Difference between revisions of "Part:BBa K4960021"
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<partinfo>BBa_K4960021 short</partinfo><br>
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enable the PVC-mediated alteration of mammalian cell energy expenditure
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===Profile===
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Name: Engineered Mitochondrial Uncoupler Pdp1NTD-EGFP-UCP1<br>
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Base Pairs: 1917 bp<br>
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Origin: <i>Photorhabdus, Aequorea Victoria, Homo Sapiens</i><br>
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Properties:enable the PVC-mediated alteration of mammalian cell energy expenditure
  
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===Usage and Biology===
 
===Usage and Biology===
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Noting the increasing demand for weight-loss drugs and the potential value and prospect of UCP1 (uncoupling protein 1) as a target for the treatment of obesity, [1] we conducted a series of design and experimental work on a modified UCP1 delivery strategy based on PVCs. Prior to this, we designed the payload in PVCs, a protein in which Pdp1NTD plays a key role in delivering protein loading into PVC, and we overexpressed UCP1 in the HEK293 cell line and added EGFP (Enhanced Green Fluorescent Protein) to see if it could target and work on the inner mitochondrial membrane.
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UCP1 is a key mitochondrial uncoupling protein in mammalian cells that regulates cellular energy expenditure and thermogenesis[1]. This part demonstrates a UCP1-based synthetic mitochondrial uncoupler that is compatable to the PVC delivery system (see the Description page of iGEM23_NUDT-CHINA wiki for more information about the PVC system). In mammalian cells, this part would be translocated into mitochondria and works as a proton transporter that disconnects oxygen consumption from ATP synthesis, thereby dissipates energy in the form of heat, leading to an increase in energy expenditure and basal metabolic rate.[2]
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===Special Design===
 
===Special Design===
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In order to prevent UCP1 from forming inclusion bodies after expression in Escherichia coli and facilitate the localization of UCP1 after transfection, we introduced EGFP protein for fusion expression with UCP1. We designed a flexible peptide linker (GGSGG) to link Pdp1NTD, UCP1 and EGFP to form a fusion protein. According to literature research, the fusion protein of UCP1 and EGFP is usually designed to attach EGFP to the C-terminal sequence of UCP1 to ensure the normal expression and function of UCP1[2] [3]( Fig. 1).
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This part includes a Pdp1NTD domain to ensure the compatability with the PVC delivery system, an EGFP domain for visualization and improving the protein production in E.coli, and a UCP1 mitochondrial uncoupling domain to enable the functionality. In [[Part:BBa_K4960022]], we have demonstrated that the Pdp1NTD-UCP1-EGFP configuration failed to effectively localize into mitochondria, possibly due to an unintended interaction between the Pdp1NTD domain and UCP1 (as highlighted in the red box in '''Figure 1a'''). To address this issue, we used AlphaFold2 to predict some of the possible structures and found that we could resolve this problem by swapping UCP1 and EGFP (as shown in '''Figure 1b'''). Consequently, we designed this part by connecting Pdp1NTD to the N-terminus EGFP and a UCP1 to the C-terminus (as depicted in '''Figure 1c''').
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Figure 1. Schematic diagram of design idea of Pdp1NTD-UCP1-EGFP. Pdp1NTD is the N-terminal domain protein of the payload part of Pdp1 in wild-type PVCs system. This N-terminal disordered region could serve as a “packaging domain”—a molecular identifier to assist the PVCs loading machinery in identifying and loading the proper payloads. Using this functional property, Pdp1NTD was connected with UCP1 and EGFP to form fusion protein, and CMV promoter and polyA termination signal were loaded for final expression.
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<img src="https://static.igem.wiki/teams/4960/wiki/21.jpg" class="figure-img img-fluid rounded"  height="200px">
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'''Figure 1. AlphaFold2 prediction of protein structure and updated schematic diagram of design ideas.''' (a). AlphaFold2 prediction of Pdp1NTD-UCP1-EGFP protein structure. The unexpected interaction between SepC and UCP1 is labeled in a red box. (b). AlphaFold2 prediction of Pdp1NTD-EGFP -UCP1 protein structure. (c). Updated schematic diagram of design ideas. In the process of designing part, we switched the original sequence of EGFP and UCP1, and carried out the same experimental treatment as a new group of experimental groups, hoping to solve the problems encountered before.<br>
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===Sequence and Feature===
  
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<span class='h3bb'>Sequence and Features</span>
 
 
<partinfo>BBa_K4960021 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4960021 SequenceAndFeatures</partinfo>
  
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===Functional test===
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==Functional test==
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This part is testing through cell experiments, that is, the SEP-UCP1-EGFP fusion protein was overexpressed on HEK293T cell lines using pcDNA3.1 as the carrier (pNC087). The purpose is to verify that the fusion protein can enter the inner membrane of mammalian mitochondria and play a normal uncoupling role to interfere with the energy metabolism of cells.
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===Method===
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'''Methods'''<br>
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To assess the function of the fusion protein, we transfected HEK-293T cells with pNC087, which encodes a PCMV promoter-driven Pdp1NTD-UCP1-EGFP protein expression cassette. Cells were imaged at 48 h post transcription to identify the cellular localization of the fusion protein. Cellular glucose consumption was then evaluated by measuring the remaining glucose levels in the cell culture medium. Results showed that instead of localizing in the mitochondria, the Pdp1NTD-UCP1-EGFP protein was localized all over the cytoplasm and nucleus (Fig. 2a). Consistent with the failed mitochondrial localization, glucose levels in the pNC087-transfected cells showed no significant difference compared to the control group transfected with pcDNA3.1(+) vector only (Fig. 2b).
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To validate the function of this part, we constructed pNC088, a CMV-driven plasmid expressing this part. We transfected HEK-293T cells with pNC088 and observed the cellular localization of the fusion protein 48 hours post-transcription by widefield fluorescent microscopy and live-cell confocal imaging. We also analyzed the glucose consumption of the transfected cells by measuring the glucose levels in the culture medium. This analysis represented the level of cellular energy expenditure. We compared the results with those of the control group cells transfected with pcDNA3.1(+) vector only.<br>
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'''Results'''<br>
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The results of both wide-field fluorescent imaging ('''Figure 2a''') and live-cell confocal imaging ('''Figure 2b''') indicated highly specific colocalization of Pdp1NTD-EGFP-UCP1 signal with mitochondria markers (MTS-mcherry, '''Figure 2c'''). Furthermore, cells that were transfected with pNC088 demonstrated a significantly higher glucose consumption rate when compared to cells transfected with the pcDNA3.1(+) vector ('''Figure 2c'''). This suggests that the energy consumption in these cells was significantly improved.
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<img src="https://static.igem.wiki/teams/4960/wiki/21-2.jpg" class="figure-img img-fluid rounded"  height="225px">
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'''Figure 2. Functionality of UCP1-based Payload Protein in HEK-293T Cells.<br>
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(a). Localization Pdp1NTD-EGFP-UCP1 in HEK-293T cells. Cells were transfected with pNC088 (PCMV-Pdp1NTD-EGFP-UCP1). (b). Cells were co-transfected with MTS-mcherry and PNC088. Photos were taken 48 h post transfection, scale bar: 100μm for wide-field microscopy and 10 μm for confocal microscopy. Data are representative images of 3 independent experiments. (c). Charactrization of cellular metabolism in HEK-293T cells transfected with either pNC088 or pcDNA3.1(+). Glucose concentration in the cell culture medium was measured 48 h after transfection; data shows mean±SD, n=3 independent experiments.<br>
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'''Compatibility with the PVC system'''
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We have also tested the compatability of this part with the PVC system using the composite [[part:BBa_K4960031]]. Please refer to the functional validation of [[Part:BBa_K4960031]] for experimental design and detailed results.
  
  
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Figure 2a. Localization of Overexpressed Pdp1NTD-UCP1-EGFP fusion protein Observed by Wide Field Microscopy.
 
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Figure 2b. Bar Chart Illustrating Glucose Consumption in pNC087 Transfected HEK-293T Cells.
 
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===Structure prediction===
 
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According to the experimental results, we conclude that the reason is that the structure of Pdp1NTD interacts with the N-terminal structure of UCP1, which makes UCP1 unable to function normally. Therefore, we used Alphafold to predict the structure of the fusion protein (Fig. 3), and the result might confirmed our idea.
 
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Figure 3. AlphaFold2-assisted Prediction of the Structure of two types of Fusion protein. The protein structure of Pdp1NTD-UCP1-EGFP and Pdp1NTD-EGFP-UCP1 were predicted by Alphafold2, and it was noted that Pdp1NTD-UCP1-EGFP overlated in the connected part of UCP1 and Pdp1NTD protein, which may be the reason why the protein could not work correctly
 
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===Functional Parameters===
 
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<partinfo>BBa_K4960021 parameters</partinfo>
 
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===References===
 
===References===
 
[1] Kolonin MG, Saha PK, Chan L, Pasqualini R, Arap W. Reversal of obesity by targeted ablation of adipose tissue. Nat Med. 2004 Jun;10(6):625-32.<br>
 
[1] Kolonin MG, Saha PK, Chan L, Pasqualini R, Arap W. Reversal of obesity by targeted ablation of adipose tissue. Nat Med. 2004 Jun;10(6):625-32.<br>
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[2] Qiu Y, Sun Y, Xu D, Yang Y, Liu X, Wei Y, Chen Y, Feng Z, Li S, Reyad-Ul Ferdous M, Zhao Y, Xu H, Lao Y, Ding Q. Screening of FDA-approved drugs identifies sutent as a modulator of UCP1 expression in brown adipose tissue. EBioMedicine. 2018 Nov;37:344-355.<br>
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[2]Kreitz J, Friedrich MJ, Guru A, Lash B, Saito M, Macrae RK, Zhang F. Programmable protein delivery with a bacterial contractile injection system. Nature. 2023 Apr;616(7956):357-364. doi: 10.1038/s41586-023-05870-7. Epub 2023 Mar 29.
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[3] Lodhi IJ, Dean JM, He A, Park H, Tan M, Feng C, Song H, Hsu FF, Semenkovich CF. PexRAP Inhibits PRDM16-Mediated Thermogenic Gene Expression. Cell Rep. 2017 Sep 19;20(12):2766-2774. <br>
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Latest revision as of 14:12, 12 October 2023


Engineered Mitochondrial Uncoupler Pdp1NTD-EGFP-UCP1
enable the PVC-mediated alteration of mammalian cell energy expenditure

Profile

Name: Engineered Mitochondrial Uncoupler Pdp1NTD-EGFP-UCP1
Base Pairs: 1917 bp
Origin: Photorhabdus, Aequorea Victoria, Homo Sapiens
Properties:enable the PVC-mediated alteration of mammalian cell energy expenditure

Usage and Biology

UCP1 is a key mitochondrial uncoupling protein in mammalian cells that regulates cellular energy expenditure and thermogenesis[1]. This part demonstrates a UCP1-based synthetic mitochondrial uncoupler that is compatable to the PVC delivery system (see the Description page of iGEM23_NUDT-CHINA wiki for more information about the PVC system). In mammalian cells, this part would be translocated into mitochondria and works as a proton transporter that disconnects oxygen consumption from ATP synthesis, thereby dissipates energy in the form of heat, leading to an increase in energy expenditure and basal metabolic rate.[2]

Special Design

This part includes a Pdp1NTD domain to ensure the compatability with the PVC delivery system, an EGFP domain for visualization and improving the protein production in E.coli, and a UCP1 mitochondrial uncoupling domain to enable the functionality. In Part:BBa_K4960022, we have demonstrated that the Pdp1NTD-UCP1-EGFP configuration failed to effectively localize into mitochondria, possibly due to an unintended interaction between the Pdp1NTD domain and UCP1 (as highlighted in the red box in Figure 1a). To address this issue, we used AlphaFold2 to predict some of the possible structures and found that we could resolve this problem by swapping UCP1 and EGFP (as shown in Figure 1b). Consequently, we designed this part by connecting Pdp1NTD to the N-terminus EGFP and a UCP1 to the C-terminus (as depicted in Figure 1c).


Figure 1. AlphaFold2 prediction of protein structure and updated schematic diagram of design ideas. (a). AlphaFold2 prediction of Pdp1NTD-UCP1-EGFP protein structure. The unexpected interaction between SepC and UCP1 is labeled in a red box. (b). AlphaFold2 prediction of Pdp1NTD-EGFP -UCP1 protein structure. (c). Updated schematic diagram of design ideas. In the process of designing part, we switched the original sequence of EGFP and UCP1, and carried out the same experimental treatment as a new group of experimental groups, hoping to solve the problems encountered before.

Sequence and Feature


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1258
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 235
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Functional test

Methods

To validate the function of this part, we constructed pNC088, a CMV-driven plasmid expressing this part. We transfected HEK-293T cells with pNC088 and observed the cellular localization of the fusion protein 48 hours post-transcription by widefield fluorescent microscopy and live-cell confocal imaging. We also analyzed the glucose consumption of the transfected cells by measuring the glucose levels in the culture medium. This analysis represented the level of cellular energy expenditure. We compared the results with those of the control group cells transfected with pcDNA3.1(+) vector only.

Results
The results of both wide-field fluorescent imaging (Figure 2a) and live-cell confocal imaging (Figure 2b) indicated highly specific colocalization of Pdp1NTD-EGFP-UCP1 signal with mitochondria markers (MTS-mcherry, Figure 2c). Furthermore, cells that were transfected with pNC088 demonstrated a significantly higher glucose consumption rate when compared to cells transfected with the pcDNA3.1(+) vector (Figure 2c). This suggests that the energy consumption in these cells was significantly improved.

Figure 2. Functionality of UCP1-based Payload Protein in HEK-293T Cells.
 (a). Localization Pdp1NTD-EGFP-UCP1 in HEK-293T cells. Cells were transfected with pNC088 (PCMV-Pdp1NTD-EGFP-UCP1). (b). Cells were co-transfected with MTS-mcherry and PNC088. Photos were taken 48 h post transfection, scale bar: 100μm for wide-field microscopy and 10 μm for confocal microscopy. Data are representative images of 3 independent experiments. (c). Charactrization of cellular metabolism in HEK-293T cells transfected with either pNC088 or pcDNA3.1(+). Glucose concentration in the cell culture medium was measured 48 h after transfection; data shows mean±SD, n=3 independent experiments.

Compatibility with the PVC system

We have also tested the compatability of this part with the PVC system using the composite part:BBa_K4960031. Please refer to the functional validation of Part:BBa_K4960031 for experimental design and detailed results.


References

[1] Kolonin MG, Saha PK, Chan L, Pasqualini R, Arap W. Reversal of obesity by targeted ablation of adipose tissue. Nat Med. 2004 Jun;10(6):625-32.
[2]Kreitz J, Friedrich MJ, Guru A, Lash B, Saito M, Macrae RK, Zhang F. Programmable protein delivery with a bacterial contractile injection system. Nature. 2023 Apr;616(7956):357-364. doi: 10.1038/s41586-023-05870-7. Epub 2023 Mar 29.