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Sphingosine-induced apoptosis is dependent on lysosomal proteases.

ABSTRACT: We propose a new mechanism for sphingosine-induced apoptosis, involving relocation of lysosomal hydrolases to the cytosol. Owing to its lysosomotropic properties, sphingosine, which is also a detergent, especially when protonated, accumulates by proton trapping within the acidic vacuolar apparatus, where most of its action as a detergent would be exerted. When sphingosine was added in low-to-moderate concentrations to Jurkat and J774 cells, partial lysosomal rupture occurred dose-dependently, starting within a few minutes. This phenomenon preceded caspase activation, as well as changes of mitochondrial membrane potential. High sphingosine doses rapidly caused extensive lysosomal rupture and ensuing necrosis, without antecedent apoptosis or caspase activation. The sphingosine effect was prevented by pre-treatment with another, non-toxic, lysosomotropic base, ammonium chloride, at 10 mM. The lysosomal protease inhibitors, pepstatin A and epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester ('E-64d'), inhibited markedly sphingosine-induced caspase activity to almost the same degree as the general caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone ('Z-VAD-FMK'), although they did not by themselves inhibit caspases. We conclude that cathepsin D and one or more cysteine proteases, such as cathepsins B or L, are important mediators of sphingosine-induced apoptosis, working upstream of the caspase cascade and mitochondrial membrane-potential changes.

SUBMITTER: Kagedal K

PROVIDER: S-EPMC1222151 | biostudies-other | 2001 Oct

REPOSITORIES: biostudies-other

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Project description:Cell entry by coronaviruses involves two principal steps, receptor binding and membrane fusion; the latter requires activation by host proteases, particularly lysosomal proteases. Despite the importance of lysosomal proteases in both coronavirus entry and cell metabolism, the correlation between lysosomal proteases and cell tropism of coronaviruses has not been established. Here, we examined the roles of lysosomal proteases in activating coronavirus surface spike proteins for membrane fusion, using the spike proteins from severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) as the model system. To this end, we controlled the contributions from receptor binding and other host proteases, thereby attributing coronavirus entry solely or mainly to the efficiency of lysosomal proteases in activating coronavirus spike-mediated membrane fusion. Our results showed that lysosomal proteases from bat cells support coronavirus spike-mediated pseudovirus entry and cell-cell fusion more effectively than their counterparts from human cells. Moreover, purified lysosomal extracts from bat cells cleave cell surface-expressed coronavirus spikes more efficiently than their counterparts from human cells. Overall, our study suggests that different lysosomal protease activities from different host species and tissue cells are an important determinant of the species and tissue tropism of coronaviruses.IMPORTANCE Coronaviruses are capable of colonizing new species, as evidenced by the recent emergence of SARS and MERS coronaviruses; they can also infect multiple tissues in the same species. Lysosomal proteases play critical roles in coronavirus entry by cleaving coronavirus surface spike proteins and activating the fusion of host and viral membranes; they also play critical roles in cell physiology by processing cellular products. How do different lysosomal protease activities from different cells impact coronavirus entry? Here, we controlled the contributions from known factors that function in coronavirus entry so that lysosomal protease activities became the only or the main determinant of coronavirus entry. Using pseudovirus entry, cell-cell fusion, and biochemical assays, we showed that lysosomal proteases from bat cells activate coronavirus spike-mediated membrane fusion more efficiently than their counterparts from human cells. Our study provides the first direct evidence supporting lysosomal proteases as a determinant of the species and tissue tropisms of coronaviruses.

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Sphingosine-induced apoptosis is dependent on lysosomal proteases.

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