acquired ESCRT puncta shortly after adding drug but not at later times (fig. S20).…”