Background3C 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.ResultsIndividual 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.Conclusions3Cpro, 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-015-0050-z) contains supplementary material, which is available to authorized users.
The multi-modular non-cellulosomal endo-1,3(4)-b-glucanase Lic16A from Clostridium thermocellum contains a so-called X module (denoted as CBMX) near the N terminus of the catalytic module (191-426 aa). Melting of X-module-containing recombinant proteins revealed an independent folding of the module. CBMX was isolated and studied as a separate fragment. It was shown to bind to various insoluble polysaccharides, including xylan, pustulan, chitin, chitosan, yeast cell wall glucan, Avicel and bacterial crystalline cellulose. CBMX thus contains a hitherto unknown carbohydrate-binding module (CBM54). It did not bind soluble polysaccharides on which Lic16A is highly active. Ca 2+ ions had effects on the binding, e.g. stimulated complex formation with chitosan, which was observed only in the presence of Ca 2+ . The highest affinity to CBMX was shown for xylan (binding constant K53.1¾10 4 M "1 ), yeast cell wall glucan (K51.4¾10 5 M "1 ) and chitin (K53.3.10 5 M "1 in the presence of Ca 2+ ). Lic16A deletion derivatives lacking CBMX had lower affinity to lichenan and laminarin and a slight decrease in optimum temperature and thermostability. However, the specific activity was not significantly affected.
Eucaryotic cytoplasmic mRNPs named when discovered "informosomes" are intensively investigated now as different mRNP granules. This review contains the data on structure and functioning of these mRNPs. The status of mRNA depends on spectrum of bound proteins and changes by signaling pathways.
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