Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro.

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.

Protease 3C of hepatitis A virus induces vacuolization of lysosomal/endosomal organelles and caspase-independent cell death.

BACKGROUND: 3C proteases, the main proteases of picornaviruses, play the key role in viral life cycle by processing polyproteins. In addition, 3C proteases digest certain host cell proteins to suppress antiviral defense, transcription, and translation. The activity of 3C proteases per se induces host cell death, which makes them critical factors of viral cytotoxicity. To date, cytotoxic effects have been studied for several 3C proteases, all of which induce apoptosis. This study for the first time describes the cytotoxic effect of 3C protease of human hepatitis A virus (3Cpro), the only proteolytic enzyme of the virus. RESULTS: Individual expression of 3Cpro induced catalytic activity-dependent cell death, which was not abrogated by the pan-caspase inhibitor (z-VAD-fmk) and was not accompanied by phosphatidylserine externalization in contrast to other picornaviral 3C proteases. The cell survival was also not affected by the inhibitors of cysteine proteases (z-FA-fmk) and RIP1 kinase (necrostatin-1), critical enzymes involved in non-apoptotic cell death. A substantial fraction of dying cells demonstrated numerous non-acidic cytoplasmic vacuoles with not previously described features and originating from several types of endosomal/lysosomal organelles. The lysosomal protein Lamp1 and GTPases Rab5, Rab7, Rab9, and Rab11 were associated with the vacuolar membranes. The vacuolization was completely blocked by the vacuolar ATPase inhibitor (bafilomycin A1) and did not depend on the activity of the principal factors of endosomal transport, GTPases Rab5 and Rab7, as well as on autophagy and macropinocytosis. CONCLUSIONS: 3Cpro, apart from other picornaviral 3C proteases, induces caspase-independent cell death, accompanying by cytoplasmic vacuolization. 3Cpro-induced vacuoles have unique properties and are formed from several organelle types of the endosomal/lysosomal compartment. The data obtained demonstrate previously undocumented morphological characters of the 3Cpro-induced cell death, which can reflect unknown aspects of the human hepatitis A virus-host cell interaction.

Cathepsin D messenger RNA is downregulated in human lung cancer.

OBJECTIVES: Lysosomal proteases cathepsins B and D (CB and CD) play a significant part in cancer progression. For many oncological diseases protein expression levels of CB and CD have been investigated and correlations with tumour characteristics revealed. Meanwhile, there is very little information concerning mRNA expression level. METHODS: In the present work, data about mRNA levels of CB and CD in human lung cancer was obtained using reverse transcription followed by real-time polymerase chain reaction. RESULTS: For the first time CD and CB mRNA in human lung cancer tumours was quantified. It was shown that CB and CD mRNA levels do not correlate with any tumour characteristics. However, in most analysed tumours, expression of CD mRNA was downregulated compared with adjacent normal tissue (p <0.0003). CONCLUSIONS: The data obtained indicate CD mRNA as a potential lung cancer marker.

Blood proteome profiling using aptamer-based technology for rejection biomarker discovery in transplantation.

Face transplantation is a promising solution for patients with devastating facial injuries who lack other satisfactory treatment options. At the same time, this type of transplantation is accompanied with high risks of acute transplant rejection. The limitations of traditional skin biopsy and the need to frequently monitor the condition of face transplant call for less invasive biomarkers to better diagnose and treat acute rejection. Discovery of peripheral serum proteins accurately reflecting the transplant status would represent a reasonable solution to meet this demand. However, to date, there is no clinical data available to address the feasibility of this approach. In this study, we used the next generation aptamer-based SOMAscan proteomics platform to profile 1305 proteins of peripheral blood serum in twenty-four samples taken from 6 patients during no-rejection, nonsevere rejection, and severe rejection episodes. Also, we provide a detailed description of biosample processing and all steps to generate and analyze the SOMAscan dataset with hope it will assist in performing biomarker discovery in other transplantation centers using this platform.

MMP3 Is a Non-invasive Biomarker of Rejection in Skin-Bearing Vascularized Composite Allotransplantation: A Multicenter Validation Study.

Background: There is unmet need for non-invasive immunomonitoring to improve diagnosis and treatment of acute rejection in vascularized composite allotransplantation (VCA). Circulating matrix metalloproteinase 3 (MMP3) was described as a candidate non-invasive biomarker to predict treatment response to acute rejection in clinical VCA. However, larger validation studies are yet to be reported to allow for more definitive conclusions. Methods: We retrospectively measured MMP3 levels using ELISA in a total of 140 longitudinal serum samples from six internal and three external face transplant recipients, as well as three internal and seven external upper extremity transplant recipients. The control groups comprised serum samples from 36 kidney transplant recipients, 14 healthy controls, and 38 patients with autoimmune skin disease. A linear mixed model was used to study the effect of rejection state (pre-transplant, no-rejection, non-severe rejection (NSR), and severe rejection) on MMP3 levels. Results: In VCA, MMP3 levels increased significantly (p < 0.001) between pre- and post-transplant no-rejection states. A further increase occurred during severe rejection (p < 0.001), while there was no difference in MMP3 levels between non-severe and no-rejection episodes. A threshold of 5-fold increase from pre-transplant levels could discriminate severe from NSR with 76% sensitivity and 81% specificity (AUC = 0.79, 95% CI = 0.65-0.92, p < 0.001). In kidney transplantation, the MMP3 levels were significantly (p < 0.001) elevated during antibody-mediated rejection but not during T-cell mediated rejection (TCMR) (p = 0.547). MMP3 levels in healthy controls and autoimmune skin disease patients were comparable with either pre-transplant or no-rejection/NSR episodes of VCA patients. Conclusion: The results of this study suggest that serum MMP3 protein is a promising marker for stratifying patients according to severity of rejection, complementary to biopsy findings.

Cytoplasmic vacuolization in cell death and survival.

Cytoplasmic vacuolization (also called cytoplasmic vacuolation) is a well-known morphological phenomenon observed in mammalian cells after exposure to bacterial or viral pathogens as well as to various natural and artificial low-molecular-weight compounds. Vacuolization often accompanies cell death; however, its role in cell death processes remains unclear. This can be attributed to studying vacuolization at the level of morphology for many years. At the same time, new data on the molecular mechanisms of the vacuole formation and structure have become available. In addition, numerous examples of the association between vacuolization and previously unknown cell death types have been reported. Here, we review these data to make a deeper insight into the role of cytoplasmic vacuolization in cell death and survival.

Alterations in gene expression of proprotein convertases in human lung cancer have a limited number of scenarios.

Proprotein convertases (PCs) is a protein family which includes nine highly specific subtilisin-like serine endopeptidases in mammals. The system of PCs is involved in carcinogenesis and levels of PC mRNAs alter in cancer, which suggests expression status of PCs as a possible marker for cancer typing and prognosis. The goal of this work was to assess the information value of expression profiling of PC genes. Quantitative polymerase chain reaction was used for the first time to analyze mRNA levels of all PC genes as well as matrix metalloproteinase genes MMP2 and MMP14, which are substrates of PCs, in 30 matched pairs of samples of human lung cancer tumor and adjacent tissues without pathology. Significant changes in the expression of PCs have been revealed in tumor tissues: increased FURIN mRNA level (p<0.00005) and decreased mRNA levels of PCSK2 (p<0.007), PCSK5 (p<0.0002), PCSK7 (p<0.002), PCSK9 (p<0.00008), and MBTPS1 (p<0.00004) as well as a tendency to increase in the level of PCSK1 mRNA. Four distinct groups of samples have been identified by cluster analysis of the expression patterns of PC genes in tumor vs. normal tissue. Three of these groups covering 80% of samples feature a strong elevation in the expression of a single gene in cancer: FURIN, PCSK1, or PCSK6. Thus, the changes in the expression of PC genes have a limited number of scenarios, which may reflect different pathways of tumor development and cryptic features of tumors. This finding allows to consider the mRNAs of PC genes as potentially important tumor markers.

Propeptides as modulators of functional activity of proteases.

Most proteases are synthesized in the cell as precursor-containing propeptides. These structural elements can determine the folding of the cognate protein, function as an inhibitor/activator peptide, mediate enzyme sorting, and mediate the protease interaction with other molecules and supramolecular structures. The data presented in this review demonstrate modulatory activity of propeptides irrespective of the specific mechanism of action. Changes in propeptide structure, sometimes minor, can crucially alter protein function in the living organism. Modulatory activity coupled with high variation allows us to consider propeptides as specific evolutionary modules that can transform biological properties of proteases without significant changes in the highly conserved catalytic domains. As the considered properties of propeptides are not unique to proteases, propeptide-mediated evolution seems to be a universal biological mechanism.