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.

The ability of protealysin, a thermolysin-like metallopeptidase from Serratia proteamaculans 94, to cleave actin and matrix metalloprotease MMP2 is reported. In globular actin, protealysin and S. proteamaculans 94 cell extracts are shown to hydrolyze the Gly42-Val43 peptide bond within the DNase-binding loop and the Gly63-Ile64 and Thr66-Ile67 peptide bonds within the nucleotide cleft of the molecule. At enzyme/substrate mass ratio of 1 : 50 and below, a 36 kDa-fragment produced by the cleavage between Gly42 and Val43 was virtually resistant to further breakdown. Judging from the results of zymography, protealysin transforms proMMP2 into a 66 kDa polypeptide characteristic of mature MMP2, indicating that protealysin can activate MMP2. Upon incubation of S. proteamaculans 94 with human larynx carcinoma Hep-2 cells intracellular bacteria were detected in about 10% of Hep-2 cells, this being the first evidence for invasion of eukaryotic cells with bacteria of this species. Thus, S. proteamaculans 94 turned out to be one more bacterial strain in which synthesis of actin-specific metalloprotease is coupled with bacterial invasion. These results are consistent with the idea of the actinase activity of bacterial metalloproteases being a factor that may promote bacterial invasion of eukaryotic cells.

Integration of a DNA copy of the human immunodeficiency virus (HIV)-1 RNA genome into the human genome is an essential step in the viral replication cycle. This process is catalysed by a viral protein, integrase (IN). The first key reaction in the overall integration process is the cleavage of a dinucleotide from each 3¢-end of the viral DNA (substrate DNA), a reaction termed 3¢-end processing. In the second step, DNA strand transfer, a pair of processed DNA ends of the same viral DNA is inserted into the host cellular DNA (target DNA). The 3¢-end processing reaction requires a conserved nucleotide sequence at the viral DNA ends, but the second reaction does not absolutely require specific sequences within the host DNA. The 3¢-processing of viral DNA extremities is the first step in the integration process catalysed by human immunodeficiency virus (HIV)-1 integrase (IN). This reaction is relatively inefficient and processed DNAs are usually detected in vitro under conditions of excess enzyme. Despite such experimental conditions, steady-state Michaelis-Menten formalism is often applied to calculate characteristic equilibrium ⁄ kinetic constants of IN. We found that the amount of processed product was not significantly affected under conditions of excess DNA substrate, indicating that IN has a limited turnover for DNA cleavage. Therefore, IN works principally in a singleturnover mode and is intrinsically very slow (single-turnover rate constant ¼ 0.004 min )1 ), suggesting that IN activity is mainly limited at the chemistry step or at a stage that precedes chemistry. Moreover, fluorescence experiments showed that IN-DNA product complexes were very stable over the time-course of the reaction. Binding isotherms of IN to DNA substrate and product also indicate tight binding of IN to the reaction product. Therefore, the slow cleavage rate and limited product release prevent or greatly reduce subsequent turnover. Nevertheless, the time-course of product formation approximates to a straight line for 90 min (apparent initial velocity), but we show that this linear phase is due to the slow single-turnover rate constant and does not indicate steady-state multiple turnover. Finally, our data ruled out the possibility that there were large amounts of inactive proteins or dead-end complexes in the assay. Most of complexes initially formed were active although dramatically slow.Abbreviations HIV, human immunodeficiency virus; IN, integrase; LTR, long terminal repeat; PIC, preintegration complex; r, anisotropy; RSV, Rous sarcoma virus.

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.

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.