Part:BBa_K5267048

P_CMV->MTNR1B->bGH_polyA

Expression of MT2 gene

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 614
  Illegal BamHI site found at 1255
  Illegal BamHI site found at 1435
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Profile

Name: P_CMV->MTNR1A->bGH_polyA
Base Pairs: 1909bp
Origin: Homo sapiens
Properties: Expression of MT1 gene

Usage and Biology

The melatonin receptors (MTs), specifically MT1 (melatonin receptor type 1) and MT2 (melatonin receptor type 2), are classified under the G protein-coupled receptor (GPCR) family A, with melatonin acting as their endogenous agonist. These receptors are pivotal in the regulation of the circadian rhythm within the human body and are intricately linked to a spectrum of vital physiological processes, including reproductive function, neuronal modulation, and immune system regulation. Furthermore, MTs represent a significant therapeutic target for the amelioration of various pathologies, such as insomnia, affective disorders, and oncological conditions.[1].

Despite the high degree of homology between human MT1 and MT2 receptors, there are considerable differences in their tissue distribution, intracellular signaling mechanisms, and physiological roles. The current dearth of selectivity in melatonin-based pharmaceuticals for either MT1 or MT2 receptors impedes the precision therapy of related disorders. Consequently, the detailed structural elucidation of the MTs agonist binding site is of paramount importance for the development of targeted pharmacotherapeutics.[2]

In light of this, we have engineered a pathway that, upon promoter activation, initiates the synthesis of the MT1 melatonin receptor protein. This strategy is instrumental in the establishment of a cellular assay system designed for the screening of melatonin receptor agonists.(Figure 1)

Figure 1. MT1 gene expression pathway.

Special design

To achieve the objective of driving the expression of the MT1 melatonin receptor, we strategically selected the CMV promoter, a robust promoter derived from the human Cytomegalovirus (CMV), known for its high transcriptional activity in eukaryotic cells. The CMV promoter has been demonstrated to be highly efficacious in facilitating the expression of lengthy and complex genes within HEK-293T cells.[3]

Utilizing the CMV promoter, we initiated the transcription of the MT1 gene within the construct of the gene expression vector, thereby enhancing the expression profile of the MT1 gene. This approach is anticipated to provide a foundation for the development of a cell-based screening platform for melatonin receptor agonists.

Function test

To substantiate the functionality of the aforementioned constructs, human embryonic kidney 293 cells (HEK293) were co-transfected with expression vectors harboring the newly engineered NanoLuc-reporter genes.

Thapsigargin (TG) is a known ER stress inducer that increases intracellular calcium (Ca2+) concentration by inhibiting the calcium atpase (SERCA pump) in the ER. This increased calcium concentration can activate a variety of cell signaling pathways, including the NFAT (nuclear factor of activated T cells) pathway, thereby analyzing the sensitivity and activation threshold of the NFAT pathway.

At the cellular level, melatonin can affect the activity of calcium channels through its receptors, leading to changes in intracellular calcium concentration. The reporting system is designed to be responsive to oscillations in intracellular Ca2+ concentrations.

The optimal configuration of the reporting pathway was ascertained by evaluating and comparing the relative luminescence unit (RLU) expression profiles of the NanoLuc reporter genes, thereby discerning the most efficacious design among the various constructs.

Method

Initially, we co-transfected HEK293T cells with an expression vector encoding the NanoLuc reporter gene, followed by the induction of an intracellular calcium ion (Ca2+) response using thapsigargin. Each experimental condition was performed in triplicate, alongside a non-transfected control group lacking NFAT (BBa_K5267049).

Upon a 48-hour exposure to thapsigargin, the luminescence intensity of the NanoLuc reporter, expressed in relative light units (RLU), was measured across all experimental groups to evaluate the transcriptional activity evoked by thapsigargin stimulation.

Subsequently, the same reporter gene was co-transfected into HEK293T cells, and the intracellular Ca2+ response was provoked by melatonin. The experiments were conducted with three replicates each, and a control group was included, which was not subjected to melatonin stimulation.

After a 24-hour melatonin stimulation period, the luminescence intensity of the NanoLuc reporter element, quantified in RLU, was assessed in all experimental groups to determine the transcriptional activity induced by melatonin treatment.

Results

Figure 3. NFAT activation in response to calcium ion signaling. (Regulation by TG)

HEK-293T cells were transfected with plasmids containing different promoters with 1×/5×/6×/7×NFAT elements respectively. Data are mean±SD of NanoLuc expression levels measured at 48 h after thapsigargin stimulation (n = 3 independent experiments).Upon a 48-hour incubation period, stimulation of the 1xNFAT promoter with 10 nM thapsigargin resulted in a mean augmentation of the NanoLuc reporter gene expression to a magnitude that was 1.96-fold superior to that ascertained in the absence of thapsigargin induction.

Figure 4. NFAT activation in response to calcium ion signaling.(Regulation by MT)

HEK-293T cells were co-transfected with melatonin receptor plasmid pCJ008(PCMV-MTNR1A) and plasmids containing different promoters with various copy numbers of NFAT elements pNC008(PNFAT_1-IgK-Nluc), pNC004(PNFAT_5-IgK-Nluc), pNC012(PNFAT_6-IgK-Nluc) and pNC010(PNFAT_7-IgK-Nluc) melatonin stimulation. Data are mean±SD of NanoLuc expression levels measured at 24 h after melatonin stimulation (n = 3 independent experiments).

In the in the P_1xNFAT->IgK->Nluc->bGH_polyA system, the introduction of 1 nM melatonin did not elicit a statistically significant increase in the mean NanoLuc expression levels compared to the system devoid of melatonin treatment.

Reference

[1] W. Zhang, T. Takahara, T. Achiha, H. Shibata, and M. Maki, “Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-Palindromic NFAT-Response Elements Facilitates Analysis of Biological Endpoint Effects of Cellular Ca2+ Mobilization,” Int. J. Mol. Sci., vol. 19, no. 2, p. 605, Feb. 2018, doi: 10.3390/ijms19020605.
[2] K. A. Strait, P. K. Stricklett, R. M. Kohan, and D. E. Kohan, “Identification of Two Nuclear Factor of Activated T-cells (NFAT)-response Elements in the 5′-Upstream Regulatory Region of the ET-1 Promoter,” J. Biol. Chem., vol. 285, no. 37, pp. 28520–28528, Sep. 2010, doi: 10.1074/jbc.M110.153189.