Correction: Plyasova et al. Penetration into Cancer Cells via Clathrin-Dependent Mechanism Allows L-Asparaginase from Rhodospirillum rubrum to Inhibit Telomerase. Pharmaceuticals 2020, 13, 286.

In the original publication [...].

L-Asparaginase Conjugates from the Hyperthermophilic Archaea Thermococcus sibiricus with Improved Biocatalytic Properties.

This study investigated the effect of polycationic and uncharged polymers (and oligomers) on the catalytic parameters and thermostability of L-asparaginase from Thermococcus sibiricus (TsA). This enzyme has potential applications in the food industry to decrease the formation of carcinogenic acrylamide during the processing of carbohydrate-containing products. Conjugation with the polyamines polyethylenimine and spermine (PEI and Spm) or polyethylene glycol (PEG) did not significantly affect the secondary structure of the enzyme. PEG contributes to the stabilization of the dimeric form of TsA, as shown by HPLC. Furthermore, neither polyamines nor PEG significantly affected the binding of the L-Asn substrate to TsA. The conjugates showed greater maximum activity at pH 7.5 and 85 °C, 10-50% more than for native TsA. The pH optima for both TsA-PEI and TsA-Spm conjugates were shifted to lower pH ranges from pH 10 (for the native enzyme) to pH 8.0. Additionally, the TsA-Spm conjugate exhibited the highest activity at pH 6.5-9.0 among all the samples. Furthermore, the temperature optimum for activity at pH 7.5 shifted from 90-95 °C to 80-85 °C for the conjugates. The thermal inactivation mechanism of TsA-PEG appeared to change, and no aggregation was observed in contrast to that of the native enzyme. This was visually confirmed and supported by the analysis of the CD spectra, which remained almost unchanged after heating the conjugate solution. These results suggest that TsA-PEG may be a more stable form of TsA, making it a potentially more suitable option for industrial use.

Enhancing the Catalytic Activity of Thermo-Asparaginase from Thermococcus sibiricus by a Double Mesophilic-like Mutation in the Substrate-Binding Region.

L-asparaginases (L-ASNases) of microbial origin are the mainstay of blood cancer treatment. Numerous attempts have been performed for genetic improvement of the main properties of these enzymes. The substrate-binding Ser residue is highly conserved in L-ASNases regardless of their origin or type. However, the residues adjacent to the substrate-binding Ser differ between mesophilic and thermophilic L-ASNases. Based on our suggestion that the triad, including substrate-binding Ser, either GSQ for meso-ASNase or DST for thermo-ASNase, is tuned for efficient substrate binding, we constructed a double mutant of thermophilic L-ASNase from Thermococcus sibiricus (TsA) with a mesophilic-like GSQ combination. In this study, the conjoint substitution of two residues adjacent to the substrate-binding Ser55 resulted in a significant increase in the activity of the double mutant, reaching 240% of the wild-type enzyme activity at the optimum temperature of 90 °C. The mesophilic-like GSQ combination in the rigid structure of the thermophilic L-ASNase appears to be more efficient in balancing substrate binding and conformational flexibility of the enzyme. Along with increased activity, the TsA D54G/T56Q double mutant exhibited enhanced cytotoxic activity against cancer cell lines with IC90 values from 2.8- to 7.4-fold lower than that of the wild-type enzyme.

Improvement of Biocatalytic Properties and Cytotoxic Activity of L-Asparaginase from Rhodospirillum rubrum by Conjugation with Chitosan-Based Cationic Polyelectrolytes.

L-asparaginases (L-ASNases, EC 3.5.1.1) are a family of enzymes that are widely used for the treatment of lymphoblastic leukemias. L-ASNase from Rhodospirillum rubrum (RrA) has a low molecular weight, low glutaminase activity, and low immunogenicity, making it a promising enzyme for antitumor drug development. In our work, the complex formation and covalent conjugation of the enzyme with synthetic or natural polycationic polymers was studied. Among non-covalent polyelectrolyte complexes (PEC), polyethyleneimine (PEI) yielded the highest effect on RrA, increasing its activity by 30%. The RrA-PEI complex had increased stability to trypsinolysis, with an inactivation constant decrease up to 10-fold compared to that of the native enzyme. The covalent conjugation of RrA with chitosan-PEI, chitosan-polyethylene glycol (chitosan-PEG), and chitosan-glycol resulted in an increase in the specific activity of L-asparagine (up to 30%). RrA-chitosan-PEG demonstrated dramatically (by 60%) increased cytotoxic activity for human chronic myeloma leukemia K562 cells in comparison to the native enzyme. The antiproliferative activity of RrA and its conjugates was significantly higher (up to 50%) than for that of the commercially available EcA at the same concentration. The results of this study demonstrated that RrA conjugates with polycations can become a promising strategy for antitumor drug development.

Molecular Analysis of L-Asparaginases for Clarification of the Mechanism of Action and Optimization of Pharmacological Functions.

L-asparaginases (EC 3.5.1.1) are a family of enzymes that catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. These proteins with different biochemical, physicochemical and pharmacological properties are found in many organisms, including bacteria, fungi, algae, plants and mammals. To date, asparaginases from E. coli and Dickeya dadantii (formerly known as Erwinia chrysanthemi) are widely used in hematology for the treatment of lymphoblastic leukemias. However, their medical use is limited by side effects associated with the ability of these enzymes to hydrolyze L-glutamine, as well as the development of immune reactions. To solve these issues, gene-editing methods to introduce amino-acid substitutions of the enzyme are implemented. In this review, we focused on molecular analysis of the mechanism of enzyme action and to optimize the antitumor activity.

Highly Active Thermophilic L-Asparaginase from Melioribacter roseus Represents a Novel Large Group of Type II Bacterial L-Asparaginases from Chlorobi-Ignavibacteriae-Bacteroidetes Clade.

L-asparaginase (L-ASNase) is a biotechnologically relevant enzyme for the pharmaceutical, biosensor and food industries. Efforts to discover new promising L-ASNases for different fields of biotechnology have turned this group of enzymes into a growing family with amazing diversity. Here, we report that thermophile Melioribacter roseus from Ignavibacteriae of the Bacteroidetes/Chlorobi group possesses two L-ASNases-bacterial type II (MrAII) and plant-type (MrAIII). The current study is focused on a novel L-ASNase MrAII that was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 70 °C and pH 9.3, with a high L-asparaginase activity of 1530 U/mg and L-glutaminase activity ~19% of the activity compared with L-asparagine. The kinetic parameters KM and Vmax for the enzyme were 1.4 mM and 5573 µM/min, respectively. The change in MrAII activity was not significant in the presence of 10 mM Ni2+, Mg2+ or EDTA, but increased with the addition of Cu2+ and Ca2+ by 56% and 77%, respectively, and was completely inhibited by Zn2+, Fe3+ or urea solutions 2-8 M. MrAII displays differential cytotoxic activity: cancer cell lines K562, Jurkat, LnCap, and SCOV-3 were more sensitive to MrAII treatment, compared with normal cells. MrAII represents the first described enzyme of a large group of uncharacterized counterparts from the Chlorobi-Ignavibacteriae-Bacteroidetes clade.

A Novel L-Asparaginase from Hyperthermophilic Archaeon Thermococcus sibiricus: Heterologous Expression and Characterization for Biotechnology Application.

L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine and food industry. Drawbacks of current commercial L-ASNases stimulate the search for better-producing sources of the enzyme, and extremophiles are especially attractive in this view. In this study, a novel L-asparaginase originating from the hyperthermophilic archaeon Thermococcus sibiricus (TsA) was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 90 °C and pH 9.0 with a specific activity of 2164 U/mg towards L-asparagine. Kinetic parameters KM and Vmax for the enzyme are 2.8 mM and 1200 µM/min, respectively. TsA is stable in urea solutions 0-6 M and displays no significant changes of the activity in the presence of metal ions Ni2+, Cu2+, Mg2+, Zn2+ and Ca2+ and EDTA added in concentrations 1 and 10 mmol/L except for Fe3+. The enzyme retains 86% of its initial activity after 20 min incubation at 90 °C, which should be enough to reduce acrylamide formation in foods processed at elevated temperatures. TsA displays strong cytotoxic activity toward cancer cell lines K562, A549 and Sk-Br-3, while normal human fibroblasts WI-38 are almost unsensitive to it. The enzyme seems to be a promising candidate for further investigation and biotechnology application.

Mechanisms of the Antiproliferative and Antitumor Activity of Novel Telomerase-Carbonic Anhydrase Dual-Hybrid Inhibitors.

Human (h) telomerase (TL; EC 2.7.7.49) plays a key role in sustaining cancer cells by means of elongating telomeric repeats at the 3' ends of chromosomes. Since TL-inhibitor (TI) stand-alone cancer therapy has been proven to be remarkably challenging, a polypharmacological approach represents a valid alternative. Here we consider a series of compounds able to inhibit both hTL and the tumor-associated carbonic anhydrases (CAs; EC 4.2.1.1) IX and XII. Compounds 7 and 9 suppressed hTL activity in both cell lysates and human colon cancer cell lines, and prolonged incubation with either 7 or 9 resulted in telomere shortening, cell cycle arrest, replicative senescence, and apoptosis. Enzyme kinetics showed that 7 and 9 are mixed-type inhibitors of the binding of DNA primers and deoxynucleoside triphosphate (dNTP) to the TL catalytic subunit hTERT, which is in agreement with docking experiments. Compound 9 showed antitumor activity in Colo-205 mouse xenografts and suppressed telomerase activity by telomere reduction.

Penetration into Cancer Cells via Clathrin-Dependent Mechanism Allows L-Asparaginase from Rhodospirillum rubrum to Inhibit Telomerase.

The anticancer effect of L-asparaginases (L-ASNases) is attributable to their ability to hydrolyze L-asparagine in the bloodstream and cancer cell microenvironment. Rhodospirillum rubrum (RrA) has dual mechanism of action and plays a role in the suppression of telomerase activity. The aim of this work was to investigate the possible mechanism of RrA penetration into human cancer cells. Labeling of widely used L-ASNases by fluorescein isothiocyanate followed by flow cytometry and fluorescent microscopy demonstrated that only RrA can interact with cell membranes. The screening of inhibitors of receptor-mediated endocytosis demonstrated the involvement of clathrin receptors in RrA penetration into cells. Confocal microscopy confirmed the cytoplasmic and nuclear localization of RrA in human breast cancer SKBR3 cells. Two predicted nuclear localization motifs allow RrA to penetrate into the cell nucleus and inhibit telomerase. Chromatin relaxation promoted by different agents can increase the ability of RrA to suppress the expression of telomerase main catalytic subunit. Our study demonstrated for the first time the ability of RrA to penetrate into human cancer cells and the involvement of clathrin receptors in this process.

Inhibition of nuclease activity by a splice-switching oligonucleotide targeting deoxyribonuclease 1 mRNA prevents apoptosis progression and prolong viability of normal human CD4+ T-lymphocytes.

The nuclease activity of deoxyribonuclease 1 (DNase I) is regulated by alternative splicing (AS) of its mRNA. The aim of this study was to define the ability of a splice-switching oligonucleotide (SSO) that base-paired with DNase I pre-mRNA to induce AS and inhibit nuclease activity in human T, B and NK lymphocytes. The SSO for DNase I could significantly downregulate the expression of full-length active DNase I and upregulate a truncated splice variant with a deleted exon 4. Such an induction of AS resulted in inhibition of nuclease activity and slowed apoptosis progression in anti-CD95/FAS stimulated lymphocytes. These results should facilitate further investigations of apoptosis regulation in lymphocytes and demonstrate that SSOs for DNase I are promising cytoprotective agents.

FTIR-based L-asparaginase activity assay enables continuous measurements in optically dense media including blood plasma.

Complex heterogeneous systems, such as micelles or blood plasma, represent a particularly challenging environment to measure the catalytic parameters of some enzymes, including l-asparaginase. Existing methods are strongly interfered by the presence of plasma proteins, amino acids, as well as other components of plasma. Here we show that FTIR spectroscopy enables continuous real-time measurement of catalytic activity of l-asparaginase, in native and in PEG-chitosan conjugated form, in aqueous solutions as well as in heterogeneous non-transparent multicomponent systems, including colloidal systems or blood plasma, with minimal or no sample preparation. The approach developed is potentially applicable to other enzymatic reactions where the spectroscopic properties of substrate and product do not allow direct measurement with absorption or fluorescence spectroscopy.

Inhibition of telomerase activity by splice-switching oligonucleotides targeting the mRNA of the telomerase catalytic subunit affects proliferation of human CD4+ T lymphocytes.

Telomerase activity is regulated at the mRNA level by alternative splicing (AS) of its catalytic subunit hTERT. The aim of this study was to define the ability of splice-switching oligonucleotides (SSOs) that pair with hTERT pre-mRNA to induce AS and inhibit telomerase activity in human CD4+ T lymphocytes. SSOs that blocked the binding of a single splicing regulatory protein, SRp20 or SRp40, to its site within intron 8 of hTERT pre-mRNA demonstrated rather moderate capacities to induce AS and inhibit telomerase. However, a SSO that blocked the interaction of both SRp20 and SRp40 proteins with pre-mRNA was the most active. Cultivation of lymphocytes with spliced hTERT and inhibited telomerase resulted in the reduction of proliferative activity without significant induction of cell death. These results should facilitate further investigation of telomerase activity regulation, and antitelomerase SSOs could become promising agents for antiproliferative cell therapy.

Endonuclease G modulates the alternative splicing of deoxyribonuclease 1 mRNA in human CD4+ T lymphocytes and prevents the progression of apoptosis.

Apoptotic endonucleases act cooperatively to fragment DNA and ensure the irreversibility of apoptosis. However, very little is known regarding the potential regulatory links between endonucleases. Deoxyribonuclease 1 (DNase I) inactivation is caused by alternative splicing (AS) of DNase I pre-mRNA skipping exon 4, which occurs in response to EndoG overexpression in cells. The current study aimed to determine the role of EndoG in the regulation of DNase I mRNA AS and the modulation of its enzymatic activity. A strong correlation was identified between the EndoG expression levels and DNase I splice variants in human lymphocytes. EndoG overexpression in CD4+ T cells down-regulated the mRNA levels of the active full-length DNase I variant and up-regulated the levels of the non-active spliced variant, which acts in a dominant-negative fashion. DNase I AS was induced by the translocation of EndoG from mitochondria into nuclei during the development of apoptosis. The DNase I spliced variant was induced by recombinant EndoG or by incubation with EndoG-digested cellular RNA in an in vitro system with isolated cell nuclei. Using antisense DNA oligonucleotides, we identified a 72-base segment that spans the adjacent segments of exon 4 and intron 4 and appears to be responsible for the AS. DNase I-positive CD4+ T cells overexpressing EndoG demonstrated decreased progression towards bleomycin-induced apoptosis. Therefore, EndoG is an endonuclease with the unique ability to inactivate another endonuclease, DNase I, and to modulate the development of apoptosis.

Contact-independent suppressive activity of regulatory T cells is associated with telomerase inhibition, telomere shortening and target lymphocyte apoptosis.

Regulatory T cells (Tregs) play a fundamental role in the maintenance of immunological tolerance by suppressing effector target T, B and NK lymphocytes. Contact-dependent suppression mechanisms have been well-studied, though contact-independent Treg activity is not fully understood. In the present study, we showed that human native Tregs, as well as induced ex vivo Tregs, can cause in vitro telomere-dependent senescence in target T, B and NK cells in a contact-independent manner. The co-cultivation of target cells with Tregs separated through porous membranes induced alternative splicing of the telomerase catalytic subunit hTERT (human Telomerase Reverse Transcriptase), which suppressed telomerase activity. Induction of the hTERT splicing variant was associated with increased expression of the apoptotic endonuclease EndoG, a splicing regulator. Inhibited telomerase in target cells co-cultivated with Tregs for a long period of time led to a decrease in their telomere lengths, cell cycle arrest, conversion of the target cells to replicative senescence and apoptotic death. Induced Tregs showed the ability to up-regulate EndoG expression, TERT alternative splicing and telomerase inhibition in mouse T, B and NK cells after in vivo administration. The results of the present study describe a novel mechanism of contact-independent Treg cell suppression that induces telomerase inhibition through the EndoG-provoked alternative splicing of hTERT and converts cells to senescence and apoptosis phenotypes.

Murine regulatory T cells induce death of effector T, B, and NK lymphocytes through a contact-independent mechanism involving telomerase suppression and telomere-associated senescence.

Regulatory T cells (Tregs) suppress the activity of effector T, B and NK lymphocytes and sustain immunological tolerance, but the proliferative activity of suppressed cells remains unexplored. In the present study, we report that mouse Tregs can induce replicative senescence and the death of responder mouse CD4+CD25- T cells, CD8+ T cells, B cells and NK cells in vitro and in vivo. Contact-independent in vitro co-cultivation with Tregs up-regulated endonuclease G (EndoG) expression and its translocation to the nucleus in responder cells. EndoG localization in the nucleus induced alternative mRNA splicing of the telomerase catalytic subunit Tert and telomerase inhibition. The lack of telomerase activity in proliferating cells led to telomere loss followed by the development of senescence and cell death. Injection of Tregs into mice resulted in EndoG-associated alternative splicing of Tert, telomerase inhibition, telomere loss, senescence development and increased cell death in vivo. The present study describes a novel contact-independent mechanism by which Tregs specify effector cell fate and provides new insights into cellular crosstalk related to immune suppression.

Inhibition of telomerase activity and induction of apoptosis by Rhodospirillum rubrum L-asparaginase in cancer Jurkat cell line and normal human CD4+ T lymphocytes.

Rhodospirillum rubrum L-asparaginase mutant E149R, V150P, F151T (RrA) down-regulates telomerase activity due to its ability to inhibit the expression of telomerase catalytic subunit hTERT. The aim of this study was to define the effect of short-term and long-term RrA exposure on proliferation of cancer Jurkat cell line and normal human CD4+ T lymphocytes. RrA could inhibit telomerase activity in dose- and time-dependent manner in both Jurkat and normal CD4+ T cells. Continuous RrA exposure of these cells resulted in shortening of telomeres followed by cell cycle inhibition, replicative senescence, and development of apoptosis. Complete death of Jurkat cells was observed at the day 25 of RrA exposure while normal CD4+ T cells died at the day 50 due to the initial longer length of telomeres. Removal of RrA from senescent cells led to a reactivation of hTERT expression, restoration telomerase activity, re-elongation of telomeres after 48 h of cultivation, and survival of cells. These findings demonstrate that proliferation of cancer and normal telomerase-positive cells can be limited by continuous telomerase inhibition with RrA. Longer telomeres of normal CD4+ T lymphocytes make such cells more sustainable to RrA exposure that could give them an advantage during anti-telomerase therapy. These results should facilitate further investigations of RrA as a potent anti-telomerase therapeutic protein.

Alternative splicing of telomerase catalytic subunit hTERT generated by apoptotic endonuclease EndoG induces human CD4+ T cell death.

Telomerase activity is regulated by alternative splicing of its catalytic subunit human Telomerase Reverse Transcriptase (hTERT) mRNA. Induction of a non-active spliced hTERT leads to inhibition of telomerase activity. However, very little is known about the mechanism of hTERT mRNA alternative splicing. The aim of this study was to determine the role of the apoptotic endonuclease EndoG in alternative splicing of hTERT and telomerase activity. A strong correlation was identified between EndoG expression levels and hTERT splice variants in human CD4+ and CD8+ T lymphocytes. Overexpression of EndoG in CD4+ T cells down-regulated the expression of the active full-length hTERT variant and up-regulated expression of the non-active spliced variant. A reduction in full-length hTERT transcripts down-regulated telomerase activity. Long-term in vitro cultivation of EndoG-overexpressing CD4+ T cells led to dramatically shortened telomeres, conversion of cells into a replicative senescence state, and activation of the BCL2/BAX-associated apoptotic pathway finally leading to cell death. These data indicated the participation of EndoG in alternative mRNA splicing of the telomerase catalytic subunit hTERT, regulation of telomerase activity and determination of cell fate.

Rhodospirillum rubruml-asparaginase targets tumor growth by a dual mechanism involving telomerase inhibition.

Rhodospirillum rubruml-asparaginase mutant RrA E149R, V150P, F151T (RrA) was previously identified to down-regulate telomerase activity along with catalyzing the hydrolysis of l-asparagine. The aim of this study was to define the effect of prolonged RrA exposure on telomerase activity, maintenance of telomeres and proliferation of cancer cells in vitro and in vivo. RrA could inhibit telomerase activity in SCOV-3, SkBr-3 and A549 human cancer cell lines due to its ability to down-regulate the expression of telomerase catalytic subunit hTERT. Telomerase activity in treated cells did not exceeded 29.63 ± 12.3% of control cells. Continuous RrA exposure of these cells resulted in shortening of telomeres followed by cell death in vitro. Using real time PCR we showed that length of telomeres in SCOV-3 cells has been gradually decreasing from 10105 ± 2530 b.p. to 1233 ± 636 b.p. after 35 days of cultivation. RrA treatment of xenograft models in vivo showed slight inhibition of tumor growth accompanied with 49.5-53.3% of decrease in hTERT expression in the all tumors. However down-regulation of hTERT expression, inhibition of telomerase activity and the loss of telomeres was significant in response to RrA administration in xenograft models. These results should facilitate further investigations of RrA as a potent therapeutic protein.

Comparative immunogenicity and structural analysis of epitopes of different bacterial L-asparaginases.

BACKGROUND: E.coli type II L-asparaginase is widely used for treatment of acute lymphoblastic leukemia. However, serious side effects such as allergic or hypersensitivity reactions are common for L-asparaginase treatment. Methods for minimizing immune response on L-asparaginase treatment in human include bioengeneering of less immunogenic version of the enzyme or utilizing the homologous enzymes of different origin. To rationalize these approaches we compared immunogenicity of L-asparaginases from five bacterial organisms and performed sequence-structure analysis of the presumable epitope regions. METHODS: IgG and IgM immune response in C57B16 mice after immunization with Wollinella succinogenes type II (WsA), Yersinia pseudotuberculosis type II (YpA), Erwinia carotovora type II (EwA), and Rhodospirillum rubrum type I (RrA) and Escherichia coli type II (EcA) L-asparaginases was evaluated using standard ELISA method. The comparative bioinformatics analysis of structure and sequence of the bacterial L-asparaginases presumable epitope regions was performed. RESULTS: We showed different immunogenic properties of five studied L-asparaginases and confirmed the possibility of replacement of EcA with L-asparaginase from different origin as a second-line treatment. Studied L-asparaginases might be placed in the following order based on the immunogenicity level: YpA > RrA, WsA ≥ EwA > EcA. Most significant cross-immunogenicity was shown between EcA and YpA. We propose that a long N-terminus of YpA enzyme enriched with charged aminoacids and tryptophan could be a reason of higher immunogenicity of YpA in comparison with other considered enzymes. Although the recognized structural and sequence differences in putative epitope regions among five considered L-asparaginases does not fully explain experimental observation of the immunogenicity of the enzymes, the performed analysis set the foundation for further research in this direction. CONCLUSIONS: The performed studies showed different immunogenic properties of L-asparaginases and confirmed the possibility of replacement of EcA with L-asparaginase from different origin. The preferable enzymes for the second line treatment are WsA, RrA, or EwA.