Recombinant intracellular Rhodospirillum rubrum L-asparaginase with low L-glutaminase activity and antiproliferative effect

Pokrovskaya, M. V.; Pokrovskiy, V. S.; Александрова, С. С.; Anisimova, N. Yu.; Андрианов, Р. М.; Treschalina, E. M.; Ponomarev, G. V.; Соколов, Н. Н.

doi:10.18097/pbmc20135902192

Cited by 18 publications

(12 citation statements)

References 23 publications

(11 reference statements)

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“…Штаммы E. coli: ХL-blue ("Stratagen", Германия) и BL21(DE3) ("Novagen", США) трансформированные плазмидой pET23 ("Invitrogen", США), несущей ген RrA, культивировали согласно условиям описанным ранее [7,8]. Индукцию синтеза рекомбинантной RrA осуществляли добавлением лактозы к экспрессирующей культуре при A 600~1 ,5 до конечной концентрации 0,2%.…”

Section: бактериальные штаммы и условия культивированияunclassified

“…Очистку рекомбинантной L-аспарагиназы RrА и её мутантных форм RrА +N17, D60K, F61L , RrА +N17, A64V, E67K , RrА +N17, E149R, V150P , RrA E149R, V150P и RrA E149R, V150P, F151T проводили как описано ранее [7,8]…”

Section: выделение L-аспарагиназunclassified

“…Ген RruA3730, клонированный в вектор pET23 (рис. 1А) между сайтами рестрикции HindIII и BamHI кодирует составной белок RrA +N17 с дополнительной N-концевой аминокислотной последовательностью MASMTGGQQMGRGSSRQ (N17) [7,8], включающей лидирующий пептид из бактериофага Т7 гена 10 (T7 tag). Присутствие T7 tag может быть использовано для мечения, очистки и характеристики белков, реакций иммуноблоттинга, иммунопреципитации и иммуноокрашивающих технологий [29].…”

Section: получение мутантных форм Rraunclassified

“…Присутствие T7 tag может быть использовано для мечения, очистки и характеристики белков, реакций иммуноблоттинга, иммунопреципитации и иммуноокрашивающих технологий [29]. Наши предварительные результаты показали, что составной RrA +N17 сохраняет каталитическую активность [7,8].…”

Section: получение мутантных форм Rraunclassified

“…Ранее нами была выделена и охарактеризована рекомбинантная L-аспарагиназа RrA, отличающаяся небольшой молекулярной массой субъединицы (18 кДa; мономер содержит 172 аминокислотных остатка), низкой гомологией с EcA и EwA. Этот фермент нетоксичен и его L-глутаминазная активность составляет менее 0,01% от L-аспарагиназной [7]. RrA является первой цитоплазматической L-аспарагиназой I типа, обладающей антипролиферативной активностью, свойственной, как считалось ранее, только периплазматическим аспарагиназам II типа (EcA и EwA и др.).…”

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Suppression of telomerase activity leukemic cells by mutant forms of Rhodospirillum rubrum L-asparaginase

Покровская

Zhdanov

Eldarov³

et al. 2017

BIOMED KHIM

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The active and stable mutant forms of short chain cytoplasmic L-asparaginase type I of Rhodospirillum rubrum (RrA): RrA+N17, D60K, F61L, RrA+N17, A64V, E67K, RrA+N17, E149R, V150P, RrAE149R, V150P and RrAE149R, V150P, F151T were obtained by the method of site-directed mutagenesis. It is established that variants RrA-N17, E149R, V150P, F151T and RrАE149R, V150P are capable to reduce an expression hTERT subunit of telomerase and, hence, activity of telomeres in Jurkat cells, but not in cellular lysates. During too time, L-asparaginasеs of Escherichia coli, Erwinia carotovora and Wolinella succinogenes, mutant forms RrА+N17, D60K, F61L and RrА+N17, A64V, E67K do not suppress of telomerase activity. The assumption of existence in structure RrA of areas (amino acids residues in the position 146-164, 1-17, 60-67) which are responsible for suppression of telomerase activity is made. The received results show that antineoplastic activity of some variants RrA is connected both with reduction of concentration of free L-asparagine, and with expression suppression of hTERT telomerase subunit, that opens new prospects for antineoplastic therapy.

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Section: бактериальные штаммы и условия культивированияunclassified

Section: выделение L-аспарагиназunclassified

Section: получение мутантных форм Rraunclassified

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Suppression of telomerase activity leukemic cells by mutant forms of Rhodospirillum rubrum L-asparaginase

Покровская

Zhdanov

Eldarov³

et al. 2017

BIOMED KHIM

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

et al. 2017

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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.

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Gene cloning, characterization, and cytotoxic activity of methionine γ‐lyase from Clostridium novyi

et al. 2017

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The exploitation of methionine-depleting enzyme methionine γ-lyase (MGL) is a promising strategy against specific cancer cells that are strongly dependent on methionine. To identify MGL from different sources with high catalytic activity and efficient anticancer action, we have expressed and characterized MGL from Clostridium novyi and compared its catalytic efficiency with the previously studied MGL from Citrobacter freundii. The purified recombinant MGL exhibits k and k /K for methionine γ-elimination reaction that are 2.4- and 1.36-fold higher than C. freundii enzyme, respectively, whereas absorption, fluorescence, and circular dichroism spectra are very similar, as expected on the basis of 87% sequence identity and high conservation of active site residues. The reactivity of cysteine residues with DTNB and iodoacetamide was investigated as well as the impact of their chemical modification on catalytic activity. This information is relevant because for increasing bioavailability and reducing immunogenity, MGL should be decorated with polyethylene glycol (PEG). It was found that Cys118 is a faster reacting residue, which results in a significant decrease in the γ-elimination activity. Thus, the protection of Cys118 before conjugation with cysteine-reacting PEG represents a valuable strategy to preserve MGL activity. The anticancer action of C. novyi MGL, evaluated in vitro against prostate (PC-3), chronic myelogenous leucemia (K562), and breast (MDA-MB-231 and MCF7) cancer cells, exhibits IC of 1.3 U mL , 4.4 U mL , 1.2 U mL , and 3.4 U mL , respectively. A higher cytotoxicity of C. novyi MGL was found against cancer cells with respect to C. freundii MGL, with the exception of PC-3, where a lower cytotoxicity was observed. © 2017 IUBMB Life, 69(9):668-676, 2017.

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Recombinant intracellular Rhodospirillum rubrum L-asparaginase with low L-glutaminase activity and antiproliferative effect

Cited by 18 publications

References 23 publications

Suppression of telomerase activity leukemic cells by mutant forms of Rhodospirillum rubrum L-asparaginase

Suppression of telomerase activity leukemic cells by mutant forms of Rhodospirillum rubrum L-asparaginase

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

Gene cloning, characterization, and cytotoxic activity of methionine γ‐lyase from Clostridium novyi

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