Part:BBa_K5267042

P_6xCRE->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.Then the cell will glow blue fluorescence.

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 118
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 391
  
• 1000
  COMPATIBLE WITH RFC[1000]

Profile

Name:P_6xCRE->IgK->Nluc->bGH_polyA
Base Pairs: 1097bp
Origin: Homo sapiens
Properties:As a reporter to show whether melatonin is accepted or not

Usage and Biology

G-protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes. G proteins are specialized proteins with the ability to bind the nucleotides guanosine triphosphate (GTP) and guanosine diphosphate (GDP). Ligand binding to the GPCR causes a change in the receptor conformation that in turn binds and activates the G-protein. The active form of the G-protein is then released from the surface of the receptor, dissociating into its subunits. Both subunits will then activate their specific effectors, causing the release of second messengers. These messengers are recognised by protein kinases leading to their activation and triggering the signaling cascade towards a cellular event.[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 G-protein coupled receptors (GPCRs), adenylate cyclase is activated via the Gα subunit, culminating in elevated levels of intracellular cAMP. Subsequently, cAMP interacts with the regulatory subunits of protein kinase A (PKA), precipitating 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 specifically binds to the coactivator cAMP response element-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]

We hope to design a reporter plasmid vector for the melatonin receptor pathway and introduce it into the recipient cells for reporting。This part is based on the cAMP-CREB pathway with MT1 acting as a cell receptor (Figure 1). P_6xCRE(BBa_K5267006) is loaded into the vector as a diagnostic element to assess the successful activation of the receptor's downstream signaling pathways., IgK (BBa_K3117006)is a signaling sequence, directing the protein into the secretory pathway , Nluc(BBa_K2728003) is engineered for optimal performance as a luminescent reporter to detect cAMP concentration [3]. If it is successfully expressed, the cell will glow blue fluorescence. bGH_polyA (BBa_K1313006)is a terminator, controlling the cessation of gene expression .

Based on the composition of this part, it functions as a cAMP concentration detection platform, and finally, this part is successfully delivered into HEK293 cell line and currently works properly when stimulated by melatonin.

< img src="https://static.igem.wiki/teams/5267/mao-parts/108bf7048666342015244d8e67a5ee3.png" class="figure-img img-fluid rounded" height="400px">

Figure 1. Schematic of cAMP-CREB signal pathway. 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.

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 P_6xCRE 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.

Method

In order to validate the composite part P_6xCRE->IgK->Nluc->bGH_polyA(BBa K5267042), 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(BBa K5267047)and the other carrying the construct P_6xCRE-> lgK -> Nluc -> bGH polyA(BBa K5267042), 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_6xCRE -> lgK -> Nluc -> bGH polyA(BBa K5267042) 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

< img src="https://static.igem.wiki/teams/5267/mao-parts/huge4xcre-f3.png" class="figure-img img-fluid rounded" height="400px">

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(4xCRE-IgK-Nluc), pNC016(5xCRE-IgK-Nluc), pNC017(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(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(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.