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

2017 ◽  
Vol 63 (1) ◽  
pp. 62-74 ◽  
Author(s):  
M.V. Pokrovskaya ◽  
D.D. Zhdanov ◽  
M.A. Eldarov ◽  
S.S. Aleksandrova ◽  
A.V. Veselovskiy ◽  
...  
Keyword(s):  
Rhodospirillum Rubrum ◽  
Erwinia Carotovora ◽  
Telomerase Activity ◽  
Jurkat Cells ◽  
Site Directed Mutagenesis ◽  
Leukemic Cells ◽  
Type I ◽  
Wolinella Succinogenes ◽  
Stable Mutant ◽  
Mutant Forms

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.

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

2017 ◽  
Vol 11 (3) ◽  
pp. 219-233 ◽  
Author(s):  
M. V. Pokrovskaya ◽  
D. D. Zhdanov ◽  
M. A. Eldarov ◽  
S. S. Aleksandrova ◽  
A. V. Veselovsky ◽  
...  
Keyword(s):  
Rhodospirillum Rubrum ◽  
Telomerase Activity ◽  
Leukemic Cells ◽  
Mutant Forms

scholarly journals Antitumor activity of L-asparaginase from Erwinia Carotovora from against different leukemic and solid tumours cell lines

2013 ◽  
Vol 59 (5) ◽  
pp. 498-513 ◽  
Author(s):  
O.Yu. Abakumova ◽  
O.V. Podobed ◽  
P.A. Karalkin ◽  
L.I. Kondakova ◽  
N.N. Sokolov
Keyword(s):  
Dna Synthesis ◽  
Tumor Cells ◽  
Solid Tumor ◽  
Human Fibroblasts ◽  
Erwinia Carotovora ◽  
Cell Number ◽  
Jurkat Cells ◽  
Leukemic Cells ◽  
Cycle Phase ◽  
Tumor Cell Death

We have studied dose- and time-dependent antitumor and cytotoxic effects of Erwinia carotovora L-asparaginase (ECAR LANS) and Escherichia coli L-asparaginase (MEDAC) on human leukemic cells and human and animal solid tumor cells. We determined the sensitivity of tumor cells to L-asparaginases, as well the effect L-asparaginases on cell growth rate, protein and DNA synthesis per se and with addition of different cytostatics. The data obtained demonstrated that ECAR LANS L-asparaginase suppressed growth of all tested solid tumor cells. Evaluation of leukemic cell number after treatment with L-asparaginases for 24, 48 and 72 h demonstrated that asparagine deficiency did not kill cells but stopped normal cell division and had no effect on protein and DNA synthesis. Cytofluorometric study of solid and leukemic cells demonstrated that the treatment with L-asparaginase for 72 h did not change cell cycle phase distribution and did not increase the number of apoptotic cells. The HL-60 cell line was only exemption. At the same time, cells treatment with L-asparaginase and doxorubicin combination leaded to increase of apoptotypical cell number to 60% for MCF7 cells, to 40% for Jurkat cells and to 99% for HL-60 cells. We have excluded apoptosis as main reason for tumor cell death after asparaginase treatment because multi resistant Jurkat/A4 cells have been asparaginase sensitive. We have not found ECAR LANS L-asparaginase effect on normal human fibroblasts growth ability and we had come to conclusion that enzyme cytotoxcisity related only with asparagine deficiency.

scholarly journals Physical-Chemical Properties of L-asparaginase Mutants From Rhodospirillum Rubrum which Showed Antitelomerase Activity

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
M.V. Pokrovskaya ◽  
S.S. Aleksandrova ◽  
A.V. Veselovsky ◽  
D.D. Zdanov ◽  
V.S. Pokrovsky ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rhodospirillum Rubrum ◽  
Chemical Properties ◽  
Telomerase Activity ◽  
Kinetic Properties ◽  
Ph Optimum ◽  
Physical Chemical ◽  
Asparaginase Activity ◽  
Terminal Amino ◽  
Mutant Forms

Rru_A3730 protein is a bacterial Rhodospirillum rubrum L-asparaginase (RrA), which is known by its anticancer activity. RrA variants with point amino acid substitutions in the region of 150 amino acids residues: RrA17N, K149E, RrAE149R, V150P, F151T, RrА17N, E149R, V150P, RrAE149R, V150P, showed antiproliferative properties, and also by their ability to suppress telomerase activity. This work is devoted to comparison of physical-chemical and catalytic properties of these mutant forms of RrA. It is shown that pH optimum is in the alkaline zone (8.5 – 9.3); L-glutaminase and D-asparaginase activity is respectively not more than 0.1% and 1.6% of L-asparaginase for all studied variants of RrA. The presence of the N17-terminal amino acid sequence MASMTGGQMGRGSSRQ of the capsid protein of bacteriophage T7 in the RrA structure leads to an increase in the thermal stability of mutant RrA analogues (from 50°C to 56°C) and their resistance to denaturation in the presence of 3 – 4 M urea. It is of Metal ions exhibit multidirectional effects on L-asparaginase activity of RrA. K+, Ca2+, Zn2+, Cs+, Co2+ in significantly affect the activity of L-asparaginase, while Mn2+, Cu2+, Fe3+ ions inhibit it. There was no correlation between antitelomerase (antiproliferative) activity and kinetic properties of mutant forms of L-asparaginase RrA.

scholarly journals Evidence for the interleukin-2 dependent expansion of leukemic cells in adult T cell leukemia

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1407-1411 ◽  
Author(s):  
M Maeda ◽  
N Arima ◽  
Y Daitoku ◽  
M Kashihara ◽  
H Okamoto ◽  
...  
Keyword(s):  
T Cell ◽  
Receptor Gene ◽  
Interleukin 2 ◽  
Leukemic Cells ◽  
Type I ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
In Vitro Proliferation ◽  
Adult T Cell Leukemia

Abstract Interleukin 2 (IL-2) receptor/Tac antigen is abnormally expressed on cells of patients with adult T cell leukemia (ATL) caused by infection with human T lymphotropic virus type I (HTLV-I). Twenty-five patients with ATL were examined to determine whether their leukemic cells continued to show IL-2-dependent proliferation. In 21 patients, the in vitro proliferation of HTLV-I-infected nonleukemic T cell clones was found to be dependent on IL-2. However, clonality analysis based on T cell receptor gene rearrangement profiles and the site of HTLV-I provirus integration revealed IL-2-dependent growth in leukemic cells in four patients with ATL. These results provide evidence for the IL-2- dependent proliferation of leukemic cells in some ATL patients.

scholarly journals Color-tuning of natural variants of heliorhodopsin

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Se-Hwan Kim ◽  
Kimleng Chuon ◽  
Shin-Gyu Cho ◽  
Ahreum Choi ◽  
Seanghun Meas ◽  
...  
Keyword(s):  
3D Structure ◽  
Site Directed Mutagenesis ◽  
Type I ◽  
Transmembrane Helices ◽  
Color Tuning ◽  
Tuning Mechanism ◽  
Spectral Changes ◽  
His Tag ◽  
Natural Variants ◽  
Retinal Chromophore

AbstractMicrobial rhodopsins are distributed through many microorganisms. Heliorhodopsins are newly discovered but have an unclear function. They have seven transmembrane helices similar to type-I and type-II rhodopsins, but they are different in that the N-terminal region of heliorhodopsin is cytoplasmic. We chose 13 representative heliorhodopsins from various microorganisms, expressed and purified with an N-terminal His tag, and measured the absorption spectra. The 13 natural variants had an absorption maximum (λmax) in the range 530–556 nm similar to proteorhodopsin (λmax = 490–525 nm). We selected several candidate residues that influence rhodopsin color-tuning based on sequence alignment and constructed mutants via site-directed mutagenesis to confirm the spectral changes. We found two important residues located near retinal chromophore that influence λmax. We also predict the 3D structure via homology-modeling of Thermoplasmatales heliorhodopsin. The results indicate that the color-tuning mechanism of type-I rhodopsin can be applied to understand the color-tuning of heliorhodopsin.

scholarly journals Molecular cloning of a novel human cDNA encoding a zinc finger protein that binds to the interleukin-3 promoter.

1994 ◽  
Vol 14 (8) ◽  
pp. 5099-5107 ◽  
Author(s):  
N Koyano-Nakagawa ◽  
J Nishida ◽  
D Baldwin ◽  
K Arai ◽  
T Yokota
Keyword(s):  
T Cell ◽  
Zinc Finger ◽  
Transcriptional Activity ◽  
Regulatory Region ◽  
Jurkat Cells ◽  
Type I ◽  
Dependent Manner ◽  
Cell Leukemia Virus Type ◽  
Transcription Activity ◽  
Interleukin 3

The CT/GC-rich region (-76 to -47) is one transcriptional regulatory region of the interleukin-3 (IL-3) gene which confers basic transcriptional activity and responds to trans-activation by human T-cell leukemia virus type I-encoded Tax. We isolated three types of cDNAs encoding Cys2/His2-type zinc finger proteins that bind to this region. Two were identical to known transcription factors, EGR1 and EGR2, and the other clone, named DB1, encoded a novel protein of 516 amino acids with six zinc finger motifs. DB1 mRNA was present in human tissues, ubiquitously. Two constitutive transcripts of 4.0 and 4.8 kb in length were present in Jurkat cells. Electrophoretic mobility shift assay, with specific antibodies, showed that DB1 constitutively binds to this region whereas EGR1 binds in a T-cell activation-dependent manner. Overexpression of DB1 in Jurkat cells had no detectable effect on the transcription activity of the IL-3 promoter, in a transient-transfection assay. EGR1 and EGR2 increased IL-3 promoter activity when the transfected cells were stimulated with phorbol-12-myristate-13-acetate and A23187. When DB1 was cotransfected with a Tax expression vector, transcription activity of the IL-3 promoter induced by Tax was significantly increased, while EGR1 and EGR2 were without effect. These results suggest that EGR1 has a role in inducible transcription of the IL-3 gene, while DB1 sustains basal transcriptional activity and also cooperates with Tax to activate the IL-3 promoter.

scholarly journals Cisplatin-induced apoptotic endonuclease EndoG inhibits telomerase activity and causes malignant transformation of human CD4+ T lymphocytes

2017 ◽  
Vol 63 (1) ◽  
pp. 13-26 ◽  
Author(s):  
D.D. Zhdanov ◽  
D.A. Vasina ◽  
E.V. Orlova ◽  
V.S. Orlova ◽  
V.S. Pokrovsky ◽  
...  
Keyword(s):  
T Cells ◽  
Alternative Splicing ◽  
Malignant Transformation ◽  
Cd4 T Cells ◽  
Replicative Senescence ◽  
Telomerase Activity ◽  
Full Length ◽  
Leukemic Cells ◽  
Proliferative Potential ◽  
Human Telomerase Reverse Transcriptase

Alternative splicing of telomerase catalytic subunit hTERT pre-mRNA (human Telomerase Reverse Transcriptase) regulates telomerase activity. Increased expression of non-active splice variant hTERT results in inhibition of telomerase. Apoptotic endonuclease EndoG is known to participate in hTERT alternative splicing. Expression of EndoG can be induced in response to DNA damages. The aim of this study was to determine the ability of a DNA-damaging compound, cisplatin, to induce EndoG and its influence on alternative splicing of hTERT and telomerase activity in human CD4+ Т lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of active full-length hTERT variant and upregulated its non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. Few cells survived and underwent malignant transformation. Transformed cells have increased telomerase activity and proliferative potential compare to initial CD4+ T cells. These cells have phenotype of T lymphoblastic leukemic cells and are able to form tumors and cause death in experimental mice.

scholarly journals Interferon-inducible miR-128 modulates HIV-1 replication by targeting TNPO3 mRNA

2017 ◽  
Author(s):  
Aurore Bochnakian ◽  
Dimitrios G Zisoulis ◽  
Adam Idica ◽  
Anjie Zhen ◽  
Vineet N KewalRamani ◽  
...  
Keyword(s):  
T Cells ◽  
Nuclear Import ◽  
Cd4 T Cells ◽  
Jurkat Cells ◽  
Type I ◽  
Coding Region ◽  
Aids Pandemic ◽  
Mrna And Protein Expression ◽  
Dual Mechanism ◽  
Hiv 1

ABSTRACTThe HIV/AIDS pandemic remains an important threat to human health. We have recently demonstrated that a novel microRNA (miR-128) represses retrotransposon (LINE-1 or L1) by a dual mechanism, by directly targeting the coding region of the L1 RNA and by repressing a required nuclear import factor (TNPO1). We have further determined that miR-128 represses the expression of all three isoforms of TNPO proteins (transportins, TNPO1,-2 and TNPO3). Here, we establish that miR-128 also controls HIV-1 replication by repressing TNPO3. TNPO3 is well established to regulate HIV-1 nuclear import and viral replication. Here, we report that the type I interferon inducible miR-128 directly targets two sites in the TNPO3 mRNA, significantly down-regulating TNPO3 mRNA and protein expression levels. Manipulation of miR-128 levels in HIV target cell lines and in primary human CD4 T-cells by over-expression or knockdown showed that modulation of TNPO3 by miR-128 affects HIV-1 replication but not MLV infection. In addition, we found that miR-128 modulation of HIV-1 replication is reduced with TNPO3-independent HIV-1 virus and in cells depleted of CPSF6, suggesting that miR-128-indued TNPO3 repression is partly required for miR-128-induced inhibition of HIV-1 replication. Finally, challenging miR-modulated Jurkat cells or primary CD4 T-cells with wildtype, replication-competent HIV-1 shows that miR-128 significantly delays spreading infection. Thus, we have established a novel role of miR-128 in anti-viral defense in human cells, inhibiting HIV-1 replication partly by targeting TNPO3.

scholarly journals Functional Importance of Hydrophobic Patches on the Ebola Virus VP35 IFN-Inhibitory Domain

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2316
Author(s):  
Nodoka Kasajima ◽  
Keita Matsuno ◽  
Hiroko Miyamoto ◽  
Masahiro Kajihara ◽  
Manabu Igarashi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ebola Virus ◽  
Structural Information ◽  
Site Directed Mutagenesis ◽  
Genome Replication ◽  
Type I ◽  
Multifunctional Protein ◽  
Functional Sites ◽  
Inhibitory Function ◽  
Inhibitory Domain

Viral protein 35 (VP35) of Ebola virus (EBOV) is a multifunctional protein that mainly acts as a viral polymerase cofactor and an interferon antagonist. VP35 interacts with the viral nucleoprotein (NP) and double-stranded RNA for viral RNA transcription/replication and inhibition of type I interferon (IFN) production, respectively. The C-terminal portion of VP35, which is termed the IFN-inhibitory domain (IID), is important for both functions. To further identify critical regions in this domain, we analyzed the physical properties of the surface of VP35 IID, focusing on hydrophobic patches, which are expected to be functional sites that are involved in interactions with other molecules. Based on the known structural information of VP35 IID, three hydrophobic patches were identified on its surface and their biological importance was investigated using minigenome and IFN-β promoter-reporter assays. Site-directed mutagenesis revealed that some of the amino acid substitutions that were predicted to disrupt the hydrophobicity of the patches significantly decreased the efficiency of viral genome replication/transcription due to reduced interaction with NP, suggesting that the hydrophobic patches might be critical for the formation of a replication complex through the interaction with NP. It was also found that the hydrophobic patches were involved in the IFN-inhibitory function of VP35. These results highlight the importance of hydrophobic patches on the surface of EBOV VP35 IID and also indicate that patch analysis is useful for the identification of amino acid residues that directly contribute to protein functions.

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