Part:BBa_K5267040
P_4xCRE->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.
Usage and Biology
In the face of increasingly frequent sleep problems, the use of melatonin treatment is an effective means. The function of melatonin is mainly mediated by two G-protein-coupled receptors, MT1 and MT2, to regulate circadian rhythms and sleep. MT1 is mainly distributed in the hypothalamus and mainly concentrated in the suprachiasmatic nucleus (SCN), which regulates rapid eye movement sleep (REM) and plays a major role in the regulation of circadian rhythm.[1] When melatonin binds to melatonin receptors in the body, it activates downstream pathways. Through literature search, we found that there are three pathways: cAMP-CREB pathway, PI3K-ERK pathway and calcium ion channel signaling pathway.[2] In the CAMP-CREB pathway, the activated PKA caused by the MT receptor is transferred to the nucleus, phosphorylation activates CREB, and binds to the cAMP response element (CRE) in the eukaryotic promoter. And the cofactor CREB-binding protein (CBP) phosphorylation synergies to regulate downstream gene transcription.。
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.
[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.
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 (Fig.1). 4xCRE-Pmin (BBa_K5267004) is loaded into the vector as signal response element, IgK (BBa_K3117006)is a signaling sequence, directing the protein into the secretory pathway , Nluc(BBa_K2728003)works as the original test report to detect cAMP concentration, [3] if it is successfully expressed, 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.
Special design
reference
[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.
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