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TURAN, KADİR

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Eczacılık Fakültesi

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TURAN

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KADİR

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Open Access
Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome
(OXFORD UNIV PRESS, 2004-01-21) TURAN, KADİR; Turan, K; Mibayashi, M; Sugiyama, K; Saito, S; Numajiri, A; Nagata, K
Mx proteins belong to the dynamin superfamily of high molecular weight GTPases and interfere with multiplication of a wide variety of viruses. Earlier studies show that nuclear mouse Mx1 and human MxA designed to be localized in the nucleus inhibit the transcription step of the influenza virus genome. Here we set a transient influenza virus transcription system using luciferase as a reporter gene and cells expressing the three RNA polymerase subunits, PB1, PB2 and PA, and NP. We used this reporter assay system and nuclear-localized MxA proteins to get clues for elucidating the anti-influenza virus activity of MxA. Nuclear-localized VP16-MxA and MxA-TAg NLS strongly interfered with the influenza virus transcription. Over-expression of PB2 led to a slight resumption of the transcription inhibition by nuclear MxA, whereas over-expression of PB1 and PA did not affect the MxA activity. Of interest is that the inhibitory activity of the nuclear MxA was markedly neutralized by over-expression of NP. An NP devoid of its C-terminal region, but containing the N-terminal RNA binding domain, also neutralized the VP16-MxA activity in a dose-dependent manner, whereas an NP lacking the N-terminal region did not affect the VP16-MxA activity. Further, not only VP16-MxA but also the wild-type MxA was found to interact with NP in influenza virus-infected cells. This indicates that the nuclear MxA suppresses the influenza virus transcription by interacting with not only PB2 but also NP.
Open Access
Heterologous expression and initial in silico characterization of a novel snakin-Z peptide
(2023-08-01) TURAN, KADİR; Teker T., Albayrak G., Turan K.
Heterologous expression of the plant-derived snakin-Z (SNK-Z) peptide was carried out inEscherichia coliBL21 and Mach 1 strains using the pET14b, and pGEX-6P-1 vectors, and its tertiary structure was determined. The most efficient production of the recombinant fusion peptide (GST/SNK-Z) in both strains was achieved by induction with 0.1 mM isopropyl β-D-1-thiogalactopyranoside at 32°C. GST/SNK-Z with 30.14kDa which yielded 6.5mg/L protein, was purified by affinity chromatography followed by dialysis. The GST tags were removed using PreScission protease. The IC50of GST/SNK-Z was calculated as 12.07 µM forStaphylococcus aureus(ATCC 25923) using nonlinear regression analysis. The secondary structure of recombinant SNK-Z consisted of α-helix and coil sites. It’s 3-D model was generated with a confidence score of -0.20 and a template modeling score of 0.69 ± 0.12. The predicted solvent accessibility and B-factor profile were detected.This is the first report of heterologous expression of SNK-Z inE. coli. Our study provides fundamental data for the large-scale production of SNK-Z, which has a high potential for use in the pharmaceutical industry because of its previously reported antimicrobial effects.
Open Access
Human sorting nexin 2 protein interacts with Influenza A virus PA protein and has a negative regulatory effect on the virus replication
(SPRINGER, 2022-01) TURAN, KADİR; Kocmar, Tugba; Caglayan, Elif; Rayaman, Erkan; Nagata, Kyosuke; Turan, Kadir
Background Replication of the influenza A viruses occurs in the cells through the viral RdRP consisting of PB1, PB2, and PA. Several cellular proteins are involved in these processes. This study aims to reveal the interaction between human SNX2 protein and the PA protein and the effects of the SNX2 on the virus replication. Results To identify potential host interacting proteins to the PA, yeast two-hybrid assay was carried out with HEK293 cell cDNA library and the PA as a bait. We focused on SNX2 protein, which interacts with the PA in the yeast cells. By using the co-immunoprecipitation assays, it has been demonstrated that the amino-terminal part of the PA was important for binding to the SNX2. Immunolocalization of the proteins in HeLa cells supported this interaction. Knockdown of the SNX2 with siRNA in the cells resulted in a significant increase in both viral transcripts and virus growth. However, the increase of SNX2 in transfected cells didn't cause a significant change in the viral RdRP activity in minireplicon assay. This may suggest that the negative effect of SNX2 on the virus replication could be saturated with its authentic intra-cellular amount. Conclusions This study revealed that the SNX2 and PA protein interact with each other in both yeast and HEK293 cells, and the SNX2 has a negative regulatory function on the virus replication. However, more knowledge is required to elucidate the action mechanism of the SNX2 on the influenza A virus replication at the molecular level.
Open Access
Effects of intra- and extracellular factors on anti-aging klotho gene expression
(FUNPEC-EDITORA, 2011) TURAN, KADİR; Turan, K.; Ata, P.
Inactivation of the klotho gene in mice causes serious systemic disorders, resembling human aging. However, at the molecular level, its action mechanisms are not well understood. The stimulatory or inhibitory effects of cis- and trans-regulatory factors on the klotho gene expression are also still unclear. We studied the effects of intra- and extracellular factors on human klotho gene expression. For this purpose, pHKP-Luc and pHKP-GFP reporter vectors were constructed with the 2.1-kbp upstream region of human klotho, covering its promoter region, using luciferase and GFP genes as the reporter. A series of vectors that have deletions in the upstream region of the klotho gene were constructed to assay cis-acting factors. Deletion of some parts of the klotho gene upstream region significantly affected reporter gene expression in HEK293 cells. p16 and p53 proteins inhibited reporter luciferase expression under the control of human klotho promoter in a dose-dependent manner. Calcium and phosphate ions stimulated klotho expression. p21, PTH, IGF-1, and angiotensin-II had no significant effect on klotho expression in HEK293 cells.
Open Access
An in silico prediction of interaction models of influenza A virus PA and human C14orf166 protein from yeast-two-hybrid screening data
(2023-06-02) TURAN, KADİR; Çağlayan E., Turan K.
The human C14orf166 protein, also known as RNA transcription, translation, and transport factor, shows positive modulatory activity on the cellular RNA polymerase II enzyme. This protein is a component of the tRNA-splicing ligase complex and is involved in RNA metabolism. It also functions in the nucleo-cytoplasmic transport of RNA molecules. The C14orf166 protein has been reported to be associated with some types of cancer. It has been shown that the C14orf166 protein binds to the influenza A virus RNA polymerase PA subunit and has a stimulating effect on viral replication. In this study, candidate interactor proteins for influenza A virus PA protein were screened with a Y2H assay using HEK293 Matchmaker cDNA. The C14orf166 protein fragments in different sizes were found to interact with the PA. The three-dimensional structures of the viral PA and C14orf166 proteins interacting with the PA were generated using the I-TASSER algorithm. The interaction models between these proteins were predicted with the ClusPro protein docking algorithm and analyzed with PyMol software. The results revealed that the carboxy-terminal end of the C14orf166 protein is involved in this interaction, and it is highly possible that it binds to the carboxyterminal of the PA protein. Although amino acid residues in the interaction area of the PA protein with the C14orf166 showed distribution from 450th to 700th position, the intense interaction region was revealed to be at amino acid positions 610–630.
Open Access
Effects of some interferon-related proteins on influenza a viruse RNA polymerase activity
(2022-01-01) TURAN, KADİR; Çağlayan E., TURAN K.
© 2022, Turkish Pharmacists Association. All rights reserved.Objectives: Interferons (IFNs) are one of the most important components of innate immunity against viruses, especially those carrying the RNA genomes such as influenza viruses. Upon viral infection, the IFNs are rapidly secreted, inducing the expression of several genes in the target cells and establishing an antiviral state. In this study, the effects of proteins encoded by some IFN-related genes on influenza A virus RNA-dependent RNA polymerase enzyme were investigated. We evaluated the importance of these proteins in the pathogenesis of different influenza A virus types. Materials and Methods: The IFN-related genes were amplified by polymerase chain reaction from the HEK293 cDNA library and cloned into pCHA expression vector. The expression of genes and subcellular localizations of the proteins were determined by Western blotting and immunofluorescence staining, respectively. The effects of IFNs-related proteins on virus RdRP enzyme were determined by influenza A virus mini-replicons. Results: The study revealed that the influenza A virus infections significantly altered the transcript level of the IFN-related CCL5, IFIT1, IFIT3, IFITM3, and OAS1 genes in HEK293 cells. It was determined that the alteration of the gene expression was also related to the virus type. The mini-replicon assays showed that the transient expression of CCL5, IFI27, OAS1, IFITM3, IFIT1, and IFIT3 have inhibitory effects on WSN and/or DkPen type virus RdRP enzymes. We observed that the proteins except OAS1 inhibited WSN type RdRP enzyme at a higher level than that of DkPen enzyme. Conclusion: It was concluded that influenza A virus infection significantly alters the IFN-related gene expression in the cells. Most of the proteins encoded from these genes showed an inhibitory effect on the virus RdRP enzymes in the HEK293 cells. The inhibition of the influenza virus RdRP with IFN-related proteins may be the result of direct or indirect interactions between the host proteins and the viral enzyme subunits.
Open Access
A SIMPLIFIED METHOD FOR THE EXTRACTION OF RECOMBINANT TAQ DNA POLYMERASE FROM ESCHERICHIA COLI
(SLOVAK UNIV AGRICULTURE NITRA, 2018-04-01) TURAN, KADİR; Turan, Kadir; Eken, Burak
DNA polymerase (Taq) enzyme isolated from Thermus aquaticus, a thermostable gram-negative bacterium, is a basic component of PCR, widely used in life sciences. The extraction and purification of this enzyme involves time-consuming and expensive steps such as precipitation of proteins with PEI and/or ammonium sulfate, column chromatography techniques, and removal of salts or other small molecular weight contaminants by dialysis. In this work, a novel and simplified method for extraction and purification of the recombinant Taq polymerase from Escherichia coli was employed, which used cold acetone instead of PEI or ammonium sulfate to precipitate the enzyme. The enzyme was efficiently recovered as active form from both the crude cell lysate and column fractions with cold acetone precipitation. This simplified method enabled us to obtain high quality Taq DNA polymerase in a much shorter time and at a lower cost.
Open Access
Human DDX56 protein interacts with influenza A virus NS1 protein and stimulates the virus replication
(SOC BRASIL GENETICA, 2021) TURAN, KADİR; Pirincal, Aysegul; Turan, Kadir
Influenza A viruses (IAV) are enveloped viruses carrying a single-stranded negative-sense RNA genome. Detection of host proteins having a relationship with IAV and revealing of the role of these proteins in the viral replication are of great importance in keeping IAV infections under control. Consequently, the importance of human DDX56, which is determined to be associated with a viral NS1 with a yeast two-hybrid assay, was investigated for IAV replication. The viral replication in knocked down cells for the DDX56 gene was evaluated. The NS1 was co-precipitated with the DDX56 protein in lysates of cells transiently expressing DDX56 and NS1 or infected with the viruses, showing that NS1 and DDX56 interact in mammalian cells. Viral NS1 showed a tendency to co-localize with DDX56 in the cells, transiently expressing both of these proteins, which supports the IP and two-hybrid assays results. The data obtained with in silico predictions supported the in vitro protein interaction results. The viral replication was significantly reduced in the DDX56-knockdown cells comparing with that in the control cells. In conclusion, human DDX56 protein interacts with the IAV NS1 protein in both yeast and mammalian cells and has a positive regulatory effect on IAV replication. However, the mechanism of DDX56 on IAV replication requires further elucidation.
Open Access
Mutations in the SARS CoV2 spike gene and their reflections on the spike protein
(2022-06-01) TURAN, KADİR; Caglayan E., TURAN K.
Objective: In this study, it was aimed to determine the mutation frequency in spike (S) genes of SARS CoV2 from six different regions of the world, their distribution on the gene and reflections of these mutations to the S protein. Materials and methods: SARS CoV2 S gene sequences originating from Asia, Africa, Europe, South America, Oceania and North America were obtained from NCBI virus database. The sequences were analyzed with Geneious and BioEdit multiple sequence alignment programs. Results and Conclusion: 865 distinct mutations on the S genes were detected in the virus samples. Among these, 59 variants with numbers of 10 and above in the virus population were detected. The D614G(A1841G) substitution was found to be the most common with an average of 88.6%. Furthermore, it was determined that S477N(G1430A) substitution in theviruses of Oceania differed from other regions with a rate of 86.7%. The average mutation frequency of the S genes from different regions was calculated as 4x10-5. The amino acid substitutions particularly in the RBD (receptor binding domain) have great importance for the virus adsorption to the cells via ACE2 (angiotensin converting enzyme 2) receptor and transmission. However, the importance of these mutations needs to be demonstrated both in silico and experimental studies.
Open Access
Psychometric properties of the Turkish version of the fear avoidance components scale in patients with chronic pain related to musculoskeletal disorders
(2023-01-01) TURAN, KADİR; SARI, ZÜBEYİR; TURAN K., SARI Z., Özden F.
Background: The fear avoidance components scale (FACS) evaluates patients’ cognitive, emotional and behavioral fear avoidance behavior. The aim of the study was to conduct the cross-cultural adaptation, reliability and validity of the Turkish version of the FACS. Methods: A prospective cross-sectional study was carried out with 208 patients (46.2 ± 11.4 years, 116 women, 92 men) diagnosed with chronic pain related to musculoskeletal disorders. Individuals were assessed with FACS, Tampa scale of kinesiophobia (TSK), Beck depression inventory (BDI), Oswestry disability index (ODI), numerical pain scale (NPS), and pain catastrophizing scale (PCS). A total of 70 patients completed the FACS for the second time 3 days later. Results: Internal consistency of the total score was excellent (Cronbach’s alpha: 0.815). There was a strong correlation between FACS and TSK and PCS (r1 0.555, r2 0.678, p < 0.001). In addition, the relationship between FACS and BDI and NPS was moderate in terms of construct validity (r1 0.357, r2 0.391, p < 0.001). FACS had a two-factor structure, as expected. The test-retest reliability of the FACS was acceptable to excellent (ICC = 0.526–0.971). Conclusion: The Turkish version of FACS is a valid and reliable questionnaire for patients with chronic pain related to musculoskeletal disorders. The FACS provides a further advantage over identical questionnaires by evaluating cognitive, behavioral and emotional fear avoidance components.