Intracellular cathepsin C levels determine sensitivity of cells to leucyl‐leucine methyl ester‐triggered apoptosis

Abstract

L‐leucyl‐leucine methyl ester (LLOMe) is a lysosomotropic detergent, which was evaluated in clinical trials in graft‐vs‐host disease because it very efficiently killed monocytic cell lines. It was also shown to efficiently trigger apoptosis in cancer cells, suggesting that the drug might have potential in anticancer therapy. Using U‐937 and THP‐1 promonocytes as models for monocytic cells, U‐87‐MG and HeLa cells as models for cancer cells, and noncancerous HEK293 cells, we show that the drug triggers rapid cathepsin C‐dependent lysosomal membrane permeabilization, followed by the release of other cysteine cathepsins into the cytosol and subsequent apoptosis. However, monocytes were found to be far more sensitive to the drug than the cancer and noncancer cells, which is most likely a consequence of the much higher intracellular levels of cathepsin C—the most upstream molecule in the pathway—in monocytic cell lines as compared to cancer cells. Overexpression of cathepsin C in HEK293 cells substantially enhances their sensitivity to the drug, consistent with the crucial role of cathepsin C. Major involvement of cysteine cathepsins B, S, and L in the downstream signaling pathway to mitochondrial cell death was confirmed in two gene ablation models, including the ablation of the major cytosolic inhibitor of cysteine cathepsins, stefin B, in primary mouse cancer cells, and simultaneous ablation of two major cathepsins, B and L, in mouse embryonic fibroblasts (MEFs). Deletion of stefin B resulted in sensitizing primary murine breast cancer cells to cell death without affecting the release of cathepsins, whereas simultaneous ablation of cathepsins B and L largely protected MEFs against cell death. However, due to the extreme sensitivity of monocytes to LLOMe, it appears that the drug may not be suitable for anticancer therapy due to risk of systemic toxicity.

Linear ubiquitination at damaged lysosomes induces local NF-κB activation and controls cell survival

“…Inhibition of caspases largely rescued LLOMe-induced cell death, confirming an important role of caspase-dependent apoptosis upon LMP induction, which is in line with reported findings that identify the induction of apoptosis upon lysosomal damage [50,71]. In many cases, LMP-induced cell death is regulated by the release of cathepsins in the cytosol [50]. In addition, cathepsins regulate apoptosis via cleavage of the pro-apoptotic protein Bid [72], the degradation of anti-apoptotic B-cell lymphoma 2 (Bcl-2) family members, like Bcl-2, Bcl-xL and Mcl-1 [73] and the release of cytochrome c and caspase activation [74].…”

“…Inhibition of caspases largely rescued LLOMe-induced cell death, confirming an important role of caspase-dependent apoptosis upon LMP induction, which is in line with reported findings that identify the induction of apoptosis upon lysosomal damage [50,71]. In many cases, LMP-induced cell death is regulated by the release of cathepsins in the cytosol [50].…”

“…Since severe LMP might eventually activate cell death pathways, like apoptosis [49], we next sought to investigate LMP-induced cell death pathways. LLOMe treatment triggered cell death which could be blocked by caspase inhibition with zVAD.fmk in both control and OTULIN KO cells (Figure 5A) , confirming that LLOMe induces caspase-dependent apoptosis [50]. Since damaged lysosomes can either be repaired or degraded via lysophagy in a highly dynamic fashion to control the balance between cell survival and lysosome-dependent apoptotic cell death [9,51], increased M1 poly-Ub-mediated NF-kB activation at damaged lysosomes might fulfill cell death-regulating functions.…”

The Key Role of Lysosomal Protease Cathepsins in Viral Infections

“…These mechanisms, which include abnormalities in their biosynthetic machinery, generating cathepsins lacking the signal peptide or truncated cathepsins with modified signal sequences, may direct cathepsin variants to the cytosol as well as to the mitochondria or nucleus. In the cytosol, cathepsins regulate apoptosis by both activating apoptotic proteases and degrading antiapoptotic proteins [ 2 , 8 , 46 , 47 ], and mediate inflammatory responses by activating inflammasome [ 32 , 33 , 34 , 35 ]. Loss of function or inactivation of cathepsins in the cytosol have been associated with pathological conditions such as neurodegenerative diseases, atherosclerosis [ 48 , 49 ], type 2 diabetes [ 50 , 51 ] kidney diseases [ 52 , 53 ], and ischemia [ 54 , 55 , 56 ].…”

Low‐level lysosomal membrane permeabilization for limited release and sublethal functions of cathepsin proteases in the cytosol and nucleus

“…It is evident that substances damaging the lysosomal membrane cause this phenomenon. Experimentally, so called lysosomotropic agents such as L‐leucyl‐L‐leucine methyl ester, which needs to be activated by cathepsin C, are widely used to initiate LMP [ 96 , 97 ]. In various pathological context ‘crystals’ that cannot be digested by the lysosome also induce LMP, cathepsin release, inflammation, and eventually immune cell death [ 45 , 46 , 98 ].…”