Analysis of the thrombopoietin receptor (MPL) promoter implicates GATA and Ets proteins in the coregulation of megakaryocyte-specific genes

he MPL gene codes for the thrombopoietin receptor, whose ligand specifically controls megakaryocytic differentiation. To understand the molecular basis for the megakaryocyte-specific expression of MPL, we analyzed the promoter of this gene. A 200 bp fragment is sufficient for high-level specific expression. This fragment can bind several trans- acting factors in vitro, including GATA-1 and members of the Ets family. GATA-1 binds with low affinity to a unique GATA motif at -70 in the MPL promoter, and destruction of this site yields only a modest decrease in expression level in HEL cells. Ets proteins also bind with low affinity to two sites. One is located at position -15 and its destruction reduces expression to 50%; the other is located immediately downstream of the GATA motif and plays a crucial role in expression of the promoter in HEL cells, as its inactivation reduces expression to 15%. Furthermore, GATA-1 and two Ets proteins, Ets-1 and Fli-1, can trans-activate the MPL promoter in heterologous cells. The effects of GATA-1 and these two Ets proteins are additive. Together with our previous results on the glycoprotein IIb (GpIIb) promoter, this study indicates a molecular basis for the coregulation of early markers of megakaryocyte differentiation.

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GATA-1 Binding to Exon 1 Directs High Level Erythroid-Specific Expression of the Human Protoporphyrinogen Oxidase Gene.

Blood ◽

10.1182/blood.v106.11.1682.1682 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1682-1682

Author(s):

Karen M.K. de Vooght ◽

Richard van Wijk ◽

Wouter W. van Solinge

Keyword(s):

Transcriptional Regulation ◽

Promoter Activity ◽

K562 Cells ◽

Promoter Strength ◽

Specific Expression ◽

Oxidase Gene ◽

Protoporphyrinogen Oxidase ◽

Exon 1 ◽

High Level

Abstract Protoporphyrinogen oxidase (PPOX), the penultimate enzyme in the heme biosynthetic pathway, catalyzes the six-electron oxidation of protoporphyrinogen IX to protoporphyrin IX. Like other heme biosynthetic proteins, PPOX is involved in synthesizing heme for red cells (erythroid-specific expression) and as a cofactor for the respiratory cytochromes (housekeeping expression). To date, little is known about transcriptional regulation of the human PPOX gene (PPOX). We established the molecular basis for erythroid-specific expression of PPOX. Using transient in vitro transfection assays in human erythroleukemic K562 cells we studied tissue-specific expression of PPOX. We found that reporter constructs lacking exon 1 showed a 75% reduction in promoter strength in K562 cells (Figure, no 1 and 2). Hence, in vitro high-level erythroid-specific expression of PPOX is dependent on the presence of exon 1. Examination of erythroid-specific regulatory elements in exon 1 revealed two GATA-1 sites, one consensus (A/T)GATA(A/G) site (GATA-1_II AGATAA) and one non-consensus site, deviating at the first nucleotide (GATA-1_I, CGATAG). To study the relative contribution of these two GATA-1 sites to erythroid-specific transcriptional regulation, we performed in vitro transfections of wild-type and mutant (GATA → GTTA) reporter plasmids in K562 cells. We found that the highest level of transcription depended on the integrity of both sites (Figure, no 5). The consensus GATA-1_II site contributed the most to promoter strength (Figure, no 4). Subsequent electrophoretic mobility shift assay and supershift experiments using K562 nuclear extracts demonstrated that both GATA sites were able to bind GATA-1 in vitro. Our experiments showed that exon 1 was dispensable for PPOX promoter activity in human hepatoma HepG2 cells. Interestingly, in HeLa human cervical carcinoma cells the presence of exon 1 decreased promoter activity. Conclusively, exon 1 of the human PPOX gene contains two GATA-1 binding sites, which are required for high level erythroid-specific expression of PPOX and, in addition, bind GATA-1 in vitro. Our results contribute to a better understanding of the molecular mechanisms involved in differential regulation of the human PPOX promoter in erythroid and non-erythroid cells. Figure Figure

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Expression of a functional recombinant vascular endothelial growth factor 165 (VEGF165) in Arabidopsis thaliana

Turkish Journal of Biochemistry ◽

10.1515/tjb-2017-0368 ◽

2018 ◽

Vol 44 (3) ◽

pp. 254-260

Author(s):

Weidong Qiang ◽

Xue Feng ◽

Yixin Li ◽

Xinxin Lan ◽

Kun Ji ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Cell Activity ◽

Downstream Processing ◽

Coding Region ◽

Specific Expression ◽

Plant Seed ◽

Huvec Cell ◽

Plant Seed Oil ◽

High Level

Abstract Objective Targeting the protein of interest to a particular tissue to achieve high-level expression is an important strategy to increase expression efficiency. The use of the plant seed oil body as a bioreactor can not only increase the amount of target protein, but also reduce the cost of downstream processing. Methods VEGF165 was expressed in Arabidopsis thaliana seeds via oilbody fusion technology. The pKO-VEGF165 vector was construted and transformed into A. thaliana seeds. T3 transgenic seeds was detected by SDS-PAGE and western blot methods. The cell activity was tested by MTT methods. Result The phaseolin promoter was used to drive seed-specific expression of the VEGF165 gene in transgenic A. thaliana. The coding region of VEGF165 was fused to the Arabidopsis oleosin sequence to target the protein to the oil bodies in the seeds of transgenic plants. The T-DNA region of recombinant plasmid pKO-VEGF165 was shifted to A. thaliana seeds via the floral-dip method. Protein was analyzed by electrophoresis and protein hybridization analyses. Finally, MTT assays showed that the oleosin-VEGF165 fusion protein played a part in the proliferation of HUVEC cells in vitro. Conclusion Oleosin-VEGF165 was successfully expressed and it had stimulated HUVEC cell proliferation activity.

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Molecular Basis of Resistance to Tribenuron in Water Starwort (Myosoton aquaticum) Populations from China

Weed Science ◽

10.1614/ws-d-12-00200.1 ◽

2013 ◽

Vol 61 (3) ◽

pp. 390-395 ◽

Cited By ~ 11

Author(s):

Weitang Liu ◽

Yaling Bi ◽

Lingxu Li ◽

Guohui Yuan ◽

Jinxin Wang

Keyword(s):

Molecular Basis ◽

Inhibitory Concentration ◽

Acetolactate Synthase ◽

Susceptible Population ◽

Documented Case ◽

Whole Plant ◽

Winter Annual ◽

High Level ◽

Plant Bioassays

Populations of water starwort, a winter annual or biennial weed in the pink family (Caryophyllaceae), can no longer be controlled by tribenuron following successive use of this herbicide over several years. Whole-plant bioassays have established that the resistant water starwort populations JS17, JS08, JS16, and JS07 showed high-level (from 203-fold to 565-fold) resistance to tribenuron. In vitro acetolactate synthase (ALS) assays revealed that resistance was due to reduced sensitivity of the ALS enzyme to tribenuron. The half-maximal inhibitory concentration (I50) values for JS17, JS08, JS16, and JS07 were 72, 71, 70, and 76 times greater, respectively, than were those of the susceptible population JS24. This altered ALS sensitivity in the resistant populations was due to a mutation in the ALS gene resulting in a Pro197to Ser substitution (JS17, JS08, and JS16) and a Pro197to Leu substitution (JS07). This study established the first documented case, to our knowledge, of evolved tribenuron resistance in water starwort and concluded that the molecular basis of resistance is due, at least in part, to a target-site modification at Pro197in the ALS gene.

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Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

10.26434/chemrxiv.12052869.v1 ◽

2020 ◽

Author(s):

James Frederich ◽

Ananya Sengupta ◽

Josue Liriano ◽

Ewa A. Bienkiewicz ◽

Brian G. Miller

Keyword(s):

Protein Interactions ◽

Neurological Diseases ◽

Adapter Proteins ◽

Protein Protein Interactions ◽

Specific Expression ◽

Individual Client ◽

Isoform Specificity ◽

Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions in vivo. Herein, we report the isoform-specificity profile of FC in vitrousing recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

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Molecular Basis of Iterative C–H Oxidation by TamI, a Multifunctional P450 Monooxygenase from the Tirandamycin Biosynthetic Pathway

10.26434/chemrxiv.12710159.v1 ◽

2020 ◽

Author(s):

Sean A. Newmister ◽

Kinshuk Raj Srivastava ◽

Rosa V. Espinoza ◽

Kersti Caddell Haatveit ◽

Yogan Khatri ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Molecular Basis ◽

Hydrophobic Interactions ◽

Cytochromes P450 ◽

Targeted Mutagenesis ◽

P450 Monooxygenase ◽

Bond Functionalization ◽

Dynamics Simulations

Biocatalysis offers an expanding and powerful strategy to construct and diversify complex molecules by C-H bond functionalization. Due to their high selectivity, enzymes have become an essential tool for C-H bond functionalization and offer complementary reactivity to small-molecule catalysts. Hemoproteins, particularly cytochromes P450, have proven effective for selective oxidation of unactivated C-H bonds. Previously, we reported the in vitro characterization of an oxidative tailoring cascade in which TamI, a multifunctional P450 functions co-dependently with the TamL flavoprotein to catalyze regio- and stereoselective hydroxylations and epoxidation to yield tirandamycin A and tirandamycin B. TamI follows a defined order including 1) C10 hydroxylation, 2) C11/C12 epoxidation, and 3) C18 hydroxylation. Here we present a structural, biochemical, and computational investigation of TamI to understand the molecular basis of its substrate binding, diverse reactivity, and specific reaction sequence. The crystal structure of TamI in complex with tirandamycin C together with molecular dynamics simulations and targeted mutagenesis suggest that hydrophobic interactions with the polyene chain of its natural substrate are critical for molecular recognition. QM/MM calculations and molecular dynamics simulations of TamI with variant substrates provided detailed information on the molecular basis of sequential reactivity, and pattern of regio- and stereo-selectivity in catalyzing the three-step oxidative cascade.

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In Vitro Effect of Chlorquine and Picroliv on Plasmodium Berghei Induced Alterations in the Activity of Adenosine Triphosphatase, Aryl Hyrocarbon Hydroxylase Enzymes and Malondialdehyde in spleen Explant Culture

Infectious Disorders - Drug Targets ◽

10.2174/1871526520666200630124419 ◽

2020 ◽

Vol 20 ◽

Author(s):

Anchal Trivedi ◽

Aparna Misra ◽

Esha Sarkar ◽

Anil K. Balapure

Keyword(s):

Drug Resistance ◽

Plasmodium Berghei ◽

Adenosine Triphosphatase ◽

Explant Culture ◽

Need To Evaluate ◽

Mda Content ◽

High Level ◽

Vitro Effect ◽

Positive Effect

Background: In recent years, great progress has been made in reducing the high level of malaria suffering worldwide. There is a great need to evaluate drug resistance reversers and consider new medicines against malaria. There are many approaches to the development of antimalarial drugs. Specific concerns must be taken in to account in these approaches, in particular there requirement for very in expensive and simple use of new therapies and the need to limit drug discovery expenses. Important ongoing efforts are the optimisation of treatment with available medications, including the use of combination therapy. The production of analogs of known agents and the identification of natural products, the use of compounds originally developed against other diseases, the assessment of overcoming drug resistance and the consideration of new therapeutic targets. Liver and spleen are the important organs which are directly associated with malarial complications. Aim: An analysis the Activity of Adenosine Triphosphatase, Aryl Hyrocarbon Hydroxylase Enzymes and Malondialdehyde in spleen Explant Culture. Objective: To determine in-Vitro Effect of Chlorquine and Picroliv on Plasmodium Berghei Induced Alterations in the Activity of Adenosine Triphosphatase, Aryl Hyrocarbon Hydroxylase Enzymes and Malondialdehyde in spleen Explant Culture. Material and method: 1-Histological preparation of spleen explants for paraplast embedding 2-Biochemicalstudies (Enzymes (Atpase, ALP&GST) and the level of protein, Malondialdehyde (MDA). Result: Splenomegalyis one of the three main diagnostic parameters of malaria infection besides fever and anaemia. Many enzymes present in the liver and spleen may also be altered or liberated under different pathological conditions. Enzymes (ATPase, ALP&GST) and the level of protein, Malondialdehyde (MDA) content was found to increase in the liver and spleen explants during malarial infection. In the liver and spleen derived from parasitized CQ treated animals, the activity of all the above enzymes (ATPase, ALP&GST) and the level of protein & MDA of liver/spleen reversed towards the normal for all the 4or3 days of incubations. Picroliv efficacy decreased with the increment of parasitaemia and at 60%parasitaemia. Conclusion: Alkalinephosphatase (ALP) was found to increase with increasing parasitaemia. After the addition of Picroliv to the medium, a decrement in the activity was observed up to day 4 of culture.A similar positive effect of Picroliv was observed on the ATPase and ALP activity of spleen explants.DNA and protein contents also increased in the parasitized liver cultured in the presence of picroliv.On the contrary, in the spleen explants DNA, protein and MDA content were found to decrease after Picroliv supplementation to the culture medium.

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Non-Viral Vectors for Gene Delivery

Nanoscience &Nanotechnology-Asia ◽

10.2174/2210681208666180110154233 ◽

2018 ◽

Vol 9 (1) ◽

pp. 4-11 ◽

Cited By ~ 5

Author(s):

Aparna Bansal ◽

Himanshu

Keyword(s):

Gene Therapy ◽

Viral Vectors ◽

Transfection Efficiency ◽

Gene Transfection ◽

Expression System ◽

Target Cells ◽

Specific Expression ◽

Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

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Overexpression of the Oral Mucosa-Specific microRNA-31 Promotes Skin Wound Closure

International Journal of Molecular Sciences ◽

10.3390/ijms20153679 ◽

2019 ◽

Vol 20 (15) ◽

pp. 3679 ◽

Cited By ~ 3

Author(s):

Lin Chen ◽

Alyne Simões ◽

Zujian Chen ◽

Yan Zhao ◽

Xinming Wu ◽

...

Keyword(s):

Wound Healing ◽

Oral Mucosa ◽

Wound Closure ◽

Expression Patterns ◽

Skin Wound ◽

Skin Wound Healing ◽

Specific Expression ◽

Keratinocyte Proliferation

Wounds within the oral mucosa are known to heal more rapidly than skin wounds. Recent studies suggest that differences in the microRNAome profiles may underlie the exceptional healing that occurs in oral mucosa. Here, we test whether skin wound-healing can be accelerating by increasing the levels of oral mucosa-specific microRNAs. A panel of 57 differentially expressed high expresser microRNAs were identified based on our previously published miR-seq dataset of paired skin and oral mucosal wound-healing [Sci. Rep. (2019) 9:7160]. These microRNAs were further grouped into 5 clusters based on their expression patterns, and their differential expression was confirmed by TaqMan-based quantification of LCM-captured epithelial cells from the wound edges. Of these 5 clusters, Cluster IV (consisting of 8 microRNAs, including miR-31) is most intriguing due to its tissue-specific expression pattern and temporal changes during wound-healing. The in vitro functional assays show that ectopic transfection of miR-31 consistently enhanced keratinocyte proliferation and migration. In vivo, miR-31 mimic treatment led to a statistically significant acceleration of wound closure. Our results demonstrate that wound-healing can be enhanced in skin through the overexpression of microRNAs that are highly expressed in the privileged healing response of the oral mucosa.

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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Allelic Exchange and Mutant Selection Demonstrate that Common Clinical embCAB Gene Mutations Only Modestly Increase Resistance to Ethambutol in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01288-09 ◽

2009 ◽

Vol 54 (1) ◽

pp. 103-108 ◽

Cited By ~ 38

Author(s):

Hassan Safi ◽

Robert D. Fleischmann ◽

Scott N. Peterson ◽

Marcus B. Jones ◽

Behnam Jarrahi ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

In Vitro Selection ◽

Gene Mutations ◽

Wild Type ◽

Mutant Selection ◽

Preferential Growth ◽

Allelic Exchange ◽

High Level ◽

Isogenic Mutants

ABSTRACT Mutations within codon 306 of the Mycobacterium tuberculosis embB gene modestly increase ethambutol (EMB) MICs. To identify other causes of EMB resistance and to identify causes of high-level resistance, we generated EMB-resistant M. tuberculosis isolates in vitro and performed allelic exchange studies of embB codon 406 (embB406) and embB497 mutations. In vitro selection produced mutations already identified clinically in embB306, embB397, embB497, embB1024, and embC13, which result in EMB MICs of 8 or 14 μg/ml, 5 μg/ml, 12 μg/ml, 3 μg/ml, and 4 μg/ml, respectively, and mutations at embB320, embB324, and embB445, which have not been identified in clinical M. tuberculosis isolates and which result in EMB MICs of 8 μg/ml, 8 μg/ml, and 2 to 8 μg/ml, respectively. To definitively identify the effect of the common clinical embB497 and embB406 mutations on EMB susceptibility, we created a series of isogenic mutants, exchanging the wild-type embB497 CAG codon in EMB-susceptible M. tuberculosis strain 210 for the embB497 CGG codon and the wild-type embB406 GGC codon for either the embB406 GCC, embB406 TGC, embB406 TCC, or embB406 GAC codon. These new mutants showed 6-fold and 3- to 3.5-fold increases in the EMB MICs, respectively. In contrast to the embB306 mutants, the isogenic embB497 and embB406 mutants did not have preferential growth in the presence of isoniazid or rifampin (rifampicin) at their MICs. These results demonstrate that individual embCAB mutations confer low to moderate increases in EMB MICs. Discrepancies between the EMB MICs of laboratory mutants and clinical M. tuberculosis strains with identical mutations suggest that clinical EMB resistance is multigenic and that high-level EMB resistance requires mutations in currently unknown loci.

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