Publications by authors named "Necla Birgul Iyison"

14 Publications

  • Page 1 of 1

Identifying and elucidating the roles of Y198N and Y204F mutations in the PAH enzyme through molecular dynamic simulations.

J Biomol Struct Dyn 2021 May 10:1-12. Epub 2021 May 10.

Faculty of Arts and Sciences, Department of Molecular Biology and Genetics, Bogazici University, Istanbul, Turkey.

Phenylketonuria is an autosomal recessive disorder caused by mutations in the phenylalanine hydroxylase gene. In phenylketonuria causes various symptoms including severe mental retardation. PAH gene of a classical Phenylketonuria patient was sequenced, and two novel heterozygous mutations, p.Y198N and p.Y204F, were found. This study aimed to reveal the impacts of these variants on the structural stability of the PAH enzyme. analyses using prediction tools and molecular dynamics simulations were performed. Mutations were introduced to the wild type catalytic monomer and full length tetramer crystal structures. Variant pathogenicity analyses predicted p.Y198N to be damaging, and p.Y204F to be benign by some prediction tools and damaging by others. Simulations suggested p.Y198N mutation cause significant fluctuations in the spatial organization of two catalytic residues in the temperature accelerated MD simulations with the monomer and increased root-mean-square deviations in the tetramer structure. p.Y204F causes noticeable changes in the spatial positioning of T278 suggesting a possible segregation from the catalytic site in temperature accelerated MD simulations with the monomer. This mutation also leads to increased root-mean-square fluctuations in the regulatory domain which may lead to conformational change resulting in inhibition of dimerization and enzyme activation. Our study reports two novel mutations in the PAH gene and gives insight to their effects on the PAH activity. MD simulations did not yield conclusive results that explains the phenotype but gave plausible insight to possible effects which should be investigated further with and studies to assess the roles of these mutations in etiology of PKU. Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2021.1921619DOI Listing
May 2021

Structural and Functional Characterization of Allatostatin Receptor Type-C of , a Potential Target for Next-Generation Pest Control Agents.

J Chem Inf Model 2021 Feb 21;61(2):715-728. Epub 2021 Jan 21.

Department of Molecular Biology and Genetics, Bogazici University, 34342 Istanbul, Turkey.

Insect neuropeptide receptors, including allatostatin receptor type C (AstR-C), a G protein-coupled receptor, are among the potential targets for designing next-generation pesticides that despite their importance in offering a new mode-of-action have been overlooked. Focusing on AstR-C of , a common pest in Mediterranean countries, by employing resonance energy transfer-based methods, we showed Gαi/o coupling and β-arrestin recruitment of the receptor at sub-nanomolar and nanomolar ranges of the endogenous ligand, AST-C, respectively. Molecular docking and molecular dynamics simulation studies revealed the importance of extracellular loop 2 in AstRC/AST-C interaction, and a combination of and approaches showed the substantial role of Q271 in G protein-dependent activation of AstR-C possibly contributing to the flexibility of the receptor's structure. The functional and structural insights obtained on AstR-C positively assist future efforts in developing environmentally friendly pest control agents that are needed urgently.
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http://dx.doi.org/10.1021/acs.jcim.0c00985DOI Listing
February 2021

Are insect GPCRs ideal next-generation pesticides: opportunities and challenges.

FEBS J 2021 Apr 27;288(8):2727-2745. Epub 2021 Jan 27.

Department of Molecular Biology and Genetics, Institute of Graduate Studies in Science and Engineering, Boğaziçi University, Istanbul, Turkey.

The increasing human population, combined with low inefficiency and adverse effects of available pesticides, has magnified the urgent need of developing next-generation pesticides. Among the available approaches, strategies targeting invertebrate G protein-coupled receptors (GPCRs) are very promising as these receptors are the targets of endogenous neuropeptides/neuromodulators that upon binding to their receptors induce profound changes in insect physiology. Therefore, exploring GPCR regulators has great potential in the development of targeted next-generation pesticides. Despite the great potential of such alternative pesticides, so far there has been only one approved compound, Amitraz, which conveys its anti-pest activity via the GPCR Octopamine receptor. Here, we review the current status of pesticide development, hazards associated with conventional pesticide compounds, alternative strategies that involve next-generation of pesticides, structural features of GPCRs, and opportunities and challenges of targeting the members of this superfamily in invertebrates to develop anti-pest agents. In conclusion, we emphasize that the potential of GPCRs cannot be utilized in full without more genomic and transcriptomic data to improve our understanding of the complex network of peptidergic signaling pathways. We argue how vital it is to obtain three-dimensional (3D) structural data on physiologically important target GPCRs and encourage the readers to use the state of the art in silico methods such as virtual screening for the discovery of new pesticide compounds.
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http://dx.doi.org/10.1111/febs.15708DOI Listing
April 2021

Pharmacological Characterization of the Stick Insect Allatostatin-C Receptor with Its Endogenous Agonist.

ACS Omega 2020 Dec 8;5(50):32183-32194. Epub 2020 Dec 8.

Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Bogazici University, Istanbul 34342, Turkey.

G protein-coupled receptors (GPCRs) play a pivotal role in regulating key physiological events in all animal species. Recent advances in collective analysis of genes and proteins revealed numerous potential neuropeptides and GPCRs from insect species, allowing for the characterization of peptide-receptor pairs. In this work, we used fluorescence resonance energy transfer (FRET)-based genetically encoded biosensors in intact mammalian cells to study the pharmacological features of the cognate GPCR of the type-C allatostatin (AST-C) peptide from the stick insect, . Analysis of multiple downstream pathways revealed that AST-C can activate the human Gi protein, and not Gs or Gq, through AST-C receptor (AlstRC). Activated AlstRC recruits β-arrestin2 independent of the Gi protein but stimulates ERK phosphorylation in a Gi protein-dependent manner. Identification of Gαi-, arrestin-, and GRK-like transcripts from revealed high evolutionary conservation at the G protein level, while β-arrestins and GRKs displayed less conservation. In conclusion, our study provides experimental and homology-based evidence on the functionality of vertebrate G proteins and downstream signaling biosensors to characterize early signaling steps of an insect GPCR. These results may serve as a scaffold for developing assays to characterize pharmacological and structural aspects of other insect GPCRs and can be used in deorphanization and pesticide studies.
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http://dx.doi.org/10.1021/acsomega.0c03382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7758886PMC
December 2020

In silico characterization of adipokinetic hormone receptor and screening for pesticide candidates against stick insect, Carausius morosus.

J Mol Graph Model 2020 12 23;101:107720. Epub 2020 Aug 23.

Computational Biology and Molecular Simulations Laboratory, Department of Biophysics, School of Medicine, Bahcesehir University, 34734, Istanbul, Turkey; Neuroscience Program, Graduate School of Health Sciences, Bahcesehir University, 34734, Istanbul, Turkey. Electronic address:

Adipokinetic hormone (AKH) is an insect neuropeptide that plays crucial roles in a variety of physiological functions such as regulation of heartbeat frequency, blood hemolymph trehalose levels, and protein synthesis. It exerts its functions through binding to its cognate G protein-coupled receptor (GPCR), named adipokinetic hormone receptor (AKHR). The aim of this study is to characterize AKHR of stick insect, Carausius morosus, which becomes an agricultural and forest pest during its outbreaks, and to screen pesticide candidates that would act through inhibition of AKHR. To this aim, the sequence of the receptor and its ligand were obtained from previously published transcriptome data and homology modeling, molecular docking, and molecular dynamics (MD) simulations were combined to find the ligand-binding pocket of AKHR. As a result, crucial residues in ligand binding were identified. These residues were located at the 6th and 7th transmembrane (TM) domains and the 2nd extracellular loop (ECL) of AKHR model. In order to propose pesticide candidates, virtual screening was performed, and candidate ligands were obtained. Considering the binding energies and the stability of the interaction between the ligand and the receptor, four hit compounds were selected. In conclusion, this study revealed a possible ligand-binding pocket of AKHR and proposed some high-affinity small-molecules to block its function, which would further facilitate pesticide design studies against the same receptor of various pests.
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http://dx.doi.org/10.1016/j.jmgm.2020.107720DOI Listing
December 2020

Whole genome sequencing of Thaumetopoea pityocampa revealed putative pesticide targets.

Genomics 2020 11 9;112(6):4203-4207. Epub 2020 Jul 9.

Department of Molecular Biology and Genetics, Institute of Graduate Studies in Science and Engineering, Bogazici University, 34342 Bebek, Istanbul, Turkey. Electronic address:

Insect neuropeptides play a major role in the regulation of the physiological processes. Due to their versatile effects on the development of insects, their corresponding receptors, which are mostly G-protein coupled receptors, are considered as ideal targets for designing next-generation pesticides. In this study, we aimed to find neuropeptide receptors of pine processionary moth (Thaumetopoea pityocampa), a pest in the Mediterranean countries, that feeds on the needles of pine trees. To this aim, Whole Genome Shotgun sequencing technique was used. de novo assembly of the genome was performed using two different assemblers, SGA and MaSuRCA. The results of two assemblers were compared, and MaSuRCA assembler showed higher N50 length. To find some target GPCRs, sequences of Drosophila melanogaster and evolutionarily close species were used as blast queries in the assembled data. Five GPCRs were chosen from the genome and their expression was confirmed in the larval stage of the insect.
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http://dx.doi.org/10.1016/j.ygeno.2020.07.017DOI Listing
November 2020

Identification of IFITM3 and MGAT1 as novel interaction partners of BRI3 by yeast two-hybrid screening.

Turk J Biol 2018 10;42(6):463-470. Epub 2018 Dec 10.

Department of Molecular Biology and Genetics, Faculty of Arts and Sciences Boğaziçi University , Bebek, İstanbul , Turkey.

BRI3 (brain protein I3) is one of the Wnt/β-catenin pathway target genes as indicated by the results of serial analysis of gene expression (SAGE) and microarray analyses performed in our laboratory. The Wnt/β-catenin signaling pathway is an evolutionarily conserved pathway, which has important functions in early vertebrate development, axis formation, cellular proliferation, and morphogenesis. Previous studies showed that BRI3 expression is upregulated at both mRNA and protein levels upon β-catenin activation by various approaches, such as lithium treatment and overexpression of Wnt ligands in Huh7 (hepatocellular carcinoma) cell lines. Moreover, with regard to the previous literature, BRI3 was found to have a very important role in the TNFα-mediated cell death pathway. In this study, we screened a human liver cDNA library by yeast two-hybrid assay using BRI3 protein as bait, with the aim of finding novel interaction partners of BRI3. Library screening by yeast mating resulted in the identification of three candidate positive clones. Among these, IFITM3 and MGAT1 proteins were confirmed as interaction partners by using cotransformation in yeast cells and coimmunoprecipitation from mammalian cell lines. Considering the poor functional characterization of BRI3 to date, identification of novel BRI3-interacting proteins is an essential first step in determining the action mechanism of BRI3 with respect to the Wnt/β-catenin pathway.
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http://dx.doi.org/10.3906/biy-1805-47DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451842PMC
December 2018

Prediction and expression analysis of G protein-coupled receptors in the laboratory stick insect, Carausius morosus.

Turk J Biol 2019 7;43(1):77-88. Epub 2019 Feb 7.

Center for Life Sciences and Technologies, Boğaziçi University , İstanbul , Turkey.

G protein-coupled receptors (GPCRs) are 7-transmembrane proteins that transduce various extracellular signals into intracellular pathways. They are the major target of neuropeptides, which regulate the development, feeding behavior, mating behavior, circadian rhythm, and many other physiological functions of insects. In the present study, we performed RNA sequencing and de novo transcriptome assembly to uncover the GPCRs expressed in the stick insect Carausius morosus. The transcript assemblies were predicted for the presence of 7-transmembrane GPCR domains. As a result, 430 putative GPCR transcripts were obtained and 43 of these revealed full-length sequences with highly significant similarity to known GPCR sequences in the databases. Thirteen different GPCRs were chosen for tissue expression analysis. Some of these receptors, such as calcitonin, inotocin, and tyramine receptors, showed specific expression in some of the tissues. Additionally, GPCR prediction yielded a novel uncharacterized GPCR sequence, which was specifically expressed in the central nervous system and ganglia. Previously, the only information about the anatomy of the stick insect was on its gastrointestinal system. This study provides complete anatomical information about the adult insect.
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http://dx.doi.org/10.3906/biy-1809-27DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426647PMC
February 2019

An in vivo RNAi mini-screen in Drosophila cancer models reveals novel potential Wnt targets in liver cancer.

Turk J Gastroenterol 2019 02;30(2):198-207

Department of Molecular Biology and Genetics, Boğaziçi University, İstanbul, Turkey;Center for Life Sciences and Technologies, Boğaziçi University, İstanbul, Turkey.

Background/aims: Aberrant activation of the Wnt/β-catenin signaling, which arises from the accumulation of mutant β-catenin in the cell, is one of the most common driving forces in hepatocellular carcinoma (HCC). We previously identified several genes that are regulated on the overexpression of β-catenin in the HCC cell line that are suggested to be novel Wnt/β-catenin targets playing effective roles in cancer. The aim of the present study was to elucidate the roles of these putative target genes in tumorigenesis with an in vivo analysis in Drosophila.

Materials And Methods: We selected 15 genes downregulated in two Drosophila cancer models.

Results: The results from the RNAi mini-screen revealed novel roles for the analyzed putative Wnt/β-catenin target genes in tumorigenesis. The downregulation of the analyzed nine genes led to tumor formation as well as metastasis in Drosophila, suggesting a tumor suppressor function. On the other hand, the knockdown of the other two genes suppressed tumor and metastasis formations and disturbed the development of the analyzed eye tissues, indicating an oncogenic or developmental role for these genes.

Conclusion: These findings could serve to identify novel subjects for cancer research in order to provide insight into the diagnostic and therapeutic processes of several cancer types.
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http://dx.doi.org/10.5152/tjg.2018.18241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6408157PMC
February 2019

MGAT1 is a novel transcriptional target of Wnt/β-catenin signaling pathway.

BMC Cancer 2018 01 8;18(1):60. Epub 2018 Jan 8.

Department of Molecular Biology and Genetics, Institute of Graduate Studies in Science and Engineering, Boğaziçi University, İstanbul, Turkey.

Background: The Wnt/β-catenin signaling pathway is an evolutionary conserved pathway, which has important functions in vertebrate early development, axis formation, cellular proliferation and morphogenesis. Additionally, Wnt/β-catenin signaling pathway is one of the most important intracellular pathways that controls cancer progression. To date most of the identified targets of this pathway are shown to harbor tumorigenic properties. We previously showed that Mannosyl glycoprotein acetylglucosaminyl-transferase (MGAT1) enzyme is among the Wnt/β-catenin signaling putative target genes in hepatocellular carcinoma cell lines (Huh7).

Methods: MGAT1 protein levels were determined by Western Blotting from Huh7 cell lines in which Wnt/β-catenin pathway was activated by means of different approaches such as LiCl treatment and mutant β-catenin overexpression. Luciferase reporter assay was used to analyze the promoter activity of MGAT1. The mRNA levels of MGAT1 were determined by quantitative real-time PCR from Huh7 cells that were treated with either Wnt agonist or GSK-3β inhibitor. Wound healing and XTT cell proliferation assays were performed in order to determine the proliferation and migration capacities of MGAT1 overexpressing stable Huh7 cells. Finally, xenograft experiments were carried out to measure the tumor formation capacities in vivo.

Results: In this study we showed that the activation of Wnt/β-catenin pathway culminates in the upregulation of MGAT1 enzyme both at transcriptional and post-transcriptional levels. We also showed that overexpression of the β-catenin gene (CTNNB1) increased the promoter activity of MGAT1. We applied a set of complementary approaches to elucidate the functional importance of MGAT1 as a vital target of Wnt/β-catenin signaling in Huh7 cells. Our analyses related to cell proliferation and migration assays showed that in comparison to the control cells, MGAT1 expressing Huh7 cells have greater proliferative and invasive capabilities. Furthermore, the stable overexpression of MGAT1 gene in Huh7 cell lines lead to a significant increase in tumor growth rate in Severe Combined Immunodeficient (SCID) mice.

Conclusions: Taken together, we showed for the first time that MGAT is a novel Wnt/β-catenin pathway target that has important implications for tumorigenesis.
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http://dx.doi.org/10.1186/s12885-017-3960-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5759366PMC
January 2018

Ligand binding pocket of a novel Allatostatin receptor type C of stick insect, Carausius morosus.

Sci Rep 2017 01 24;7:41266. Epub 2017 Jan 24.

Boğaziçi University Department of Molecular Biology and Genetics, Istanbul, 34342, Turkey.

Allatostatins (AST) are neuropeptides with variable function ranging from regulation of developmental processes to the feeding behavior in insects. They exert their effects by binding to cognate GPCRs, called Allatostatin receptors (AlstR), which emerge as promising targets for pesticide design. However, AlstRs are rarely studied. This study is the first reported structural study on AlstR-AST interaction. In this work, the first C type AlstR from the stick insect Carausius morosus (CamAlstR-C) was identified and its interaction with type C AST peptide was shown to be physically consistent with the experimental results. The proposed structure of CamAlstR-C revealed a conserved motif within the third extracellular loop, which, together with the N-terminus is essential for ligand binding. In this work, computational studies were combined with molecular and nano-scale approaches in order to introduce an unknown GPCR-ligand system. Consequently, the data obtained provided a reliable target region for future agonist/inverse agonist studies on AlstRs.
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http://dx.doi.org/10.1038/srep41266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5259779PMC
January 2017

MENA is a transcriptional target of the Wnt/beta-catenin pathway.

PLoS One 2012 17;7(5):e37013. Epub 2012 May 17.

Department of Molecular Biology and Genetics, Bogazici University, Istanbul, Turkey.

Wnt/β-catenin signalling pathway plays important roles in embryonic development and carcinogenesis. Overactivation of the pathway is one of the most common driving forces in major cancers such as colorectal and breast cancers. The downstream effectors of the pathway and its regulation of carcinogenesis and metastasis are still not very well understood. In this study, which was based on two genome-wide transcriptomics screens, we identify MENA (ENAH, Mammalian enabled homologue) as a novel transcriptional target of the Wnt/β-catenin signalling pathway. We show that the expression of MENA is upregulated upon overexpression of degradation-resistant β-catenin. Promoters of all mammalian MENA homologues contain putative binding sites for Tcf4 transcription factor--the primary effector of the Wnt/β-catenin pathway and we demonstrate functionality of these Tcf4-binding sites using luciferase reporter assays and overexpression of β-catenin, Tcf4 and dominant-negative Tcf4. In addition, lithium chloride-mediated inhibition of GSK3β also resulted in increase in MENA mRNA levels. Chromatin immunoprecipitation showed direct interaction between β-catenin and MENA promoter in Huh7 and HEK293 cells and also in mouse brain and liver tissues. Moreover, overexpression of Wnt1 and Wnt3a ligands increased MENA mRNA levels. Additionally, knock-down of MENA ortholog in D. melanogaster eyeful and sensitized eye cancer fly models resulted in increased tumor and metastasis formations. In summary, our study identifies MENA as novel nexus for the Wnt/β-catenin and the Notch signalling cascades.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037013PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3355175PMC
January 2013

Analysis of the Wnt/B-catenin/TCF4 pathway using SAGE, genome-wide microarray and promoter analysis: Identification of BRI3 and HSF2 as novel targets.

Cell Signal 2010 Oct 9;22(10):1523-35. Epub 2010 Jun 9.

Department of Molecular Biology and Genetics, Bogazici University, Istanbul, Turkey.

The Wnt signaling pathway is involved in many differentiation events during embryonic development and can lead to tumor formation after aberrant activation of its components. beta-catenin, a cytoplasmic component, plays a major role in the transduction of canonical Wnt signaling. The aim of this study was to identify novel genes that are regulated by active beta-catenin/TCF signaling in hepatocellular carcinoma-derived Huh7 cells with high (transfected) and low beta-catenin/TCF activities. High TCF activity Huh7 cells led to earlier and larger tumor formation when xenografted into nude mice. SAGE (Serial Analysis of Gene Expression), genome-wide microarray and in silico promoter analysis were performed in parallel, to compare gene expression between low and high beta-catenin/TCF activity clones, and also those that had been rescued from the xenograft tumors. SAGE and genome-wide microarray data were compared and contrasted. BRI3 and HSF2 were identified as novel targets of Wnt/beta-catenin signaling after combined analysis and confirming experiments including qRT-PCR, ChIP, luciferase assay and lithium treatment.
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http://dx.doi.org/10.1016/j.cellsig.2010.05.021DOI Listing
October 2010

Prenatal expressions of hyperpolarization-activated cyclic-nucleotide-gated channel (HCN) genes in dysplastic hippocampi in rats.

Turk Neurosurg 2008 Oct;18(4):327-35

Department of Neurosurgery, Bagcilar Training and Research Hospital, Istanbul, Turkey.

Aim: Hyperpolarization-activated cyclic nucleotide-gated (HCN or h-channel) channels mediate hyperpolarization-activating currents in the hippocampus and neocortex. The aim of this study is to present prenatal h-channel gene expressions (HCN1 and HCN2; HCN1-Entrez-Gene ID: 84390; HCN2- Entrez Gene ID: 114244) in dysplastic hippocampal pyramidal neurons induced by in utero irradiation in rats.

Materials And Methods: Time-pregnant Wistar albino rats were irradiated and the dysplastic hippocampus in their 2 month-old litters was studied. Gene expression was studied by RNA extraction and polymerase chain reaction methods.

Results: None of the rats showed seizure activity. mRNA levels of HCN1 and HCN2 genes were decreased especially in the CA1 and CA3 pyramidal neurons in the hippocampi of experimental rats; however, the differences were not significant compared to controls. In CA2, mRNA levels of both genes were increased and this rise did not reach significant level. The CA4 sub-region showed a different pattern of expression: HCN1 increased but HCN2 decreased insignificantly compared to controls.

Conclusion: Our results demonstrated that dysplastic neurons showed decreased levels of mRNA expression of HCN1 and HCN2 genes, in particularly CA1 and CA3 pyramidal neurons. The rationale for how these changes contribute to epileptogenesis in dysplastic tissues still requires further studies.
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October 2008