Dina Safina

Structural Organization of Precursors of Thermolysin-like Proteinases

The primary structures of the full-length precursors of thermolysin-like proteinases (TLPs) were systemically analyzed. Structural comparison of the precursor amino-terminal regions (ATRs) removed during maturation allowed us to divide the family into two groups: peptidases with short (about 50 amino acids) and long (about 200 amino acids) ATRs. The accumulation of mutations in the ATRs of both types proved to correlate with that in the catalytic domains. No classical signal peptides were identified in the short ATRs, but they contained a conserved PPL-motif near the initiation methionine. The functional role of the short ATRs and PPL-motif is currently unclear. The C-terminal regions (CTRs) of TLP precursors, which are often removed during maturation, too, are found in about a half of precursors with long ATRs, but occur more rarely in precursors with short ATRs. CTRs in TLP precursors contain previously identified conserved domains typical for many other proteins and likely underlie the interaction with high molecular weight substrates.

Derivation, Characterization, and Stable Transfection of Induced Pluripotent Stem Cells from Fischer344 Rats

The rat represents an important animal model that, in many respects, is superior to the mouse for dissecting behavioral, cardiovascular and other physiological pathologies relevant to humans. Derivation of induced pluripotent stem cells from rats (riPS) opens the opportunity for gene targeting in specific rat strains, as well as for the development of new protocols for the treatment of different degenerative diseases. Here, we report an improved lentivirus-based hit-and-run riPS derivation protocol that makes use of small inhibitors of MEK and GSK3. We demonstrate that the excision of proviruses does not affect either the karyotype or the differentiation ability of these cells. We show that the established riPS cells are readily amenable to genetic manipulations such as stable electroporation. Finally, we propose a genetic tool for an improvement of riPS cell quality in culture. These data may prompt iPS cell-based gene targeting in rat as well as the development of iPS cell-based therapies using disease models established in this species.

Oligomeric organization of recombinant human neurotrophins expressed in Escherichia coli cells

Genes of human neurotrophins NGF, BDNF, NT-3 were cloned, and the corresponding proteins and their fragments were expressed in Escherichia coli BL-21 (DE3λ) cells. Their intracellular localization was determined. The conditions for isolation and purification of the target recombinant proteins and for folding of BDNF and NT-3 precursors were selected. The recombinant proprecursors of human neurotrophines have been shown to possess complex oligomeric structure.

Molecular Mechanisms of Stabilization of Proteolytic Enzymes: A Model of Thermolysin-Like Microbial Metalloproteases

The molecular mechanisms that ensure the stability of proteolytic proteins are discussed. The autolytic pathway of protease degradation is emphasized. Experiments aimed at increasing the thermal stability of thermolysin-like metalloproteases are comprehensively described.

The regulation of Bacillus intermedius glutamyl endopeptidase biosynthesis in the recombinant Bacillus subtilis strain AJ73 during sporulation

The growth of the recombinant Bacillus subtilis strain AJ73 carrying the Bacillus intermedius 3-19 glutamyl endopeptidase gene on a multicopy plasmid and the effect of some nutrients on the efficiency of extracellular glutamyl endopeptidase production in the stationary growth phase were studied. In this phase, the concentration of glutamyl endopeptidase in the culture liquid peaked at the 48th and 78th hours of cultivation and depended on the composition of the cultivation medium. Unlike the synthesis of glutamyl endopeptidase in the trophophase (i.e., during vegetative growth), which was suppressed by glucose, the synthesis of this enzyme during sporulation was resistant to glucose present in the cultivation medium. A multifactorial experimental design allowed optimal proportions between the concentrations of major nutrients (peptone and inorganic phosphate) to be determined. Inorganic phosphate and ammonium ions augmented the production of glutamyl endopeptidase by 30–150%, and complex organic substrates, such as casein and gelatin, enhanced the production of glutamyl endopeptidase by 50–100%. During sporulation, the production of glutamyl endopeptidase was stimulated by some bivalent cations (Ca2+, Mg2+, and Co2+) and inhibited by others (Zn2+, Fe2+, and Cu2+). The inference is drawn that the regulatory mechanisms of glutamyl endopeptidase synthesis during vegetative growth and sporulation are different.

Effect of neurotrophin-3 precursor on glutamate-induced calcium homeostasis deregulation in rat cerebellum granule cells

Neurotrophin‐3 (NT‐3) belongs to the family of highly conserved dimeric growth factors that controls the differentiation and activity of various neuronal populations. Mammals contain both the mature (NT‐3) and the precursor (pro‐NT‐3) forms of neurotrophin. Members of the neurotrophin family are involved in the regulation of calcium homeostasis in neurons; however, the role of NT‐3 and pro‐NT‐3 in this process remains unclear. The current study explores the effects of NT‐3 and pro‐NT‐3 on disturbed calcium homeostasis and decline of mitochondrial potential induced by a neurotoxic concentration of glutamate (Glu; 100 µM) in the primary culture of rat cerebellar granule cells. In this Glu excitotoxicity model, mature NT‐3 had no effect on the induced changes in Ca2+ homeostasis. In contrast, pro‐NT‐3 decreased the period of delayed calcium deregulation (DCD) and concurrent strong mitochondrial depolarization. According to the amplitude of the increase in the intracellular free Ca2+ concentration ([Ca2+]i) and Fura‐2 fluorescence quenching by Mn2+ within the first 20 sec of exposure to Glu, pro‐NT‐3 had no effect on the initial rate of Ca2+ entry into neurons. During the lag period preceding DCD, the mean amplitude of [Ca2+]i rise was 1.2‐fold greater in the presence of pro‐NT‐3 than in the presence of Glu alone (1.67 ± 0.07 and 1.39 ± 0.04, respectively, P < 0.05). The Glu‐induced changes in Са2+ homeostasis in the presence of pro‐NT‐3 likely are due to the decreased rate of Са2+ removal from the cytosol during the DCD latency period.

Cytotoxic effect of co-expression of human hepatitis A virus 3C protease and bifunctional suicide protein FCU1 genes in a bicistronic vector

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.

Comparative evaluation of the transgene expression efficiency provided by the model genetic constructs of different structure

Comparative evaluation of transgene expression efficiency provided by genetic constructs of different structures is an important stage in the development of new and optimization of existing expression vectors. However, there is no universal approach at present for correct comparison of expression efficiency of vectors of different structure. The goal of this work was to develop an experimental system for comparative evaluation of the expression efficiency provided by nonviral genetic vectors of various size and topology in human cell cultures. This system is based on the gene for the green fluorescence protein used as a reporter, flow cytometry methods for the evaluation of expression levels, and quantitative PCR for the adequate selection of transfection conditions. This system was tested with two model constructs: a linear DNA molecule and a circular plasmid.

Involvement of Propeptides in Formation of Catalytically Active Metalloproteinase from Thermoactinomyces sp.

The metalloproteinase from Thermoactinomyces sp. 27a (Mpr) represents secretory thermolysin-like metalloproteinases of the M4 family. The Thermoactinomyces enzyme is synthesized as a precursor consisting of a signal peptide, N-terminal propeptide, mature region, and C-terminal propeptide. The functional molecule lacks the signal peptide, N-terminal propeptide, and C-terminal propeptide, which indicates the processing of these regions. Until now, it remained unclear if the N-terminal propeptide is involved in the formation and functioning of Mpr, and the role of the C-terminal propeptide was also unclear. In this work, a Bacillus subtilis AJ73 strain expressing Mpr without the C-terminal propeptide- encoding region being involved has been obtained. The absence of the Mpr C-terminal propeptide had no significant effect on the formation of the functional molecule and did not interfere with the protease secretion in B. subtilis AJ73 cells. Strains producing the N-terminal propeptide, mature region, and mature region covalently bound to the N-terminal propeptide were generated from Escherichia coli BL-21(DE3) cells. Functionally active Mpr forms could be produced only in the presence of the N-terminal propeptide, either covalently bound to the mature region (in cis) or as a separate molecule (in trans). Thus, the Mpr three-dimensional structure is formed according to the propeptide-assisted mechanism with no requirement of a covalent bond between the N-terminal propeptide and mature region. Moreover, Mpr variants generated in cis and in trans differed in their specificity for certain synthetic substrates.