Difference between revisions of "Part:BBa K5267001"

 
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<partinfo>BBa_K5267001 short</partinfo>
 
<partinfo>BBa_K5267001 short</partinfo>
  
<p> The mammalian MT1 melatonin receptor is a G protein-coupled receptor (GPCR) primarily coupled to the Gi/o protein family. This receptor plays a crucial role in regulating circadian rhythms and sleep-wake cycles by responding to melatonin, a hormone produced by the pineal gland. The MT1 receptor has a seven-transmembrane domain structure characteristic of GPCRs and is involved in inhibiting adenylate cyclase activity, leading to decreased levels of cAMP. It also participates in other signaling pathways, including the activation of phospholipase C and the regulation of intracellular calcium levels. Structural studies of MT1 reveal unique features such as a "lid-like" structure in the extracellular loop 2 (ECL2) that influences ligand binding and selectivity. This part is essential for projects involving the study of circadian biology, sleep regulation, and the pharmacological targeting of melatonin receptors. </p>
+
<p>The mammalian MT1 melatonin receptor is a G protein-coupled receptor (GPCR). MT1 plays a crucial role in regulating circadian rhythms and sleep-wake cycles by responding to melatonin, a hormone produced by the pineal gland. </p>
 +
<p>MT1 has a seven-transmembrane domain structure characteristic of GPCRs. Structural studies of MT1 reveal unique features, such as a "lid-like" structure in the extracellular loop 2 (ECL2) that influences ligand binding and selectivity. </p>
 +
<p>Upon melatonin-mediated activation of the melatonin receptor MT1, several pathways, such as the cAMP-PKA pathways and Ca2+ signaling pathways, are activated, thereby influencing gene expression related to cellular processes such as metabolism, growth, and apoptosis.</p
 +
>
 +
 
 
<!-- -->
 
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===Sequence and Features===
+
==Sequence and Features==
 
<partinfo>BBa_K5267001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5267001 SequenceAndFeatures</partinfo>
  
===Profile===
+
==Profile==
 
Name: MTNR1a
 
Name: MTNR1a
<br> Base Pairs: 1050bp
+
<br>Base Pairs: 1050bp
<br> Origin: Homo sapiens
+
<br>Origin: Homo sapiens
<br> Properties: A GPCR that responds to melatonin
+
<br>Properties: A GPCR that responds to melatonin
<br> Short description: MTNR1a
+
<br>Short description: MTNR1a
<br> Full description: The part encodes a 7-transmembrane receptor, which responses to melatonin.
+
<br>Full description: The part encodes a 7-transmembrane melatonin receptor MTNR1a, which responses to melatonin.
  
  
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<!-- Add more about the biology of this part here -->
 
<!-- Add more about the biology of this part here -->
 
===Usage and Biology===
 
===Usage and Biology===
<p> This part of gene encodes one of two high affinity forms of a receptor for melatonin, the primary hormone secreted by the pineal gland. This receptor is a G-protein coupled, 7-transmembrane receptor, a rhodopsin-like class A receptor that is responsible for melatonin effects on mammalian circadian rhythm and reproductive alterations affected by day length. The receptor is an integral membrane protein that is resadily detectable and localized to two specific regions of the brain. The hypothalamic suprachiasmatic nucleus appears to be involved in circadian rhythm while the hypophysial pars tuberalis may be responsible for the reproductive effects of melatonin.[1] </p>
+
<p>This part encodes MT1, one of the two high-affinity forms of the melatonin receptor, and is the core component of cell-based screening platform for melatonin receptor agonist. The MT1 receptor is a G-protein coupled, 7-transmembrane receptor, a rhodopsin-like class A receptor responsible for melatonin's effects on mammalian circadian rhythm and reproductive alterations influenced by day length. The receptor is an integral membrane protein that is readily detectable and localized to two specific regions of the brain: the hypothalamic suprachiasmatic nucleus, which is involved in circadian rhythm regulation, and the hypophysial pars tuberalis, which may be responsible for the reproductive effects of melatonin.[1]</p>
 
+
<p>Introducing this part into mammalian cells enables the cells to express MT1, allowing them to sense the stimulation of melatonin or activation of melatonin receptor by drugs.</p>
<p> In the human body, melatonin (N-acetyl-5-methoxytryptamine) is a widespread neurohormone with roles in circadian rhythm regulation, antioxidative protection and several other functions. It binds to the ligand binding pocket of melatonin receptor with high affinity in the human body.[2] </p>
+
<p>In the human body, melatonin (N-acetyl-5-methoxytryptamine) is a widespread neurohormone with roles in circadian rhythm regulation, antioxidative protection, and several other functions. It binds to the ligand-binding pocket of the melatonin receptor with high affinity.[2] The figure from Okamoto, H. H (2024) shows the overall structure of MT1 in both its activated and inactivated forms, and the position of the ligand-binding pocket of MT1, where melatonin binds to activate downstream gene pathways.[2]</p>
 
+
<p>In our project, this part, together with downstream synthetic reporter system such as P_5xCRE>IgK->Nluc->bGH_polyA(BBa_K5267041),P_7xNFAT->IgK->Nluc->bGH_polyA (BBa_K5267044), enables mammalian cells to respond to drugs that can activate the melatonin receptor. The part lays the foundational basis for a cell-based screening platform and subsequent experiments for screening melatonin receptor agonists.</p>
<p> The figure from Okamoto, H. H., Cecon, E., Nureki, O., Rivara, S., & Jockers, R. (2024) shows the overall structure of MT1, both activated structure and inactivated structure, and the position of the ligand binding pocket of MT1, where allows melatonin binds to it and activate of downstream gene pathways.[2] </p>
+
  
 
<html>
 
<html>
  
 
<figure class="figure">
 
<figure class="figure">
<div style="width=100%;height=auto;align-items:center">
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<div style="width=100%; height=auto; text-align: center">
<img src="https://static.igem.wiki/teams/5267/mao-parts/mt1-figure-1.jpg" class="figure-img img-fluid rounded"  height="400px">
+
<img src="https://static.igem.wiki/teams/5267/runtimeerror/mtnr1a-1.png" class="figure-img img-fluid rounded"  height="800px">
 +
</div>
 
</figure>
 
</figure>
 
</html>
 
</html>
  
  
<p> Figure: Overall structures of MT1 (A: inactive state [PDB ID: 6ME2], H: active state [PDB ID: 7DB6]), J: active state [PDB ID: 7VH0]). (B) Top view (left) and side view (right) of MT1 in an inactive state [PDB ID: 6ME2]. (C) Overall TM6 movement during receptor activation of MT1 (inactive state: [PDB ID: 6ME2] and active state: [PDB ID: 7DB6]). (E) Ligand binding site of crystal structures of MT1 (left top: [PDB ID: 6ME2], left bottom: [PDB ID: 6ME3], right top: [PDB ID: 6ME4], right bottom: [PDB ID: 6ME5]). (I) Ligand binding site of cryo‐EM structures of MT1 (left: [PDB ID: 7DB6], middle: [PDB ID: 7VGY], right: [PDB ID: 7VGZ]).[2] </p>
+
<p> '''Figure 1. Overall structures of MT1 (A) Inactive state [PDB ID: 6ME2]. (B) Top view (left) and side view (right) of MT1 in an inactive state [PDB ID: 6ME2]. (C) Overall TM6 movement during receptor activation of MT1 (inactive state: [PDB ID: 6ME2] and active state: [PDB ID: 7DB6]). (D) Ligand binding site of crystal structures of MT1 (left top: [PDB ID: 6ME2], left bottom: [PDB ID: 6ME3], right top: [PDB ID: 6ME4], right bottom: [PDB ID: 6ME5]). (E) active state [PDB ID: 7DB6]. (F) Ligand binding site of cryo‐EM structures of MT1 (left: [PDB ID: 7DB6], middle: [PDB ID: 7VGY], right: [PDB ID: 7VGZ]).[2]''' </p>
 
<html>
 
<html>
  
 
<figure class="figure">
 
<figure class="figure">
<div style="width=100%;height=auto;align-items:center">
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<div style="width=100%; height=auto; text-align: center">
<img src="https://static.igem.wiki/teams/5267/mao-parts/mt1-figrue2.jpg" class="figure-img img-fluid rounded"  height="200px">
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<img src="https://static.igem.wiki/teams/5267/mao-parts/mt1-figrue2.jpg" class="figure-img img-fluid rounded"  height="270px">
 +
</div>
 
</figure>
 
</figure>
 
</html>
 
</html>
  
 
<p>[2]</p>
 
<p>[2]</p>
<p> Figure: (A) Overview of structural changes during activation of MT1 (inactive state: [PDB ID: 6ME2], active state: [PDB ID: 7DB6]). (B) Conformational changes from the ligand binding site to the PIF motif. (C) Conformational changes from the Na+ binding site to the DRY (NRY in MTRs) motif.[2] </p>
+
<p> '''Figure 2 (A) Overview of structural changes during activation of MT1 (inactive state: [PDB ID: 6ME2], active state: [PDB ID: 7DB6]). (B) Conformational changes from the ligand binding site to the PIF motif. (C) Conformational changes from the Na+ binding site to the DRY (NRY in MTRs) motif.[2]''' </p>
  
<p> The MTNR1a part is cloned after the CMV promotor and before the bGH polyA sequence in the frame of Sleeping Beauty transposable element. After transfection into HEK293 cells, the P<sub>CMV</sub>->MTNR1a->BGH polyA device could stably exist in the genome of HEK293 cells. </p>
+
<p> To construct a MTNR1a-expressing plasmid, the MTNR1a part is cloned downstream of the CMV promoter and upstream of the bGH polyA sequence within the framework of the Sleeping Beauty transposable element. After transfection into HEK293 cells, the PCMV->MTNR1a->bGH polyA construct can integrate stably into the genome of HEK293 cells </p>
  
===Functional Validation===
+
==Functional Validation==
<p> In order to validate the basic part MTNR1a, which can express MTNR1a 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)[2], activating the expression of gene with CRE (cAMP response element) promotors. </p>
+
  
<p> 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 P<sub>CMV</sub> -> MTNR1a -> bGH polyA and the other carrying the construct P<sub>4xCRE</sub> -> P _ min -> lgK -> Nluc -> bGH polyA, 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 pLeo694 plasmid carrying the P<sub>CMV</sub> -> MTNR1a -> bGH_polyA 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. </p>
+
<p>According to previous studies, upon melatonin-mediated activation of the melatonin receptor MT1, several pathways, such as cAMP-PKA pathways and Ca2+ signaling pathways are activated. </p>
  
<p> The experimental results are as follows: </p>
+
<p> Based on the mechanism, we therefore utilized two distinct methodologies to validate the activation of Melatonin receptor, specifically focusing on the downstream signaling transduction of cAMP-PKA pathway and the Ca2+ signaling pathway. </p>
 +
 
 +
===Characterization strategy 1===
 +
<p> In order to validate the basic part MTNR1a, which can express MTNR1a receptor in mammalian cell chassis, we first focus on The cAMP-PKA pathway.</p>
 +
<p>'''The cAMP-PKA pathway''' is activated when melatonin binds to the MT1. This binding activates the associated G proteins, which in turn stimulate adenylate cyclase to convert ATP into cyclic adenosine monophosphate (cAMP). The increase in cAMP levels activates protein kinase A (PKA), which phosphorylates various target proteins, leading to a cascade of downstream effects. For instance, PKA can modulate the activity of transcription factors such as cAMP response element-binding protein (CREB), thereby influencing gene expression related to cellular processes such as metabolism, growth, and apoptosis. To assess the activation of cAMP-PKA pathway, synthetic CRE promoter  P4xCRE -> P _ min -> lgK -> Nluc -> bGH polyA (Composite part: BBa_K5267040) was used. CREB activation can therefore activate the NanoLuc expression in BBa_K5267040 with CREB binding to 4xCRE promoter.</p>
 +
<p>For such matter, part expressing MTNR1A (Basic part: BBa_K5267047) and the synthetic CRE promoter were co-transfected into HEK293 cells, and the melatonin was added post-transfection. 48 hours post-transfection, the NanoLuc activity was assessed to characterize the activation of the cAMP-PKA pathway.</p>
 +
===Result===
 +
The experimental results are as follows:
 
<html>
 
<html>
  
 
<figure class="figure">
 
<figure class="figure">
<div style="width=100%;height=auto;align-items:center">
+
<div style="width=100%; height=auto; text-align: center">
<img src="https://static.igem.wiki/teams/5267/mao-parts/mt1-figure3.jpg" class="figure-img img-fluid rounded"  height="400px">
+
<img src="https://static.igem.wiki/teams/5267/mao-parts/mt1gaoqing.png" class="figure-img img-fluid rounded"  height="400px">
 +
</div>
 
</figure>
 
</figure>
 
</html>
 
</html>
  
<p> Figure: The HEK293 stable cell line co-transfected by plasmid A and B, or plasmid A and C at the transfection ratio of 50ng : 100ng, stimulated by 1nM melatonin, and Nluc expression is tested 48 hours later. The results are shown above.
+
<p> '''Figure 3 The HEK293 stable cell line co-transfected by pNC099(
<br> plasmid A: plasmid carrying PCRE4->IgK->Nluc->bGH_polyA in the frame of Sleeping Beauty transposable element
+
carrying PCRE4->IgK->Nluc->bGH_polyA in the frame of Sleeping Beauty transposable element.)and pNC099(carrying PCMV->MTNR1a->bGH_polyA in the frame of Sleeping Beauty transposable element), or pNC099 and pcDNA3.1(+).  at the transfection ratio of 50ng : 100ng, stimulated by 1nM melatonin, and NanoLuc expression is assessed 48 hours post transfection.
<br> plasmid B: plasmid carrying P<sub>CMV</sub>->MTNR1a->bGH_polyA in the frame of Sleeping Beauty transposable element
+
)''' </p>
<br> plasmid C: pcDNA3.1(+)</p>
+
Upon activation of melatonin receptors (MTs) by melatonin, the activity of NanoLuc was significantly elevated compared to the control group without melatonin stimulation. This result demonstrates that the part is capable of sensing melatonin stimulation and subsequently activating CRE-promoter-initiated transcription as expected.
 +
===Characterization strategy 2===
  
 
<p> We have also validated the part by detecting the change of Ca<sup>2+</sup> concentration, for it has also been reported that the Ca<sup>2+</sup> is up-regulated after the activation of MT1. </p>
 
<p> We have also validated the part by detecting the change of Ca<sup>2+</sup> concentration, for it has also been reported that the Ca<sup>2+</sup> is up-regulated after the activation of MT1. </p>
  
<p> We used GCaMP, an ultra-sensitive fluorescent protein for imaging Ca<sup>2+</sup>[4], to detect the change of intracellular Ca<sup>2+</sup> concentration. And used Thapsigargin to stimulate the cell as a positive control, a drug that is reported to cause endoplasmic reticulum stress, which will lead to the up-regulation of Ca<sup>2+</sup> in the cytoplasm[5]. </p>
+
<p> For such matter, we used GCaMP (Composite part: BBa_K3755041), an ultra-sensitive protein-based sensor that allows visualization of Ca2+ dynamics within living cells[4]. Thapsigargin was used as a positive control to stimulate the cells, as it is known to induce endoplasmic reticulum stress, leading to an up-regulation of Ca2+ in the cytoplasm [3]</p>
 +
 
 +
===Result 2===
  
 
<p> The results are as follows: </p>
 
<p> The results are as follows: </p>
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<html>
 
<html>
 
<figure class="figure">
 
<figure class="figure">
<div style="width=100%;height=auto;align-items:center">
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<div style="width=100%; height=auto; text-align: center">
<img src="https://static.igem.wiki/teams/5267/runtimeerror/2.png"  height="400px">
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<img src="https://static.igem.wiki/teams/5267/mao-parts/mt2gaoqing2.png"  height="300px">
 +
</div>
 
</figure>
 
</figure>
 
</html>
 
</html>
  
<p> Figure:
+
<p> '''Figure 4 Real-time Ca<sup>2+</sup> fluorescence intensity of different groups of cell after the stimulation of melatonin or thapsigargin.'''
<br> The yellow group represents the cell only transfected by plasmid carrying P<sub>CMV</sub>->GCaMP and stimulated by 100 μM Melatonin, as the blank control.
+
</p>
<br> The blue group represents the cell co-transfected by plasmid carrying P<sub>CMV</sub>->GCaMP and plasmid carrying P<sub>CMV</sub>->MTNR1a, and stimulated by Melatonin, as the experimental group.
+
<br> The red group represents the cell only transfected by plasmid carrying P<sub>CMV</sub>->GCaMP and stimulated by thapsigargin, as the positive control.
+
<br> The change of Ca<sup>2+</sup> fluorescence intensity of the three groups are as follows. </p>
+
  
<p> The result indicates that the up-regulation of intracellular Ca<sup>2+</sup> concentration downstream the MTNR1a activation is strong but temporary, which may explain the reason why MTNR1a - Ca<sup>2+</sup> pathway did not work. </p>
+
<p> The results showed that the addition of both melatonin and thapsigargin induced a significant increase in the Ca<sup>2+</sup> fluorescent signal. This demonstrates that our construct is capable of activating the Ca<sup>2+</sup> signaling pathway upon melatonin stimulation as expected.</p>
  
<p> The results indicate that the part works well. </p>
+
<p> These findings indicate that the construct functions effectively in the mammalian cell chassis, and its activation by either thapsigargin or melatonin successfully leads to the activation of melatonin receptor-related pathways as anticipated. </p>
  
===References===
+
==References==
 
<p> [1] N. database, "Gene [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004 – [cited 2024 Sep 01]. Available from: https://www.ncbi.nlm.nih.gov/gene/," 2004. </p>
 
<p> [1] N. database, "Gene [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004 – [cited 2024 Sep 01]. Available from: https://www.ncbi.nlm.nih.gov/gene/," 2004. </p>
  
 
<p> [2] H. H. Okamoto, E. Cecon, O. Nureki, S. Rivara, and R. Jockers, “Melatonin receptor structure and signaling,” Journal of Pineal Research, vol. 76, no. 3, 2024. </p>
 
<p> [2] H. H. Okamoto, E. Cecon, O. Nureki, S. Rivara, and R. Jockers, “Melatonin receptor structure and signaling,” Journal of Pineal Research, vol. 76, no. 3, 2024. </p>
  
<p> [3] M. A. Ayoub, A. Levoye, P. Delagrange, and R. Jockers, “Preferential Formation of MT1/MT2 Melatonin Receptor Heterodimers with Distinct Ligand Interaction Properties Compared with MT2 Homodimers,” Molecular Pharmacology, vol. 66, no. 2, pp. 312-321, 2004. </p>
+
<p> [3] A. Abdullahi, M. Stanojcic, A. Parousis, D. Patsouris, and M. G. Jeschke, “Modeling Acute ER Stress in Vivo and in Vitro,” Shock, vol. 47, no. 4, pp. 506–513, Apr. 2017, doi: 10.1097/SHK.0000000000000759. </p>
 
+
<p> [4] T.-W. Chen et al., “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature, vol. 499, no. 7458, pp. 295–300, Jul. 2013, doi: 10.1038/nature12354. </p>
+
 
+
<p> [5] A. Abdullahi, M. Stanojcic, A. Parousis, D. Patsouris, and M. G. Jeschke, “Modeling Acute ER Stress in Vivo and in Vitro,” Shock, vol. 47, no. 4, pp. 506–513, Apr. 2017, doi: 10.1097/SHK.0000000000000759. </p>
+

Latest revision as of 12:42, 2 October 2024


Mammalian MT1 melatonin receptor, Gi-coupled GPCR.

The mammalian MT1 melatonin receptor is a G protein-coupled receptor (GPCR). MT1 plays a crucial role in regulating circadian rhythms and sleep-wake cycles by responding to melatonin, a hormone produced by the pineal gland.

MT1 has a seven-transmembrane domain structure characteristic of GPCRs. Structural studies of MT1 reveal unique features, such as a "lid-like" structure in the extracellular loop 2 (ECL2) that influences ligand binding and selectivity.

Upon melatonin-mediated activation of the melatonin receptor MT1, several pathways, such as the cAMP-PKA pathways and Ca2+ signaling pathways, are activated, thereby influencing gene expression related to cellular processes such as metabolism, growth, and apoptosis.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 629
    Illegal BamHI site found at 809
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Profile

Name: MTNR1a
Base Pairs: 1050bp
Origin: Homo sapiens
Properties: A GPCR that responds to melatonin
Short description: MTNR1a
Full description: The part encodes a 7-transmembrane melatonin receptor MTNR1a, which responses to melatonin.


Usage and Biology

This part encodes MT1, one of the two high-affinity forms of the melatonin receptor, and is the core component of cell-based screening platform for melatonin receptor agonist. The MT1 receptor is a G-protein coupled, 7-transmembrane receptor, a rhodopsin-like class A receptor responsible for melatonin's effects on mammalian circadian rhythm and reproductive alterations influenced by day length. The receptor is an integral membrane protein that is readily detectable and localized to two specific regions of the brain: the hypothalamic suprachiasmatic nucleus, which is involved in circadian rhythm regulation, and the hypophysial pars tuberalis, which may be responsible for the reproductive effects of melatonin.[1]

Introducing this part into mammalian cells enables the cells to express MT1, allowing them to sense the stimulation of melatonin or activation of melatonin receptor by drugs.

In the human body, melatonin (N-acetyl-5-methoxytryptamine) is a widespread neurohormone with roles in circadian rhythm regulation, antioxidative protection, and several other functions. It binds to the ligand-binding pocket of the melatonin receptor with high affinity.[2] The figure from Okamoto, H. H (2024) shows the overall structure of MT1 in both its activated and inactivated forms, and the position of the ligand-binding pocket of MT1, where melatonin binds to activate downstream gene pathways.[2]

In our project, this part, together with downstream synthetic reporter system such as P_5xCRE>IgK->Nluc->bGH_polyA(BBa_K5267041),P_7xNFAT->IgK->Nluc->bGH_polyA (BBa_K5267044), enables mammalian cells to respond to drugs that can activate the melatonin receptor. The part lays the foundational basis for a cell-based screening platform and subsequent experiments for screening melatonin receptor agonists.


Figure 1. Overall structures of MT1 (A) Inactive state [PDB ID: 6ME2]. (B) Top view (left) and side view (right) of MT1 in an inactive state [PDB ID: 6ME2]. (C) Overall TM6 movement during receptor activation of MT1 (inactive state: [PDB ID: 6ME2] and active state: [PDB ID: 7DB6]). (D) Ligand binding site of crystal structures of MT1 (left top: [PDB ID: 6ME2], left bottom: [PDB ID: 6ME3], right top: [PDB ID: 6ME4], right bottom: [PDB ID: 6ME5]). (E) active state [PDB ID: 7DB6]. (F) Ligand binding site of cryo‐EM structures of MT1 (left: [PDB ID: 7DB6], middle: [PDB ID: 7VGY], right: [PDB ID: 7VGZ]).[2]

[2]

Figure 2 (A) Overview of structural changes during activation of MT1 (inactive state: [PDB ID: 6ME2], active state: [PDB ID: 7DB6]). (B) Conformational changes from the ligand binding site to the PIF motif. (C) Conformational changes from the Na+ binding site to the DRY (NRY in MTRs) motif.[2]

To construct a MTNR1a-expressing plasmid, the MTNR1a part is cloned downstream of the CMV promoter and upstream of the bGH polyA sequence within the framework of the Sleeping Beauty transposable element. After transfection into HEK293 cells, the PCMV->MTNR1a->bGH polyA construct can integrate stably into the genome of HEK293 cells

Functional Validation

According to previous studies, upon melatonin-mediated activation of the melatonin receptor MT1, several pathways, such as cAMP-PKA pathways and Ca2+ signaling pathways are activated.

Based on the mechanism, we therefore utilized two distinct methodologies to validate the activation of Melatonin receptor, specifically focusing on the downstream signaling transduction of cAMP-PKA pathway and the Ca2+ signaling pathway.

Characterization strategy 1

In order to validate the basic part MTNR1a, which can express MTNR1a receptor in mammalian cell chassis, we first focus on The cAMP-PKA pathway.

The cAMP-PKA pathway is activated when melatonin binds to the MT1. This binding activates the associated G proteins, which in turn stimulate adenylate cyclase to convert ATP into cyclic adenosine monophosphate (cAMP). The increase in cAMP levels activates protein kinase A (PKA), which phosphorylates various target proteins, leading to a cascade of downstream effects. For instance, PKA can modulate the activity of transcription factors such as cAMP response element-binding protein (CREB), thereby influencing gene expression related to cellular processes such as metabolism, growth, and apoptosis. To assess the activation of cAMP-PKA pathway, synthetic CRE promoter P4xCRE -> P _ min -> lgK -> Nluc -> bGH polyA (Composite part: BBa_K5267040) was used. CREB activation can therefore activate the NanoLuc expression in BBa_K5267040 with CREB binding to 4xCRE promoter.

For such matter, part expressing MTNR1A (Basic part: BBa_K5267047) and the synthetic CRE promoter were co-transfected into HEK293 cells, and the melatonin was added post-transfection. 48 hours post-transfection, the NanoLuc activity was assessed to characterize the activation of the cAMP-PKA pathway.

Result

The experimental results are as follows:

Figure 3 The HEK293 stable cell line co-transfected by pNC099( carrying PCRE4->IgK->Nluc->bGH_polyA in the frame of Sleeping Beauty transposable element.)and pNC099(carrying PCMV->MTNR1a->bGH_polyA in the frame of Sleeping Beauty transposable element), or pNC099 and pcDNA3.1(+). at the transfection ratio of 50ng : 100ng, stimulated by 1nM melatonin, and NanoLuc expression is assessed 48 hours post transfection. )

Upon activation of melatonin receptors (MTs) by melatonin, the activity of NanoLuc was significantly elevated compared to the control group without melatonin stimulation. This result demonstrates that the part is capable of sensing melatonin stimulation and subsequently activating CRE-promoter-initiated transcription as expected.

Characterization strategy 2

We have also validated the part by detecting the change of Ca2+ concentration, for it has also been reported that the Ca2+ is up-regulated after the activation of MT1.

For such matter, we used GCaMP (Composite part: BBa_K3755041), an ultra-sensitive protein-based sensor that allows visualization of Ca2+ dynamics within living cells[4]. Thapsigargin was used as a positive control to stimulate the cells, as it is known to induce endoplasmic reticulum stress, leading to an up-regulation of Ca2+ in the cytoplasm [3]

Result 2

The results are as follows:

Figure 4 Real-time Ca2+ fluorescence intensity of different groups of cell after the stimulation of melatonin or thapsigargin.

The results showed that the addition of both melatonin and thapsigargin induced a significant increase in the Ca2+ fluorescent signal. This demonstrates that our construct is capable of activating the Ca2+ signaling pathway upon melatonin stimulation as expected.

These findings indicate that the construct functions effectively in the mammalian cell chassis, and its activation by either thapsigargin or melatonin successfully leads to the activation of melatonin receptor-related pathways as anticipated.

References

[1] N. database, "Gene [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004 – [cited 2024 Sep 01]. Available from: https://www.ncbi.nlm.nih.gov/gene/," 2004.

[2] H. H. Okamoto, E. Cecon, O. Nureki, S. Rivara, and R. Jockers, “Melatonin receptor structure and signaling,” Journal of Pineal Research, vol. 76, no. 3, 2024.

[3] A. Abdullahi, M. Stanojcic, A. Parousis, D. Patsouris, and M. G. Jeschke, “Modeling Acute ER Stress in Vivo and in Vitro,” Shock, vol. 47, no. 4, pp. 506–513, Apr. 2017, doi: 10.1097/SHK.0000000000000759.