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Human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause in vitro caspase-independent cell death, accompanied by previously undescribed cytoplasmic vacuolization with abnormal fusion of various types of endosomal-lysosomal organelles. Yeasts are unicellular eukaryotes widely used as a biological model to study fundamental cellular processes. In particular, yeast models of cell death are useful due to the similarity of regulated cell death mechanisms between yeast and humans. This, together with the functional similarity of yeast vacuoles and the lysosomal/endosomal compartment of mammalian cells, suggests yeasts to be a promising model to study molecular mechanisms of 3Cpro action. Here, we expressed 3Cpro and its mutant inactivated form (3Cmut) in Saccharomyces cerevisiae using pYES2 vector. It was shown that expression of 3Cpro, but not 3Cmut, caused cells to lose the ability to proliferate, but maintained viability, thus indicating the enzyme exerts a cytostatic effect. In addition, using vacuolar dyes and light and electron microscopy, it was found that 3Cpro causes vacuole distortion and fragmentation, accompanied by the abnormal vacuolar staining. In summary, the effects of the 3Cpro on yeast cells resemble the response of mammalian cells. Therefore, the experimental system based on S. cerevisiae can be used for further studies of 3Cpro action.

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., Measurement(s)biomarker • graft rejection AETechnology Type(s)proteomics platformFactor Type(s)transplant rejection severitySample Characteristic - OrganismHomo sapiens Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.10548122

International audience; 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.

Studies of the molecular mechanisms of esophageal cancer development have to be carried out on sufficient amount of tumor material, obtained under conditions of controlled exposure to carcinogenic factors. Esophageal cancer models on laboratory animals serve an indispensable source of this material. One of these models is esophageal cancer induction in rats by N-nitroso compound precursors. Despite adequate reproduction of human esophageal cancer, this model in fact has not been used since the 1990ies. Re-examination of esophageal cancer model, induced by N-nitrososarcosine ethyl ester precursors, is carried out and its efficiency in induction of squamous cell carcinoma is confirmed.

A method for the in vitro evaluation of the toxic effects occurring in human cell lines upon the expression of genes from a range of pathogens is proposed. The method is based on the transient expression of the genes in the HEK293 cell line. Induction of cell death upon the expression of the gene coding for protease 3C from the human hepatitis A virus has been demonstrated for the first time using the method proposed. Expression of the gene coding for protease 2A from human poliovirus has also been shown to induce cell death, while cathepsins B and D did not have a cytotoxic effect on the culture used.

Recent reports on various cancer models demonstrate a great potential of cytosine deaminase/5-fluorocytosine suicide system in cancer therapy. However, this approach has limited success and its application to patients has not reached the desirable clinical significance. Accordingly, the improvement of this suicide system is an actively developing trend in gene therapy. The purpose of this study was to explore the cytotoxic effect observed after co-expression of hepatitis A virus 3C protease (3C) and yeast cytosine deaminase/uracil phosphoribosyltransferase fusion protein (FCU1) in a bicistronic vector. A set of mono- and bicistronic plasmid constructs was generated to provide individual or combined expression of 3C and FCU1. The constructs were introduced into HEK293 and HeLa cells, and target protein synthesis as well as the effect of 5-fluorocytosine on cell death and the time course of the cytotoxic effect was studied. The obtained vectors provide for the synthesis of target proteins in human cells. The expression of the genes in a bicistronic construct provide for the cytotoxic effect comparable to that observed after the expression of genes in monocistronic constructs. At the same time, co-expression of FCU1 and 3C recapitulated their cytotoxic effects. The combined effect of the killer and suicide genes was studied for the first time on human cells in vitro. The integration of different gene therapy systems inducing cell death (FCU1 and 3C genes) in a bicistronic construct allowed us to demonstrate that it does not interfere with the cytotoxic effect of each of them. A combination of cytotoxic genes in multicistronic vectors can be used to develop pluripotent gene therapy agents.

// Andrey V. Shubin 1,2,3,* , Ilya V. Demidyuk 1,* , Alexey A. Komissarov 1 , Lola M. Rafieva 1 and Sergey V. Kostrov 1 1 Laboratory of Protein Engineering, Institute of Molecular Genetics, Moscow, Russia 2 Laboratory of Chemical Carcinogenesis, N.N. Blokhin Russian Cancer Research Center, Moscow, Russia 3 Laboratory of Biologically Active Nanostructures, N.F. Gamaleya Institute of Epidemiology and Microbiology, Moscow, Russia * These authors contributed equally to this work Correspondence to: Andrey V. Shubin, email: // Keywords : regulated cell death, vacuolization, microbial toxins, viruses Received : February 27, 2016 Accepted : June 06, 2016 Published : June 17, 2016 Abstract 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.

Here, we review the problems of neurotrophic factors' folding, the role of its precursors (proneurotrophins) and the contribution of elements deleted during its maturation (propeptides) in biological functioning of these growth factors.

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.

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

Glutamyl endopeptidase from Bacillus intermedius(BIGEP) is a secretory serine proteinase specificallyhydrolyzing peptide bonds involving a-carboxyl groupsof glutamic and aspartic acids. In this work, differentBIGEP forms (full-length precursor, precursor withoutsignal peptide and mature part) were expressed inEscherichia coli and the process of enzyme maturationwas studied in vitro. BIGEP precursor renaturation leadsto autocatalytic hydrolysis of the propeptide at Glu(216).At the same time, the enzyme activation requires thecomplete removal of the prosequence by other protein-ases. The mature part of BIGEP cannot be activated,which indicates that the propeptide is required for theactive protein formation. The data obtained allowed us toapply directed mutagenesis of the processing site toobtain a BIGEP form that matured autocatalytically.This approach makes it possible to produce the enzymewithout extrinsic proteinases, which is a prerequisite forusing it in limited hydrolysis of proteins and peptides.Keywords: activation/gene expression/glutamylendopeptidase/precursor maturation/protein renaturationIntroductionGlutamyl endopeptidase from Bacillus intermedius (BIGEP) isa secretory serine proteinase of the structural family of chymo-trypsin. BIGEP specifically hydrolyzes bonds involvinga-carboxyl groups of glutamic and aspartic acids. Previously,the BIGEP gene was cloned, sequenced and expressed in alaboratory strain of Bacillus subtilis (Rebrikov et al., 1999).The enzyme was described (Leshchinskaya et al., 1997),directed mutagenesis of the substrate-binding site was per-formed (Demidyuk et al., 2004) and the enzyme three-dimensional structure was determined (Meijers et al., 2004).Proteolytic enzymes with a narrow substrate specificityincluding BIGEP are suitable for limited hydrolysis of pro-teins and peptides, which makes them indispensable in massspectrometry-based proteomic studies. Such enzymes areused to determine the primary structure and domain organiz-ation as well as to identify proteins. They can also be appliedin sequence-specific hydrolysis of fusion proteins and theproduction of biologically active peptides.Heterologous expression is the most efficient approach toproduce proteins for both research and applied purposes. At thesame time, the proper protein folding problem appears whenthis technique used. Most secretory proteinases (includingBIGEP) are synthesized as precursors, and their propeptide isrequired for proper folding in many of them (Shinde andInouye, 2000; Demidiuk et al., 2003). At the same time, itshould be removed for active enzyme production. If the clea-vage is autocatalytic, spontaneous maturation is commonlyobserved after renaturation of a proteinase expressed in a heter-ologous system (Ikemura and Inouye, 1988; Silen et al., 1989;Marie-Claire et al., 1998). However, some enzymes, typicallyproteinases with a narrow substrate specificity (like BIGEP),cannot cleave off their propeptide, and an additional proteolyticenzyme is required. At the same time, extrinsic proteinases areundesirable in experiments, since their removal is redundantand sometimes non-trivial. An admixture of a heterologousactivating enzyme is inadmissible in limited proteolysis thatrelies on highlyspecific proteinases.The BIGEP precursor includes the signal peptide, propeptideand mature moiety. The propeptide can be autocatalyticallyremoved in glutamyl endopeptidases only if a Glu–Xaa bondis present in the processing site (Stennicke et al., 1996; Parket al., 2004);otherwise, otherenzymes are required for the acti-vation (Park et al., 2004;Trachuk et al., 2005;Nickerson et al.,2007; Nemoto et al., 2008). During the BIGEP maturation, aLys–Val bond is hydrolyzed and, hence, this enzyme is likelyactivated by another proteinase in vivo. By analogy with gluta-myl endopeptidases from Bacillus licheniformis (Trachuket al., 2005) and Staphylococcus aureus (V8 protease)(Nemoto et al., 2008), one could expect that the BIGEPexpressed in a heterologous system requires to be activated byanother enzyme with the corresponding specificity.In this work, different BIGEP forms were expressed inE.coli cells, and the enzyme maturation process was studiedin vitro. The obtained data allowed us to propose an approachto produce proteinases with a narrow substrate specificityinvolving no extrinsic proteolytic enzymes.Materials and methods

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

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. Electronic supplementary material The online version of this article (doi:10.1186/s12860-015-0050-z) contains supplementary material, which is available to authorized users.