Part:BBa_K5267040
P_4xCRE->IgK->Nluc->bGH_polyA
P_4×CRE->IgK->Nluc->bGH_polyA
The cAMP/PKA/CREB pathway is a well-established signaling pathway in mammalian cells, regulated by various upstream pathways such as G-protein-coupled receptors (GPCRs). Upon activation of GPCRs, the cAMP/PKA/CREB pathways are triggered, ultimately leading to the phosphorylation of the endogenous transcription factor CREB. This phosphorylation allows CREB to bind to cAMP response elements (CRE) and regulate the expression of downstream genes.
The composite part P_4×CRE->IgK->Nluc->bGH_polyA (Part:BBa K5267040) is designed to sense and characterize the status of the cAMP/PKA/CREB pathway based on its signaling transduction mechanism. Activation of this pathway results in increased phosphorylation of CREB, which then binds to four tandem repeats of the CRE site within the P_4CRE promoter, activating the expression of the reporter gene NanoLuc luciferase.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 238
- 1000COMPATIBLE WITH RFC[1000]
Profile
Name:P_4×CRE->IgK->Nluc->bGH_polyA
Base Pairs: 944bp
Origin: Homo sapiens
Properties:Enables sensing and characterizing the status (such as activation/inhibition) of the cAMP/PKA/CREB pathway
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_4×CRE ([1]): A synthetic promoter that assess the activation status of the cAMP/PKA/CREB signaling pathways, with 4 tandem repeats of CRE serving as a binding site for phosphorylated CREB.
2. IgK (BBa_K3117006): A signal sequence directing the protein into the secretory pathway.
3. Nluc (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 (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
The composite part P_4×CRE->IgK->Nluc->bGH_polyA (BBa_K5267040) is essential for evaluating the responsiveness of the melatonin signaling pathway. Standard approaches for studying signaling pathways typically involve cloning the response element of the relevant transcription factor into a luciferase reporter gene vector, such as Pcre-luc.[3] Due to the limited transcriptional impact of a single response element, we incorporated multiple tandem copies of the element near the reporter gene's genomic location. This amplification enhanced the initiation efficacy of the signaling cascade. Therefore, we constructed the Pmin_4×CRE sequence, which includes a 5′ minimal promoter incorporating 4 tandem repeats of CREs.[4] (Figure 2)
Function Test
Method
Forskolin, a known activator of adenylate cyclase (AC), is used to stimulate the cAMP signaling pathway2. We used forskolin as a stimulant and constructed plasmids carrying the reporter gene NanoLuc and different CRE copies (4×CRE, 5×CRE, and 6×CRE; BBa_K5267040, BBa_K5267041, BBa_K5267042), such as our composite part 4×CRE_Pmin-IgK->Nluc->bGH_polyA (BBa_K5267040). These plasmids were co-transfected with PCMV->MTNR1A->bGH_polyA (BBa_K5267047) into HEK293T cells. The cells were then stimulated with forskolin to determine whether forskolin-induced activation of the cAMP/PKA/CREB signaling pathway can be sensed and characterized by our composite part P_4×CRE -> IgK -> Nluc -> bGH_polyA.
To further validate and evaluate the function of the composite part P_4×CRE -> IgK -> Nluc -> bGH polyA (Part: BBa_K5267040), we co-transfected it with MTNR1A (BBa_K5267001) into HEK-293T cell lines. This setup aims to mimic the melatonin receptor-mediated cAMP/PKA/CREB signaling transduction pathway. The melatonin-activated MTNR1A receptors should activate CREB, thereby binding to the 4×CREs and initiating NanoLuc expression from the composite part.
Theoretically, upon stimulation with forskolin or melatonin, the cAMP/PKA/CREB signaling pathway is triggered, ultimately leading to the phosphorylation of the endogenous transcription factor CREB and activation of the Pcre, thereby regulating the expression of downstream genes.
Result
We measured the expression of NanoLuc in both forskolin-treated and untreated groups 48 hours post-stimulation. The results confirmed that the cAMP/PKA/CREB signaling pathway was activated by forskolin as expected. Platforms with different tandem repeats of CRE exhibited varying fold changes in NanoLuc expression. Notably, cells transfected with P_4×CRE->IgK->Nluc->bGH polyA (BBa_K5267040) showed the highest fold change (~8.62 fold) compared to the control. These results demonstrate the correct responsiveness of the cAMP signaling pathway (Figure 3).
Figure 4. Characterization of composite part P_4×CRE->IgK->Nluc->bGH_polyA. (A) Co-expression of MTNR1A and Pcre-promoter variants enables robust transcriptional activation upon melatonin stimulation. Melatonin stimulation was added to HEK-293T cells co-transfected with PCMV-MTNR1A and P_4xCRE-IgK-Nluc, P_5×CRE-IgK-Nluc, P_6×CRE-IgK-Nluc respectively. (B) Co-expression of MTNR1A and Pcre-promoter variants enables robust transcriptional activation upon melatonin stimulation. HEK-293T cells were co-transfected with PCMV-MTNR1A and P_4×CRE-IgK-Nluc under various transfection ratio (100:25/100:50/100:100/100:200). (C) Step-response dynamics of HEKMT cells under melatonin treatment. HEK-293T cell lines expressing PCMV-MTNR1A and P_4×CRE-IgK-Nluc was stimulated with various concentrations of melatonin (0.1nM /0.5nM /0.7nM /1nM /10nM /30nM) . Data are mean±SD of NanoLuc expression levels measured 24 h after melatonin stimulation (n = 3 independent experiments)
Moreover, we utilized melatonin receptor-mediated activation of the cAMP/PKA/CREB pathway as a strategy to test the functionalities of the composite part P_4×CRE->IgK->Nluc->bGH polyA. The results showed a significant increase in NanoLuc expression in the melatonin-stimulated group compared to the unstimulated group (Figure 4A and B), indicating that the composite part can correctly respond to melatonin stimulation as expected. Notably, the NanoLuc expression level in cells transfected with P_4×CRE -> IgK -> Nluc -> bGH polyA (BBa_K5267040) was the highest among all experimental groups, with the highest fold change (~8.62-fold) compared to the control.
To further characterize this composite part, we generated a stable HEKMT cell line carrying both the Pcre-Nluc and PCMV-MTNR1A expression cassettes using the Sleeping Beauty transposon system, as shown in Figure 3C. The results showed dose-dependent responses upon stimulation with different concentrations of melatonin. Therefore, we selected this composite part as the optimal component and constructed a cell-based screening platform based on P_4×CRE->IgK->Nluc->bGH polyA (BBa_K5267040) for our iGEM project.
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.
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