Difference between revisions of "Part:BBa K364201"

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<partinfo>BBa_K364201 parameters</partinfo>
 
<partinfo>BBa_K364201 parameters</partinfo>
 
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Improved by NOFLS_YZ
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===Improvement===
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Biology
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Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily. Its typical functional domains include DNA binding domain and ligand binding domain. After binding its ligand, FXR forms a heterodimer with another nuclear receptor retinoid X receptor, and then the receptor dimer binds to the FXR response element (FXRE) located in the promoter region of the FXR target gene, thereby, regulating the transcription of these genes.
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===Engineering Success===
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Transactivation of FXRE by FXR-RXRα complex
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Part BBa_K3522013 consists of a FXRE fragment and luciferase gene. When presence of FXR and RXRα, the FXRE is activated, subsequently, the luciferase gene is transcribed. Such a transactivation assay cam be used to verify the antagonistic effect of H7 on FXR.
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Finally, transactivation assay was further carried out to verify the antagonistic effect of H7 on FXR. As shown in Figure 3, FXR agonist GW4064 efficiently activated reporter gene expression, and H7 antagonized GW4064-induced reporter gene stimulation in transactivation assay. Thus, these results confirmed the antagonism of H7 against FXR transactivation activity.
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[[File:T--NOFLS_YZ-BBa_K3522013 Fig 1.jpg|500px|thumb|center|Figure 1]]
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                            Figure 1. H7 as an FXR antagonist inhibited transactivation activity.
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HEK293T cells were transiently transfected with pcDNA3.1a-FXR, pcDNA3.1a-RXRα, pGL3-FXRE-Luc and pRL-SV40. After 6 h, cells were treated with different concentrations of H7 with GW4064 for 24 h. Then transactivation activity of FXR was detected by luciferase reporter assay. GW4064: FXR agonist, GS: FXR antagonist. All data were presented as mean ± S.E.M (*P<0.05, **P< 0.01, ***P< 0.001).

Revision as of 16:37, 27 October 2020

Human RXR DBD

RXRα is a member of the nuclear receptor superfamily that mediates signaling by 9-cis retinoic acid, a vitamin A (retinol) derivative. Along with other members of the RXR family, RXRα plays roles in a variety of processes including embryonic patterning and organogenesis, cell proliferation and differentiation. RXRs commonly function as heterodimers with other members of the nuclear receptor superfamily. RXR-α is expressed in mulitple organs in many major physiological systems (central nervous system (CNS), endocrine, metabolic, gastrointestinal, reproductive and cardiopulmonary), with particularly high levels in the liver, preputial gland, skeletal muscle and skin. RXR-α variants have been associated with psychiatric (schizophrenia, autism, alcoholism, bipolar disorder and attention deficit hyperactivity disorder (ADHD) and immune (psoriasis) disorders.

More on RXRalpha here: [http://en.wikipedia.org/wiki/RXRA].

RXRalpha Uniprot: [http://www.uniprot.org/uniprot/P19793].

View the RXRa DBD crystal structure here: [http://www.pdb.org/pdb/explore/jmol.do?structureId=1RXR].

Picture of human RXR DNA binding domain

Description of the RXRa DBD crystal structure with reference here: [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WK7-45S499G-7J&_user=10&_coverDate=08%2F14%2F1998&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=70873168b504b6edc1afa579ccc60a29&searchtype=a].


Picture of gel electrophoresis: hRXRα DBD in pSB1C3 resulting an insert of 198 bp. Circular pSB1C3-hRXRα DBD can be seen as control on the gel.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]




Improved by NOFLS_YZ

Improvement

Biology Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily. Its typical functional domains include DNA binding domain and ligand binding domain. After binding its ligand, FXR forms a heterodimer with another nuclear receptor retinoid X receptor, and then the receptor dimer binds to the FXR response element (FXRE) located in the promoter region of the FXR target gene, thereby, regulating the transcription of these genes.

Engineering Success

Transactivation of FXRE by FXR-RXRα complex Part BBa_K3522013 consists of a FXRE fragment and luciferase gene. When presence of FXR and RXRα, the FXRE is activated, subsequently, the luciferase gene is transcribed. Such a transactivation assay cam be used to verify the antagonistic effect of H7 on FXR. Finally, transactivation assay was further carried out to verify the antagonistic effect of H7 on FXR. As shown in Figure 3, FXR agonist GW4064 efficiently activated reporter gene expression, and H7 antagonized GW4064-induced reporter gene stimulation in transactivation assay. Thus, these results confirmed the antagonism of H7 against FXR transactivation activity.

Figure 1
                            Figure 1. H7 as an FXR antagonist inhibited transactivation activity.

HEK293T cells were transiently transfected with pcDNA3.1a-FXR, pcDNA3.1a-RXRα, pGL3-FXRE-Luc and pRL-SV40. After 6 h, cells were treated with different concentrations of H7 with GW4064 for 24 h. Then transactivation activity of FXR was detected by luciferase reporter assay. GW4064: FXR agonist, GS: FXR antagonist. All data were presented as mean ± S.E.M (*P<0.05, **P< 0.01, ***P< 0.001).