Part:BBa_K5267041

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 103
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 376
1000
COMPATIBLE 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.

Figure 1 Concrete schematic diagram of BBa_K5267041 composite part

Special design

The composite part P_5xCRE->IgK->Nluc->bGH_polyA (Part: 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 P_min_5xCRE sequence, which includes a 5′ minimal promoter incorporating 5 tandem repeats of CREs.[4] (Figure 2)

Figure 2. Schematic of cAMP-CREB Signaling Transduction. When melatonin binds to the MT1, it activates adenylate cyclase (AC), which in turn regulates the level of the second messenger cAMP. The increase in cAMP activates protein kinase A (PKA), which amplifies the signal. PKA then catalyzes the phosphorylation of CREB in the nucleus, thereby binding to the P_4xCRE/P_5xCRE/P_6xCRE and initiate the transcription of Nluc

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 t4xCRE_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_4xCRE -> IgK -> Nluc -> bGH_polyA.

To further validate and evaluate the function of the composite part P_5xCRE -> IgK -> Nluc -> bGH polyA (Part: BBa_K5267041), we co-transfected it with MTNR1A (Basic Part: 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 5xCREs 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

Figure 2. CREB activation in response to cAMP signaling. (a) Schematic diagram of MT1 receptor activating the downstream cAMP pathway. (b) Melatonin stimulation applied to HEK-293T cells co-transfected with pCJ008(PCMV-MTNR1A) and pNC005(P_4xCRE-IgK-Nluc), pNC016(P_5xCRE-IgK-Nluc), pNC017(P_6xCRE-IgK-Nluc) respectively. Data are mean±SD of NanoLuc expression levels measured 24 h after melatonin stimulation (n = 3 independent experiments). (c) HEK-293T cells were co-transfected with pCJ008(PCMV-MTNR1A) and pNC005(P_4xCRE-IgK-Nluc) under various transfection ratio(100:25/100:50/100:100/100:200). Data are mean±SD of NanoLuc expression levels measured at 24 h after melatonin stimulation (n = 3 independent experiments). (d) Stable HEK-293T cells co-transfected with pCJ008(PCMV-MTNR1A) and pNC005(P_4xCRE-IgK-Nluc) stimulated with various concentrations of melatonin(0.1nM /0.5nM /0.7nM /1nM /10nM /30nM) . Data are mean±SD of NanoLuc expression levels measured at different time points

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.