scholarly journals Differential activity and expression of human 5β-reductase (AKR1D1) splice variants

2020 ◽  
Author(s):  
Nathan Appanna ◽  
Elena Gangitano ◽  
Niall J Dempster ◽  
Karen Morris ◽  
Sherly George ◽  
...  
Keyword(s):  
Human Liver ◽  
Specific Activity ◽  
Splice Variants ◽  
Steroid Hormone ◽  
Hepatoma Cell ◽  
Receptor Activation ◽  
The Body ◽  
Hek293 Cells ◽  
Luciferase Reporter ◽  
Functional Protein

AbstractSteroid hormones, including glucocorticoids and androgens, exert a wide variety of effects in the body across almost all tissues. The steroid A-ring 5β-reductase (AKR1D1) is expressed in human liver and testes, and three splice variants have been identified (AKR1D1-001, AKR1D1-002, AKR1D1-006). Amongst these, AKR1D1-002 is the best described; it modulates steroid hormone availability and catalyses an important step in bile acid synthesis. However, specific activity and expression of AKR1D1-001 and AKR1D1-006 are unknown.AKR1D1-002, AKR1D1-001 and AKR1D1-006 were measured in human liver biopsies and human hepatoma cell lines by qPCR. Three-dimensional (3D) structures of AKR1D1 variants were determined using in silico approaches. AKR1D1 variants were over-expressed in HEK293 cells, and successful overexpression confirmed by qPCR and western blotting. Steroid hormone clearance was measured by mass spectrometry and ELISA, and steroid receptor activation determined by luciferase reporter assays.AKR1D1-002 and AKR1D1-001 are expressed in human liver, and only AKR1D1-006 is expressed in human testes. Following over-expression in HEK293 cells, AKR1D1-001 and AKR1D1-006 protein levels were lower than AKR1D1-002, but significantly increased following treatment with the proteasomal inhibitor, MG-132. AKR1D1-002 efficiently metabolised glucocorticoids and androgens and decreased receptor activation. AKR1D1-001 and AKR1D1-006 poorly metabolised dexamethasone, but neither protein metabolised cortisol, prednisolone or testosterone.We have demonstrated the differential expression and role of AKR1D1 splice variants to regulate steroid hormone clearance and receptor activation. AKR1D1-002 is the predominant functional protein in steroidogenic and metabolic tissues. In addition, AKR1D1-001 and AKR1D1-006 may have a limited role in the regulation of synthetic glucocorticoid action.

scholarly journals Differential activity and expression of human 5β-reductase (AKR1D1) splice variants

2021 ◽  
Author(s):  
Nathan Appanna ◽  
Hylton Gibson ◽  
Elena Gangitano ◽  
Niall J Dempster ◽  
Karen Morris ◽  
...  
Keyword(s):  
Human Liver ◽  
Specific Activity ◽  
Splice Variants ◽  
Steroid Hormone ◽  
Hepatoma Cell ◽  
Receptor Activation ◽  
The Body ◽  
Hek293 Cells ◽  
Luciferase Reporter ◽  
Functional Protein

Steroid hormones, including glucocorticoids and androgens, exert a wide variety of effects in the body across almost all tissues. The steroid A-ring 5β-reductase (AKR1D1) is expressed in human liver and testes, and three splice variants have been identified (AKR1D1-001, AKR1D1-002, AKR1D1-006). Amongst these, AKR1D1-002 is the best described; it modulates steroid hormone availability and catalyses an important step in bile acid biosynthesis. However, specific activity and expression of AKR1D1-001 and AKR1D1-006 are unknown. Expression of AKR1D1 variants were measured in human liver biopsies and hepatoma cell lines by qPCR. Their three-dimensional (3D) structures were predicted using in silico approaches. AKR1D1 variants were over-expressed in HEK293 cells, and successful overexpression confirmed by qPCR and western blotting. Cells were treated with either cortisol, dexamethasone, prednisolone, testosterone or androstenedione, and steroid hormone clearance was measured by mass spectrometry. Glucocorticoid and androgen receptor activation were determined by luciferase reporter assays. AKR1D1-002 and AKR1D1-001 are expressed in human liver, and only AKR1D1-006 is expressed in human testes. Following over-expression, AKR1D1-001 and AKR1D1-006 protein levels were lower than AKR1D1-002, but significantly increased following treatment with the proteasomal inhibitor, MG-132. AKR1D1-002 efficiently metabolised glucocorticoids and androgens and decreased receptor activation. AKR1D1-001 and AKR1D1-006 poorly metabolised dexamethasone, but neither protein metabolised cortisol, prednisolone, testosterone or androstenedione. We have demonstrated the differential expression and role of AKR1D1 variants in steroid hormone clearance and receptor activation in vitro. AKR1D1-002 is the predominant functional protein in steroidogenic and metabolic tissues. In addition, AKR1D1-001 and AKR1D1-006 may have a limited, steroid-specific role in the regulation of dexamethasone action.

scholarly journals Identification of two novel chicken GHRH receptor splice variants: implications for the roles of aspartate 56 in the receptor activation and direct ligand–receptor interaction

2007 ◽  
Vol 195 (3) ◽  
pp. 525-536 ◽  
Author(s):  
Crystal Ying Wang ◽  
Yajun Wang ◽  
Amy Ho Yan Kwok ◽  
Frederick C Leung
Keyword(s):  
Ligand Binding ◽  
Splice Variants ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Receptor Activation ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Splice Acceptor Site ◽  
Receptor Interaction ◽  
Acceptor Site

In this study, two novel GHRHR receptor splice variants, named chicken GHRHR-v1 (cGHRHR-v1) and cGHRHR-v2 respectively, were identified from chicken pituitary using RT-PCR assay. cGHRHR-v1 is characterized by an N-terminal deletion of 36 amino acid residues, including an aspartate at position 56 (Asp56) conserved in G protein-coupled receptor B-I subfamily. cGHRHR-v2 is a carboxyl-terminal truncated receptor variant with four putative transmembrane domains, which arose from alternative use of a splice acceptor site on intron 8. Using the pGL3-CRE-luciferase reporter system, the functionality of the two variants was examined in Chinese hamster ovary cells. cGHRHR-v1 was shown to be capable of transmitting signal upon agonist stimulation, but cGHRHR-v2 could not. Both GHRH and pituitary adenylate cyclase-activating peptide (PACAP) could activate cGHRHR-v1 at high dosages (GHRH ≥10−8 M; PACAP ≥10−6 M) and GHRH was much more potent than PACAP, suggesting that cGHRHR-v1 is a functional membrane-spanning receptor with an impairment in high-affinity ligand binding, rather than in receptor activation and ligand-binding specificity. This finding also points out the possibility that Asp56 is not a critical determinant for receptor activation and direct ligand–receptor interaction. To substantiate this hypothesis, using site-directed mutagenesis, two receptor mutants with replacement of Asp56 by Ala or Gly were generated. Expectedly, chicken or human GHRH could still activate both receptor mutants with reduced potencies (about 2- to 14-fold less potent). Taken together, our findings not only suggest that cGHRHR variants may play a role in controlling normal pituitary functions, but also support that Asp56 is nonessential for receptor activation and direct ligand–receptor interaction.

scholarly journals Equilibrative Nucleoside Transporters Mediate the Import of Nicotinamide Riboside and Nicotinic Acid Riboside into Human Cells

2021 ◽  
Vol 22 (3) ◽  
pp. 1391
Author(s):  
Andrey Kropotov ◽  
Veronika Kulikova ◽  
Kirill Nerinovski ◽  
Alexander Yakimov ◽  
Maria Svetlova ◽  
...  
Keyword(s):  
Nicotinic Acid ◽  
Mammalian Cells ◽  
Experimental Models ◽  
The Body ◽  
Nucleoside Transporters ◽  
Hek293 Cells ◽  
Nucleoside Transporter ◽  
Vitamin B3 ◽  
Animal Cells ◽  
Nicotinamide Riboside

Nicotinamide riboside (NR), a new form of vitamin B3, is an effective precursor of nicotinamide adenine dinucleotide (NAD+) in human and animal cells. The introduction of NR into the body effectively increases the level of intracellular NAD+ and thereby restores physiological functions that are weakened or lost in experimental models of aging and various pathologies. Despite the active use of NR in applied biomedicine, the mechanism of its transport into mammalian cells is currently not understood. In this study, we used overexpression of proteins in HEK293 cells, and metabolite detection by NMR, to show that extracellular NR can be imported into cells by members of the equilibrative nucleoside transporter (ENT) family ENT1, ENT2, and ENT4. After being imported into cells, NR is readily metabolized resulting in Nam generation. Moreover, the same ENT-dependent mechanism can be used to import the deamidated form of NR, nicotinic acid riboside (NAR). However, NAR uptake into HEK293 cells required the stimulation of its active utilization in the cytosol such as phosphorylation by NR kinase. On the other hand, we did not detect any NR uptake mediated by the concentrative nucleoside transporters (CNT) CNT1, CNT2, or CNT3, while overexpression of CNT3, but not CNT1 or CNT2, moderately stimulated NAR utilization by HEK293 cells.

scholarly journals The AaCBF4-AaBAM3.1 module enhances freezing tolerance of kiwifruit (Actinidia arguta)

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Shihang Sun ◽  
Chungen Hu ◽  
Xiujuan Qi ◽  
Jinyong Chen ◽  
Yunpeng Zhong ◽  
...  
Keyword(s):  
Cold Stress ◽  
Freezing Tolerance ◽  
Luciferase Reporter ◽  
Dna Interactions ◽  
Functional Protein ◽  
Actinidia Arguta ◽  
Protein Dna Interactions ◽  
Similar Phenotype ◽  
Reporter Assays

AbstractBeta-amylase (BAM) plays an important role in plant resistance to cold stress. However, the specific role of the BAM gene in freezing tolerance is poorly understood. In this study, we demonstrated that a cold-responsive gene module was involved in the freezing tolerance of kiwifruit. In this module, the expression of AaBAM3.1, which encodes a functional protein, was induced by cold stress. AaBAM3.1-overexpressing kiwifruit lines showed increased freezing tolerance, and the heterologous overexpression of AaBAM3.1 in Arabidopsis thaliana resulted in a similar phenotype. The results of promoter GUS activity and cis-element analyses predicted AaCBF4 to be an upstream transcription factor that could regulate AaBAM3.1 expression. Further investigation of protein-DNA interactions by using yeast one-hybrid, GUS coexpression, and dual luciferase reporter assays confirmed that AaCBF4 directly regulated AaBAM3.1 expression. In addition, the expression of both AaBAM3.1 and AaCBF4 in kiwifruit responded positively to cold stress. Hence, we conclude that the AaCBF-AaBAM module is involved in the positive regulation of the freezing tolerance of kiwifruit.

Expression of a novel FGF in the Xenopus embryo. A new candidate inducing factor for mesoderm formation and anteroposterior specification

Development ◽  
1992 ◽  
Vol 114 (3) ◽  
pp. 711-720 ◽  
Author(s):  
H.V. Isaacs ◽  
D. Tannahill ◽  
J.M. Slack
Keyword(s):  
Specific Activity ◽  
Biological Properties ◽  
The Body ◽  
Mesoderm Induction ◽  
Gastrula Stage ◽  
Blastula Stage ◽  
Mesoderm Formation ◽  
Low Levels

We have cloned and sequenced a new member of the fibroblast growth factor family from Xenopus laevis embryo cDNA. It is most closely related to both mammalian kFGF (FGF-4) and FGF-6 but as it is not clear whether it is a true homologue of either of these genes we provisionally refer to it as XeFGF (Xenopus embryonic FGF). Two sequences were obtained, differing by 11% in derived amino acid sequence, which probably represent pseudotetraploid variants. Both the sequence and the behaviour of in vitro translated protein indicates that, unlike bFGF (FGF-2), XeFGF is a secreted molecule. Recombinant XeFGF protein has mesoderm-inducing activity with a specific activity similar to bFGF. XeFGF mRNA is expressed maternally and zygotically with a peak during the gastrula stage. Both probe protection and in situ hybridization showed that the zygotic expression is concentrated in the posterior of the body axis and later in the tailbud. Later domains of expression were found near the midbrain/hindbrain boundary and at low levels in the myotomes. Because of its biological properties and expression pattern, XeFGF is a good candidate for an inducing factor with possible roles both in mesoderm induction at the blastula stage and in the formation of the anteroposterior axis at the gastrula stage.

Abstract P501: Editing of Myosin Phosphatase Gene as a Novel Approach for Vasodilator Sensitization and Lowering of Blood Pressure

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Mariam Meddeb ◽  
Jeanine Ursitti ◽  
John Reho ◽  
Steven A Fisher
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Leucine Zipper ◽  
Splice Variants ◽  
Species Conservation ◽  
Mesenteric Arteries ◽  
Hek293 Cells ◽  
Myosin Phosphatase ◽  
Novel Approach

Myosin Phosphatase (MP) is the primary effector of vascular smooth muscle (VSM) relaxation and a key end target of signaling pathways that regulate vessel tone. Regulated splicing of alternative Exon24 (E24) of Myosin Phosphatase Regulatory/ Targeting subunit (MYPT1) sets vasodilator sensitivity. Skipping E24 codes for a Mypt1 isoform that contains a C-terminal leucine zipper (LZ) motif required for cGK1α binding and NO/cGMP activation of MP resulting in vasodilation. Inclusion of 31 nt E24 shifts the reading frame coding for a Mypt1 isoform with a distinct C-terminus (LZ-) that is unresponsive to NO/cGMP. We are using two editing approaches to test the function of Mypt1 E24 splice variants in the control of BP in vivo. First, LoxP sites were inserted in introns flanking E24, crossed with smMHCCre ER , and treated with Tamoxifen to achieve smooth muscle-specific cKO of E24 (SMcKO E24), thereby converting Mypt1 to the LZ+ isoform. E24 cKO mice had mean BP that was 15 + 3 mmHg lower than control (n=3-5; p<0.05). Mesenteric arteries from these mice were significantly more sensitive to DEA/NO mediated relaxation (EC 50 : 2.1+0.5 nM vs 18.2+5.6 μM; n=5-6, p<0.05). We now are developing CRISPR/CAS9 editing of Mypt1 for translation into humans with hypertension. Guide(g)RNAs targeting E24 were designed using Benchling.com and selected for further study based on predicted efficacy, specificity (>10%,>60%) and cross-species conservation. Plasmids were generated by sub-cloning of oligonucleotides into the parent pX601 plasmid for the purpose of co-expression of gRNA and saCas9. These plasmids were transfected into HEK293 cells singly and in combinations and Mypt1 gene editing assayed by PCR, Surveyor nuclease assays and sequencing of genomic DNA. Single gRNAs yielded deletions of 1-3 nt. Combinations yielded deletions of 104-334 nt that removed >80% of E24 with an efficiency of editing that varied from 10% (gRNAs 6+9 and 5+9) to 40% (gRNAs 6+11 and 5+11). We have now generated AAVgE24 and are testing their efficiency of editing of VSM in vivo. These studies support that AAV mediated CRISPR/Cas9 editing of Mypt1 E24 could be a novel strategy for vasodilator sensitization and effective lowering of blood pressure in humans.

Progesterone enhances target gene transcription by receptor free of heat shock proteins hsp90, hsp56, and hsp70

1991 ◽  
Vol 11 (10) ◽  
pp. 4998-5004
Author(s):  
M K Bagchi ◽  
S Y Tsai ◽  
M J Tsai ◽  
B W O'Malley
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Heat Shock Protein ◽  
Transcriptional Activation ◽  
Target Gene ◽  
Steroid Hormone ◽  
Receptor Activation ◽  
Target Cells ◽  
Dependent Manner ◽  
Shock Proteins

Steroid receptors regulate transcription of target genes in vivo and in vitro in a steroid hormone-dependent manner. Unoccupied progesterone receptor exists in the low-salt homogenates of target cells as a functionally inactive 8 to 10S complex with several nonreceptor components such as two molecules of 90-kDa heat shock protein (hsp90), a 70-kDa heat shock protein (hsp70), and a 56-kDa heat shock protein (hsp56). Ligand-induced dissociation of receptor-associated proteins such as hsp90 has been proposed as the mechanism of receptor activation. Nevertheless, it has not been established whether, beyond release of heat shock proteins, the steroidal ligand plays a role in modulating receptor activity. To examine whether the release of these nonreceptor proteins from receptor complex results in a constitutively active receptor, we isolated an unliganded receptor form essentially free of hsp90, hsp70, and hsp56. Using a recently developed steroid hormone-responsive cell-free transcription system, we demonstrate for the first time that the dissociation of heat shock proteins is not sufficient to generate a functionally active receptor. This purified receptor still requires hormone for high-affinity binding to a progesterone response element and for efficient transcriptional activation of a target gene. When an antiprogestin, Ru486, is bound to the receptor, it fails to promote efficient transcription. We propose that in the cell, in addition to the release of receptor-associated inhibitory proteins, a distinct hormone-mediated activation event must precede efficient gene activation.

A generic microfluidic biosensor of G protein-coupled receptor activation – impedance measurements of reversible morphological changes of reverse transfected HEK293 cells on microelectrodes

RSC Advances ◽  
2015 ◽  
Vol 5 (65) ◽  
pp. 52563-52570 ◽  
Author(s):  
Saurabh K. Srivastava ◽  
Rajesh Ramaneti ◽  
Margriet Roelse ◽  
Hien Duy Tong ◽  
Elwin X. Vrouwe ◽  
...  
Keyword(s):  
G Protein ◽  
Morphological Changes ◽  
Receptor Activation ◽  
Membrane Receptors ◽  
Hek293 Cells ◽  
G Protein Coupled Receptor ◽  
Impedance Measurements ◽  
Transfected Cells ◽  
Microfluidic Biosensor ◽  
G Protein Coupled

Flowcell with micro-IDEs (250–500 μm) covered with both stable and reverse transfected cells overexpressing membrane receptors to demonstrate impedance responses to serial injections of analyte.

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