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Revision as of 11:37, 2 October 2024
P_5xCRE>IgK->Nluc->bGH_polyA
When melatonin binds to the MT1 receptor, the activation of adenylate cyclase (AC) is activated, regulating the level of the second messenger cAMP, while activating protein kinase to further amplify the signal. It catalyzes the phosphorylation of CREB in the nucleus and regulates the expression of downstream genes.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 103
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 376
- 1000COMPATIBLE WITH RFC[1000]
Profile
Name:P_5xCRE->IgK->Nluc->bGH_polyA
Base Pairs: 1082bp
Origin: Homo sapiens
Properties:As a reporter to show whether melatonin is accepted or not
Usage and Biology
G-protein-coupled receptors (GPCRs) represent the largest and most diverse group of membrane receptors in eukaryotes. G proteins are specialized proteins that can bind the nucleotides guanosine triphosphate (GTP) and guanosine diphosphate (GDP). When a ligand binds to a GPCR, it induces a conformational change in the receptor, which subsequently activates the associated G protein. The active form of the G protein is released from the receptor and dissociates into its subunits. These subunits then activate specific effectors, leading to the release of second messengers that are recognized by protein kinases, triggering a signaling cascade that results in various cellular events.[1]
Cyclic adenosine monophosphate (cAMP) serves as a pivotal second messenger that transduces extracellular signals to intracellular effectors by modulating its concentration within the cell. Upon ligand binding to GPCRs, adenylate cyclase is activated via the Gα subunit, resulting in elevated levels of intracellular cAMP. Subsequently, cAMP interacts with the regulatory subunits of protein kinase A (PKA), leading to the dissociation of its catalytic subunits. The liberated catalytic subunits of PKA translocate to the nucleus, where they phosphorylate the transcription factor cAMP response element-binding protein (CREB). Phosphorylated CREB then binds to the coactivator CREB-binding protein (CBP), forming a complex that recognizes and binds to the cAMP response element (CRE) within the regulatory regions of target genes, thereby initiating transcriptional activation. [2]
This cAMP/PKA/CREB pathway is a well-established and common signaling pathway in mammalian cells, regulated by various importance upstream pathways such as GPCRs we describe above. Therefore, in the view of mammalian cell synthetic biology, a synthetic gene circuit that enables sensing and characterizing the status (such as activation/inhibition) of the cAMP/PKA/CREB pathway can be very useful that only provide valuable insights into their functionality and but also offer toolkits for potential biotechnological applications..
To achieve the function described above, we present this composite that can directly sense and accurately characterize the status of the cAMP/PKA/CREB pathway in mammalian cells.
This composite part consisting of:
1. P_5xCRE (Part: BBa_K5267005): A synthetic promoter that assess the activation status of the cAMP/PKA/CREB signaling pathways, with 5 tandem repeats of CRE serving as a binding site for phosphorylated CREB.
2. IgK (Part: BBa_K3117006): A signal sequence directing the protein into the secretory pathway.
3. Nluc (Part: BBa_K2728003): An engineered luminescent reporter that detects cAMP concentration. When expressed, it results in blue fluorescence, indicating the activation status of the cAMP/PKA/CREB pathway.
4. bGH_polyA (Part: BBa_K1313006): Functions as a terminator, ensuring proper cessation of gene expression.
By incorporating this composite part into mammalian cell chassis, such as the HEK293 cell line, we can build a cAMP/PKA/CREB pathway characterization platform that effectively senses and monitors the activation of the pathway, providing detectable reporter signals. This synthetic gene circuit can be a valuable tool for studying signaling pathways and can be applied in various biotechnological and therapeutic research areas.
Special design
This basic part is an important element for testing whether the downstream pathway of melatonin responds successfully. At present, the commonly used method to study the signaling pathway is to clone the response element of the transcription factor corresponding to the signaling pathway into the luciferase reporter gene vector, that is, pCRE-luc .[3] However, the expression effect of a single responder is weak，so multiple tandem repeats of the same responder element are usually inserted upstream of the reporter gene (the 5 '-UTR region) to enhance the activation of the signaling pathway. By searching through literature, we constructed the 4xCRE-Pmin sequence，It contains a 5′ minimal promoter incorporating 4 multimerized palin-dromic CREs with the sequence 5′-AGCC[TGACGTCC]GAG-3′. (CRE denoted in brackets)，with the exception of a single nucleotide substitution (C for A) within the CRE[4]，which may strengthen gene expression downstream.
Function Test
Method
In order to validate the composite part P_5*CRE->IgK->Nluc->bGH_polyA(Part:BBa_K5267041), which can express receptor in mammalian cells, we designed a controlled cell experiment, according to the natural gene pathway in human SCN cells that the activated MTNR1a receptors could activate several gene pathways which all lead to the activation of CREB (cAMP response element binding protein)[5], activating the expression of gene with CRE (cAMP response element) promotors. We prepared two dishes of HEK-293T cell lines, each with the same passage number, identical viability, and a cell count of 500,000. One dish served as the experimental group, and the other as the control group. The experimental group was co-transfected with pLeo694 plasmids, one carrying the construct PCMV -> MTNR1a -> bGH polyA(Part:BBa_K5267047) and the other carrying the construct P_5xCRE-> lgK -> Nluc -> bGH polyA(Part:BBa_K5267041), in a 100 ng to 50 ng ratio (the optimal transfection ratio validated by experiments). The control group was transfected only with 50 ng of the plasmid carrying the P_5xCRE-> lgK -> Nluc -> bGH polyA (Part:BBa_K5267041)construct. After transfection, both groups of cells were stimulated with 1 nM melatonin, and samples were collected at 24 and 48 hours to detect Nluc expression.
Result
reference
[1] Q. Wang et al., "Structural basis of the ligand binding and signaling mechanism of melatonin receptors," Nat Commun, vol. 13, no. 1, p. 454, Jan 24 2022, doi: 10.1038/s41467-022-28111-3.
[2] A. J. Shaywitz and M. E. Greenberg, "CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals," Annu Rev Biochem, vol. 68, pp. 821-61, 1999, doi: 10.1146/annurev.biochem.68.1.821.
[3] C. Kemmer, D. A. Fluri, U. Witschi, A. Passeraub, A. Gutzwiller, and M. Fussenegger, "A designer network coordinating bovine artificial insemination by ovulation-triggered release of implanted sperms," J Control Release, vol. 150, no. 1, pp. 23-9, Feb 28 2011, doi: 10.1016/j.jconrel.2010.11.016.
[4] O. G. Chepurny and G. G. Holz, "A novel cyclic adenosine monophosphate responsive luciferase reporter incorporating a nonpalindromic cyclic adenosine monophosphate response element provides optimal performance for use in G protein coupled receptor drug discovery efforts," J Biomol Screen, vol. 12, no. 5, pp. 740-6, Aug 2007, doi: 10.1177/1087057107301856.
[5] H. H. Okamoto, E. Cecon, O. Nureki, S. Rivara, and R. Jockers, "Melatonin receptor structure and signaling," J Pineal Res, vol. 76, no. 3, p. e12952, Apr 2024, doi: 10.1111/jpi.12952.