Processive kinetics in the three-step lanosterol 14α-demethylation reaction catalyzed by human cytochrome P450 51A1

Journal of Biological Chemistry

Published On 2023/7/1

Cytochrome P450 (P450, CYP) family 51 enzymes catalyze the 14α-demethylation of sterols, leading to critical products used for membranes and the production of steroids, as well as signaling molecules. In mammals, P450 51 catalyzes the 3-step, 6-electron oxidation of lanosterol to form (4β,5α)-4,4-dimethyl-cholestra-8,14,24-trien-3-ol (FF-MAS). P450 51A1 can also use 24,25-dihydrolanosterol (a natural substrate in the Kandutsch-Russell cholesterol pathway). 24,25-Dihydrolanosterol and the corresponding P450 51A1 reaction intermediates, the 14α-alcohol and -aldehyde derivatives of dihydrolanosterol, were synthesized to study the kinetic processivity of the overall 14α-demethylation reaction of human P450 51A1. A combination of steady-state kinetic parameters, steady-state binding constants, dissociation rates of P450-sterol complexes, and kinetic modeling of the time course of oxidation of a P450 …

Journal

Journal of Biological Chemistry

Published On

2023/7/1

Volume

299

Issue

7

Authors

Fred Guengerich

Fred Guengerich

Vanderbilt University

Position

Professor of Biochemistry

H-Index(all)

175

H-Index(since 2020)

51

I-10 Index(all)

0

I-10 Index(since 2020)

0

Citation(all)

0

Citation(since 2020)

0

Cited By

0

Research Interests

Enzymology

drug metabolism

cytochrome P450

mutagenesis

University Profile Page

Kevin D. McCarty

Kevin D. McCarty

Vanderbilt University

Position

H-Index(all)

4

H-Index(since 2020)

4

I-10 Index(all)

0

I-10 Index(since 2020)

0

Citation(all)

0

Citation(since 2020)

0

Cited By

0

Research Interests

Biochemistry

University Profile Page

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Kevin D. McCarty

Kevin D. McCarty

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ACS catalysis

Oxygen-18 Labeling Defines a Ferric Peroxide (Compound 0) Mechanism in the Oxidative Deformylation of Aldehydes by Cytochrome P450 2B4

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Fred Guengerich

Fred Guengerich

Vanderbilt University

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Fat mass obesity-associated protein (FTO) is a DNA/RNA demethylase involved in the epigenetic regulation of various genes and is considered a therapeutic target for obesity, cancer, and neurological disorders. Here, we aimed to design novel FTO-selective inhibitors by merging fragments of previously reported FTO inhibitors. Among the synthesized analogues, compound 11b, which merges key fragments of Hz (3) and MA (4), inhibited FTO selectively over alkylation repair homologue 5 (ALKBH5), another DNA/RNA demethylase. Treatment of acute monocytic leukemia NOMO-1 cells with a prodrug of 11b decreased the viability of acute monocytic leukemia cells, increased the level of the FTO substrate N6-methyladenosine in mRNA, and induced upregulation of MYC and downregulation of RARA, which are FTO target genes. Thus, Hz (3)/MA (4) hybrid analogues represent an entry into a new class of FTO …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Chemical Research in Toxicology

In Vivo and In Vitro Induction of Cytochrome P450 3A4 by Thalidomide in Humanized-Liver Mice and Experimental Human Hepatocyte HepaSH cells

Autoinduction of cytochrome P450 (P450) 3A4-mediated metabolism of thalidomide was investigated in humanized-liver mice and human hepatocyte-derived HepaSH cells. The mean plasma ratios of 5-hydroxythalidomide and glutathione adducts to thalidomide were significantly induced (3.5- and 6.0-fold, respectively) by thalidomide treatment daily at 1000 mg/kg for 3 days and measured at 2 h after the fourth administration (on day 4). 5-Hydroxythalidomide was metabolically activated by P450 3A4 in HepaSH cells pretreated with 300 and 1000 μM thalidomide, and 5,6-dihydroxythalidomide was detected. Significant induction of P450 3A4 mRNA expression (4.1-fold) in the livers of thalidomide-treated mice occurred. Thalidomide exerts a variety of actions through multiple mechanisms following bioactivation by induced human P450 3A enzymes.

Fred Guengerich

Fred Guengerich

Vanderbilt University

Formation of potentially toxic metabolites of drugs in reactions catalyzed by human drug-metabolizing enzymes

Data are presented on the formation of potentially toxic metabolites of drugs that are substrates of human drug metabolizing enzymes. The tabular data lists the formation of potentially toxic/reactive products. The data were obtained from in vitro experiments and showed that the oxidative reactions predominate (with 96% of the total potential toxication reactions). Reductive reactions (e.g., reduction of nitro to amino group and reductive dehalogenation) participate to the extent of 4%. Of the enzymes, cytochrome P450 (P450, CYP) enzymes catalyzed 72% of the reactions, myeloperoxidase (MPO) 7%, flavin-containing monooxygenase (FMO) 3%, aldehyde oxidase (AOX) 4%, sulfotransferase (SULT) 5%, and a group of minor participating enzymes to the extent of 9%. Within the P450 Superfamily, P450 Subfamily 3A (P450 3A4 and 3A5) participates to the extent of 27% and the Subfamily 2C (P450 2C9 and P450 2C19 …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Journal of Biological Chemistry

Proteomics, modeling, and fluorescence assays delineate cytochrome b5 residues involved in binding and stimulation of cytochrome P450 17A1 17, 20-lyase

Cytochrome b5 (b5) is known to stimulate some catalytic activities of cytochrome P450 (P450, CYP) enzymes, although mechanisms still need to be defined. The reactions most strongly enhanced by b5 are the 17,20-lyase reactions of P450 17A1 involved in steroid biosynthesis. We had previously used a fluorescently labeled human b5 variant (Alexa 488-T70C-b5) to characterize human P450 17A1-b5 interactions, but subsequent proteomic analyses indicated that lysines in b5 were also modified with Alexa 488 maleimide in addition to Cys-70, due to disulfide dimerization of the T70C mutant. A series of b5 variants were constructed with Cys replacements for the identified lysine residues and labeled with the dye. Fluorescence attenuation and the function of b5 in the steroid lyase reaction depended on the modified position. Apo-b5 (devoid of heme group) studies revealed the lack of involvement of the b5 heme in …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Angewandte Chemie

Oxygen‐18 Labeling Reveals a Mixed Fe− O Mechanism in the Last Step of Cytochrome P450 51 Sterol 14α‐Demethylation

The 14α‐demethylation step is critical in eukaryotic sterol biosynthesis, catalyzed by cytochrome P450 (P450) Family 51 enzymes, for example, with lanosterol in mammals. This conserved three‐step reaction terminates in a C−C cleavage step that generates formic acid, the nature of which has been controversial. Proposed mechanisms involve roles of P450 Compound 0 (ferric peroxide anion, FeO2−) or Compound I (perferryl oxygen, FeO3+) reacting with either the aldehyde or its hydrate, respectively. Analysis of 18O incorporation into formic acid from 18O2 provides a means of distinguishing the two mechanisms. Human P450 51A1 incorporated 88 % 18O (one atom) into formic acid, consistent with a major but not exclusive FeO2− mechanism. Two P450 51 orthologs from amoeba and yeast showed similar results, while two orthologs from pathogenic trypanosomes showed roughly equal contributions of both …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Journal of Biological Chemistry

Ninety-eight semesters of cytochrome P450 enzymes and related topics—What have I taught and learned?

This Reflection article begins with my family background and traces my career through elementary and high school, followed by time at the University of Illinois, Vanderbilt University, the University of Michigan, and then for 98 semesters as a Vanderbilt University faculty member. My research career has dealt with aspects of cytochrome P450 enzymes, and the basic biochemistry has had applications in fields as diverse as drug metabolism, toxicology, medicinal chemistry, pharmacogenetics, biological engineering, and bioremediation. I am grateful for the opportunity to work with the Journal of Biological Chemistry not only as an author but also for 34 years as an Editorial Board Member, Associate Editor, Deputy Editor, and interim Editor-in-Chief. Thanks are extended to my family and my mentors, particularly Profs. Harry Broquist and Minor J. Coon, and the more than 170 people who have trained with me. I have …

Fred Guengerich

Fred Guengerich

Vanderbilt University

ACS catalysis

Oxygen-18 Labeling Defines a Ferric Peroxide (Compound 0) Mechanism in the Oxidative Deformylation of Aldehydes by Cytochrome P450 2B4

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Fred Guengerich

Fred Guengerich

Vanderbilt University

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Kevin D. McCarty

Kevin D. McCarty

Vanderbilt University

Angewandte Chemie

Oxygen‐18 Labeling Reveals a Mixed Fe− O Mechanism in the Last Step of Cytochrome P450 51 Sterol 14α‐Demethylation

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Fred Guengerich

Fred Guengerich

Vanderbilt University

Journal of Biological Chemistry

The multistep oxidation of cholesterol to pregnenolone by human cytochrome P450 11A1 is highly processive

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2023/11/24

Article Details
Fred Guengerich

Fred Guengerich

Vanderbilt University

Journal of Biological Chemistry

Processive kinetics in the three-step lanosterol 14α-demethylation reaction catalyzed by human cytochrome P450 51A1

Cytochrome P450 (P450, CYP) family 51 enzymes catalyze the 14α-demethylation of sterols, leading to critical products used for membranes and the production of steroids, as well as signaling molecules. In mammals, P450 51 catalyzes the 3-step, 6-electron oxidation of lanosterol to form (4β,5α)-4,4-dimethyl-cholestra-8,14,24-trien-3-ol (FF-MAS). P450 51A1 can also use 24,25-dihydrolanosterol (a natural substrate in the Kandutsch-Russell cholesterol pathway). 24,25-Dihydrolanosterol and the corresponding P450 51A1 reaction intermediates, the 14α-alcohol and -aldehyde derivatives of dihydrolanosterol, were synthesized to study the kinetic processivity of the overall 14α-demethylation reaction of human P450 51A1. A combination of steady-state kinetic parameters, steady-state binding constants, dissociation rates of P450-sterol complexes, and kinetic modeling of the time course of oxidation of a P450 …

Kevin D. McCarty

Kevin D. McCarty

Vanderbilt University

Steroid 17α-hydroxylase/17, 20-lyase (cytochrome P450 17A1)

Cytochrome P450 (P450) 17A1 plays a key role in steroidogenesis, in that this enzyme catalyzes the 17α-hydroxylation of both pregnenolone and progesterone, followed by a lyase reaction to cleave the C-20 land C-21 carbons from each steroid. The reactions are important in the production of both glucocorticoids and androgens. The enzyme is critical in humans but is also a drug target in treatment of prostate cancer. Detailed methods are described for the heterologous expression of human P450 17A1 in bacteria, purification of the recombinant enzyme, reconstitution of the enzyme system in the presence of cytochrome b5, and chromatographic procedures for sensitive analyses of reaction products. Historic assay approaches are reviewed. Some information is also provided about outstanding questions in the research field, including catalytic mechanisms and searches for selective inhibitors.

Fred Guengerich

Fred Guengerich

Vanderbilt University

Nucleic Acids Research

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To perform double-stranded DNA passage, type II topoisomerases generate a covalent enzyme-cleaved DNA complex (i.e. cleavage complex). Although this complex is a requisite enzyme intermediate, it is also intrinsically dangerous to genomic stability. Consequently, cleavage complexes are the targets for several clinically relevant anticancer and antibacterial drugs. Human topoisomerase IIα and IIβ and bacterial gyrase maintain higher levels of cleavage complexes with negatively supercoiled over positively supercoiled DNA substrates. Conversely, bacterial topoisomerase IV is less able to distinguish DNA supercoil handedness. Despite the importance of supercoil geometry to the activities of type II topoisomerases, the basis for supercoil handedness recognition during DNA cleavage has not been characterized. Based on the results of benchtop and rapid-quench flow kinetics experiments, the forward …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Xenobiotica

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Fred Guengerich

Fred Guengerich

Vanderbilt University

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Vanderbilt University

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Fred Guengerich

Fred Guengerich

Vanderbilt University

International Journal of Molecular Sciences

Identification of Three Human POLH Germline Variants Defective in Complementing the UV-and Cisplatin-Sensitivity of POLH-Deficient Cells

DNA polymerase (pol) η is responsible for error-free translesion DNA synthesis (TLS) opposite ultraviolet light (UV)-induced cis-syn cyclobutane thymine dimers (CTDs) and cisplatin-induced intrastrand guanine crosslinks. POLH deficiency causes one form of the skin cancer-prone disease xeroderma pigmentosum variant (XPV) and cisplatin sensitivity, but the functional impacts of its germline variants remain unclear. We evaluated the functional properties of eight human POLH germline in silico-predicted deleterious missense variants, using biochemical and cell-based assays. In enzymatic assays, utilizing recombinant pol η (residues 1—432) proteins, the C34W, I147N, and R167Q variants showed 4- to 14-fold and 3- to 5-fold decreases in specificity constants (kcat/Km) for dATP insertion opposite the 3’-T and 5′-T of a CTD, respectively, compared to the wild-type, while the other variants displayed 2- to 4-fold increases. A CRISPR/Cas9-mediated POLH knockout increased the sensitivity of human embryonic kidney 293 cells to UV and cisplatin, which was fully reversed by ectopic expression of wild-type pol η, but not by that of an inactive (D115A/E116A) or either of two XPV-pathogenic (R93P and G263V) mutants. Ectopic expression of the C34W, I147N, and R167Q variants, unlike the other variants, did not rescue the UV- and cisplatin-sensitivity in POLH-knockout cells. Our results indicate that the C34W, I147N, and R167Q variants—substantially reduced in TLS activity—failed to rescue the UV- and cisplatin-sensitive phenotype of POLH-deficient cells, which also raises the possibility that such hypoactive germline POLH variants may …

Fred Guengerich

Fred Guengerich

Vanderbilt University

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Journal of Biological Chemistry

Abstract 1713 Shaping antiviral strategies with potent and selective non-covalent SARS-CoV-2 papain-like protease inhibitors

SARS-CoV-2 continues to elude containment efforts and remains a significant global health challenge. There is a critical need for drugs that employ multiple mechanisms of action, ensuring a more robust and versatile approach to effectively manage both current and emerging coronavirus infections. The papain-like protease (PLpro) domain of Nsp3 is essential for viral replication in human coronaviruses (HCoV). PLpro also contributes to the COVID-19-associated ‘cytokine storm’by cleaving ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host proteins and disrupting host immune responses. Consequently, PLpro is a promising target for small-molecule therapeutics. In this study, we have refined non-covalent inhibitors of SARS-CoV-1 PLpro and SARS-CoV-2 PLpro from primary literature, resulting in a lead inhibitor with improved cytotoxicity values and specificity towards SARS-CoV-1 and SARS …

Geoff Horsman

Geoff Horsman

Wilfrid Laurier University

Journal of Biological Chemistry

Abstract 1559" Stuffed" Epimerase Domains in Pyochelin-like Natural Product Biosynthesis may be Defunct Methyltransferases and not Catalytically Required for Activity

Nonribosomal peptide synthetases (NRPSs) are used by bacteria, fungi, and plants to generate secondary metabolites called nonribosomal peptides (NRPs). These bioactive NRPs also have medicinal applications as antibiotics, anticancer drugs, and immunosuppressants. A common feature of NRPs is the need for further tailoring for bioactivity such as epimerization, methylation, reduction, hydroxylation, heterocylization, etc. The inclusion of amino acids with D-stereocenters, which have been found to be important for the chemical functionality of the peptide, and as a way to evade protease degradation typically arise by the action of individual epimerase tailoring domains that have been well studied. Interestingly, 2-hydroxyphenylthiazoline natural products do not use common epimerase tailoring domains but have been hypothesized to employ noncanonical epimerase domains that are embedded, or'stuffed' …

Stephanie Archer-Hartmann

Stephanie Archer-Hartmann

University of Georgia

Journal of Biological Chemistry

Abstract 2338 Development of Methods for Identification and Quantification of Glycoconjugates

Glycoconjugates are ubiquitous and of tremendous significance in biological systems. To understand their function, it is critical to be able to accurately elucidate their precise structure, even when only minute amounts can be obtained from cells or tissues. We will present new analytical tools for the analysis of glycosphingolipids (GSLs) and other glycoconjugates. GSLs are a group of glycolipids with a glycan head glycosidically linked to the C-1 hydroxyl group of a ceramide tail. While some diversity in GSLs is due to the ceramide group, the primary structural and functional categorizations are based upon glycan variability with impacts in cell adhesion, signaling, proliferation, endocytosis, intracellular transport, inflammation, and apoptosis. This study adopts a comparative approach for identification and quantification of GSLs. Therefore, two methods for analyzing GSLs were compared. In the first experiment, isolated …

David Wilson

David Wilson

James Cook University

Journal of Biological Chemistry

Structural analysis of a U-superfamily conotoxin containing a mini-granulin fold: Insights into key features that distinguish between the ICK and granulin folds

We are entering an exciting time in structural biology where artificial intelligence can be used to predict protein structures with greater accuracy than ever before. Extending this level of accuracy to the predictions of disulfide-rich peptide structures is likely to be more challenging, at least in the short term, given the tight packing of cysteine residues and the numerous ways that the disulfide bonds can potentially be linked. It has been previously shown in many cases that several disulfide bond connectivities can be accommodated by a single set of NMR-derived structural data without significant violations. Disulfide-rich peptides are prevalent throughout nature, and arguably the most well-known are those present in venoms from organisms such as cone snails. Here, we have determined the first three-dimensional structure and disulfide connectivity of a U-superfamily cone snail venom peptide, TxVIIB. TxVIIB has a VI …

Nathan Alder

Nathan Alder

University of Connecticut

Journal of Biological Chemistry

Abstract 1951 Conserved and specific cardiolipin-mitochondrial ADP/ATP carrier interactions assume structural and functional roles

Notch receptor activation is regulated by the intramembrane protease γ-secretase, which cleaves and liberates the Notch intracellular domain (Nicd) that regulates gene transcription. Here, we identify Divalent metal transporter 1 (Dmt1, Slc11A2) as a novel and essential regulator of Notch signalling. Cells lacking Dmt1 exhibit defects in Notch signaling, accompanied by disruptions in autophagosomal and endolysosomal functions. Dmt1 deficiency leads to elevated Fe2+ levels, disturbed ferritin dynamics, heightened cytoplasmic and mitochondrial reactive oxygen species (ROS), increased lipid peroxidation, and altered autophagic flux. Consequently, these changes culminate in reduced lysosomal activity, impeding the degradation of Lamp1 and cleaved Notch1 (NICD1) proteins. Notably, Dmt1 exists in two isoforms, with and without an iron response element (ire). Our investigation reveals that silencing Dmt1-ire …

Miriam L. Greenberg

Miriam L. Greenberg

Wayne State University

Journal of Biological Chemistry

Abstract 2303 Cardiolipin at the epicenter of energy metabolism–implications for Barth syndrome

Cardiolipin (CL), the signature lipid of the mitochondrial membrane, is crucial for optimal mitochondrial function and bioenergetics. Perturbation of CL metabolism due to mutation of the TAFAZZIN (TAZ) gene leads to the life-threatening disorder, Barth syndrome (BTHS). TAZ encodes the transacylase tafazzin (Taz) responsible for incorporating unsaturated acyl chains into CL. While the clinical phenotypes of BTHS, including dilated cardiomyopathy and skeletal myopathy, point to mitochondrial bioenergetic defects, metabolic dysregulation is also a key pathological component. Taz-deficient cells exhibit abnormal levels of metabolites associated with the tricarboxylic acid (TCA) cycle. Consistent with this, we have identified inhibition of pyruvate dehydrogenase (PDH), the gatekeeper enzyme for TCA cycle carbon influx, as a key deficiency in various BTHS models. Further, we determined that deficient PDH activity can …

Catherine Drennan

Catherine Drennan

Massachusetts Institute of Technology

Journal of Biological Chemistry

Abstract 1607 Characterization of a glycyl radical enzyme activating enzyme (GRE-AE) that equips anaerobes to functionalize inert hydrocarbons

Glycyl radical enzyme activating-enzymes (GRE-AEs) are members of the radical S-adenosyl-L-methionine (rSAM) superfamily and activate their cognate glycyl radical enzymes (GREs) by post-translational installation of the radical cofactor. Upon activation, GREs perform chemically challenging transformations that are inaccessible without the use of a radical cofactor. 4-isopropylbenzylsuccinate synthase (IBSS) is a GRE that activates inert hydrocarbons by CH bond functionalization. First, IBSS activating-enzyme (IbsAE) installs a glycyl radical cofactor on IBSS, which then allows IBSS to catalyze the addition of p-cymene to fumarate in a hydroalkylation reaction to form 4-isopropylbenzylsuccinate. Of the thousands of bioinformatically identified GRE-AEs, only one activase–pyruvate formate-lyase activating enzyme (PFL-AE)–has been structurally characterized. Comparatively, PFL-AE is markedly different, as it …

JUAN CAMILO HERNANDEZ ORTIZ

JUAN CAMILO HERNANDEZ ORTIZ

Universidad del Valle

Journal of Biological Chemistry

Abstract 1358 Scaling relationships in animal nervous systems

Scaling relationships are functions of the form Y= aXb, where Y is a response variable,“a” is a normalization constant, X is a predictor variable (usually body mass) and “b” is an exponent. These functions describe scale-invariant, often universal, behavior and underlying features associated with the complex structure and functioning of biological systems. In this study, we explore the scaling relationships between the number of neurons and glial cells in the nervous systems of animals. We compiled data from the literature for different taxa, encompassing both decentralized and centralized (with brain) nervous systems and implemented simple linear regression analysis to fit power-law relationships (in logarithmic scale). Although we found varying exponent values for different taxa, through appropriate scaling, we were able to collapse data across all groups. Our findings reveal universality in the structure and …

David De Sancho

David De Sancho

Universidad del País Vasco

Journal of Biological Chemistry

High-Throughput virtual search of small-molecules for controlling the mechanical stability of human CD4

Protein mechanical stability determines the function of a myriad of proteins, especially proteins from the extracellular matrix. Failure to maintain protein mechanical stability may result in diseases and disorders such as cancer, cardiomyopathies, or muscular dystrophy. Thus, developing mutation-free approaches to enhance and control the mechanical stability of proteins using pharmacology-based methods, may have important implications in drug development and discovery. Here, we present the first approach that employs computational High-Throughput Virtual Screening (HTVS) and Molecular Docking to search for small-molecules in chemical libraries that function as mechano-regulators of the stability of human CD4, receptor of HIV-1. Using single-molecule force spectroscopy we prove that these small-molecules can increase the mechanical stability of CD4D1D2 domains over 4-fold in addition to modifying …

jeetain mittal

jeetain mittal

Lehigh University

Journal of Biological Chemistry

Abstract 1965 Revealing the structural basis for self-association of the RNA binding protein EWS

Ewing sarcoma RNA-binding protein 1 (EWS) is a member of the FET (FUS, EWS, TAF15) family of RNA binding proteins that function in transcription, splicing, and DNA damage repair. Mutations in FET proteins are linked to various neurodegenerative diseases, including frontal temporal dementia and amyotrophic lateral sclerosis, and the FET proteins participate in oncogenic fusions that cause numerous pediatric malignancies. EWS, like all FET family members, harbors extensive intrinsically disordered regions that lack a defined structure and facilitate multivalent and highly dynamic homo-and heterotypic interactions. Through a chromosomal translocation event, the intrinsically disordered N-terminal low complexity domain of EWS (EWSLCD) becomes fused to the FLI1 DNA-binding domain to yield the prototypical FET oncogenic fusion EWS::-FLI1, the driving mutation of Ewing sarcoma. The EWSLCD forms …

mete ozkurt

mete ozkurt

Eskisehir Osmangazi Üniversitesi

Journal of Biological Chemistry

Abstract 1229''Targeting Notch, IL-1 and Leptin crosstalk may have promising approach in HCT-15 colorectal cancer cells''

Objective The interaction between NILCO signaling and VEGF is associated with metastasis and decreased survival rate. Previous studies have shown that leptin plays a pivotal role in the crosstalk between NILCO and angiogenic VEGF. Additionally, it has been shown that matrix metalloproteinases (MMPs) are induced by leptin, causing tumor cells to spread from the basement membrane and contributing to tumor angiogenesis. In this study, we aimed to determine the effect of targeting NILCO in human colorectal cancer (CRC). Methods HCT-115 cells were transfected with Notch, IL-1, and leptin small interfering RNAs (siRNA) using lipofectamine 2000 reagent according to the manufacturers' instructions. Notch1, Cas-9 and VEGFA mRNA gene levels were determined by qRT-PCR; leptin, VEGF-A and TIPM-1 protein levels were determined by WB, and leptin, VEGF-A and IL-1β protein levels were determined by …