Abstract
Regulated cell death (RCD) is a fundamental process common to nearly all living beings and essential for the development and tissue homeostasis in animals and humans. A wide range of molecules can induce RCD, including a number of viral proteolytic enzymes. To date, numerous data indicate that picornaviral 3C proteases can induce RCD. In most reported cases, these proteases induce classical caspase-dependent apoptosis. In contrast, the human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause caspase-independent cell death accompanied by previously undescribed features. Here, we expressed 3Cpro in HEK293, HeLa, and A549 human cell lines to characterize 3Cpro-induced cell death morphologically and biochemically using flow cytometry and fluorescence microscopy. We found that dead cells demonstrated necrosis-like morphological changes including permeabilization of the plasma membrane, loss of mitochondrial potential, as well as mitochondria and nuclei swelling. Additionally, we showed that 3Cpro-induced cell death was efficiently blocked by ferroptosis inhibitors and was accompanied by intense lipid peroxidation. Taken together, these results indicate that 3Cpro induces ferroptosis upon its individual expression in human cells. This is the first demonstration that a proteolytic enzyme can induce ferroptosis, the recently discovered and actively studied type of RCD.
Highlights
Regulated cell death (RCD) is a fundamental physiological process common to most living beings [1]
Two genetic constructs were derived from the pCI vector: pCI-3C for the constitutive expression of active hepatitis A virus 3C protease (3Cpro) and pCI-3Cmut expressing the mutant enzyme with no proteolytic activity due to the Cys172-Ala substitution in the active site (3Cmut) (Figure 1A) [13,14]
We recently demonstrated that human hepatitis A virus 3C protease (3Cpro) induces cell death with previously undescribed morphological features
Summary
Regulated cell death (RCD) is a fundamental physiological process common to most living beings [1]. RCD is crucial for the development and tissue homeostasis, while abnormal RCD causes a wide range of diseases [2]. A wide range of molecules can induce RCD including certain viral proteolytic enzymes. Similar to other virus proteins, viral proteases are multifunctional. In addition to the main function of processing viral proteins, they can cleave cellular proteins. This can inhibit the transcription of cellular genes and cellular mRNA translation, block nuclear transport, and suppress the antiviral immune response [4,5].
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