Difference between revisions of "Part:BBa K4960022"
Line 5: Line 5:
  
 
===Usage and Biology===
 
===Usage and Biology===
−
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.
+
Our project aims to design a system that can effectively deliver the uncoupling protein UCP1 into adipocytes via PVCs. For this, we are using BBa_K4960022 as the basic part to enables the delivery of UCP1 by serving as the payload for the PVC delivery system. UCP1 is a naturally occurring mitochondrial uncoupler protein found in brown adipose tissue of mammals. It works by transporting protons across the mitochondrial membrane, inducing a process of mitochondrial uncoupling that disconnects oxygen consumption from ATP synthesis. This uncoupling process results in the dissipation of energy in the form of heat, leading to an increase in energy expenditure and basal metabolic rate.
 
<!-- -->
 
<!-- -->
  
 
===Special Design===
 
===Special Design===
−
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).
+
In PVCpnf, the N-terminus domain of Pdp1 (Pdp1NTD) could serve as a “packaging domain”—a molecular identifier to assist the PVCs loading machinery in identifying and loading the proper payloads. Hence, a Pdp1NTD domain must be supplemented to the N-terminus of UCP1 in order to efficiently load UCP1 into the PVCs. In addition, we also decided to fuse an EGFP tag to provide a potential visualization signal and improve the solubility of UCP1 in the E. coli expression system. Hence, we designed this part by connecting Pdp1NTD to the N-terminus of UCP1 and an EGFP to the C-terminus (Figure )
 
<html>
 
<html>
  
 
<figure class="figure">
 
<figure class="figure">
−
<img src="https://static.igem.wiki/teams/4960/wiki/basic-part/ucp1-egfp-1.png" class="figure-img img-fluid rounded"  height="300px">
+
<img src="https://static.igem.wiki/teams/4960/wiki/basic-part/ucp1-egfp-2.png" class="figure-img img-fluid rounded"  height="300px">
  
 
</figure>
 
</figure>
Line 20: Line 20:
  
 
Figure 1. Schematic diagram of design idea of Pdp1NTD-UCP1-EGFP. <br>
 
Figure 1. Schematic diagram of design idea of Pdp1NTD-UCP1-EGFP. <br>
−
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.
 
−
===Functional text===
 
−
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.
 
  
 
===Sequence and Features===
 
===Sequence and Features===
Line 30: Line 27:
 
<!-- -->
 
<!-- -->
  
 +
===Functional text===
 +
===Methods===
  
−
===Method===
+
To validate the function of this part, we constructed pNC087, a CMV-driven Pdp1NTD-UCP1-EGFP expressing plasmid. We transfected HEK-293T cells with pNC087 and observed the cellular localization of the fusion protein 48 hours post-transcription by widefield fluorescent microscopy. 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.
−
 
+
−
In PVCpnf, the N-terminus domain of Pdp1 (Pdp1NTD) is critical for the efficient loading of payload proteins into the PVCs. Hence, to efficiently load mitochondrial uncoupler proteins (UCP1 in our case) into the PVCs, an N-terminus Pdp1NTD domain must be supplemented. In addition, we also decided to fuse an EGFP tag to provide a potential visualization signal and improve the solubility of UCP1 in the E. coli expression system.
+
−
 
+
−
Since the UCP1-EGFP construct has been previously reported in transgenic mice [4], we started by constructing a Pdp1NTD-UCP1-EGFP construct as an initial test (Figure 2a). To validate the protein function in the best possible scenario, we transfected the HEK-293T cells with pNC087, a mammalian expression vector carrying the PCMV-Pdp1NTD-UCP1-EGFP cassette. Cells were imaged at 48 h post transfection to validate the subcellular location of the Pdp1NTD-UCP1-EGFP fusion protein. Unfortunately, results showed that instead of localizing in the mitochondria, the Pdp1NTD-UCP1-EGFP protein was localized all over the cytoplasm and nucleus (Figure 2a), suggesting the interaction between UCP1 and key chaperone proteins enabling its mitochondria translocation was compromised by the protein fusion. Moreover, by evaluating the remaining glucose level in the culture medium, we analyzed the glucose consumption of the cells transfected with either pNC087 or pcDNA3.1(+) control plasmid, which, in a way, represented the level of cellular energy expenditure. 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 (Figure 2b).
+
−
 
+
−
To understand how the fusion of Pdp1NTD and EGFP affected the function of UCP1, we performed structural prediction using AlphaFold2. Interestingly, we observed an unexpected interaction between the Pdp1NTD domain and UCP1 (Figure 2c, red box), which could possibly change the local protein structure and affect the translocation and function of UCP1.  
+
  
 +
===Results===
 +
Unfortunately, the results showed that instead of localizing in the mitochondria, the Pdp1NTD-UCP1-EGFP protein was present all over the cytoplasm and nucleus (Figure 2a). This suggested that the interaction between UCP1 and key chaperone proteins enabling its mitochondria translocation was compromised by the protein fusion. The glucose levels in the pNC087-transfected cells showed no significant difference compared to the control group transfected with pcDNA3.1(+) vector only (Figure 2b).
  
 +
To understand how the fusion of Pdp1NTD and EGFP affected the function of UCP1, we performed structural prediction using AlphaFold2. Interestingly, we observed an unexpected interaction between the Pdp1NTD domain and UCP1 (Figure 2c, red box), which could possibly change the local protein structure and affect the translocation and function of UCP1.
 
<html>
 
<html>
  
Line 58: Line 54:
 
[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>
 
[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>
 
[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>
 
[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>
−
[4] Bates, R., Huang, W., & Cao, L. (2020). Adipose Tissue: An Emerging Target for Adeno-associated Viral Vectors. Mol Ther Methods Clin Dev, 19, 236-249.<br>
 

Revision as of 13:50, 11 October 2023


Pdp1NTD-3*GGSGG-UCP1-2*GGSGG-EGFP


Usage and Biology

Our project aims to design a system that can effectively deliver the uncoupling protein UCP1 into adipocytes via PVCs. For this, we are using BBa_K4960022 as the basic part to enables the delivery of UCP1 by serving as the payload for the PVC delivery system. UCP1 is a naturally occurring mitochondrial uncoupler protein found in brown adipose tissue of mammals. It works by transporting protons across the mitochondrial membrane, inducing a process of mitochondrial uncoupling that disconnects oxygen consumption from ATP synthesis. This uncoupling process results in the dissipation of energy in the form of heat, leading to an increase in energy expenditure and basal metabolic rate.

Special Design

In PVCpnf, the N-terminus domain of Pdp1 (Pdp1NTD) could serve as a “packaging domain”—a molecular identifier to assist the PVCs loading machinery in identifying and loading the proper payloads. Hence, a Pdp1NTD domain must be supplemented to the N-terminus of UCP1 in order to efficiently load UCP1 into the PVCs. In addition, we also decided to fuse an EGFP tag to provide a potential visualization signal and improve the solubility of UCP1 in the E. coli expression system. Hence, we designed this part by connecting Pdp1NTD to the N-terminus of UCP1 and an EGFP to the C-terminus (Figure )

Figure 1. Schematic diagram of design idea of Pdp1NTD-UCP1-EGFP.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 505
  • 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 text

Methods

To validate the function of this part, we constructed pNC087, a CMV-driven Pdp1NTD-UCP1-EGFP expressing plasmid. We transfected HEK-293T cells with pNC087 and observed the cellular localization of the fusion protein 48 hours post-transcription by widefield fluorescent microscopy. 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

Unfortunately, the results showed that instead of localizing in the mitochondria, the Pdp1NTD-UCP1-EGFP protein was present all over the cytoplasm and nucleus (Figure 2a). This suggested that the interaction between UCP1 and key chaperone proteins enabling its mitochondria translocation was compromised by the protein fusion. The glucose levels in the pNC087-transfected cells showed no significant difference compared to the control group transfected with pcDNA3.1(+) vector only (Figure 2b).

To understand how the fusion of Pdp1NTD and EGFP affected the function of UCP1, we performed structural prediction using AlphaFold2. Interestingly, we observed an unexpected interaction between the Pdp1NTD domain and UCP1 (Figure 2c, red box), which could possibly change the local protein structure and affect the translocation and function of UCP1.

Figure 2. Functionality of UCP1-based Payload Protein in HEK-293T Cells.
(a) Localization of UCP1-based payload protein Pdp1NTD-UCP1-EGFP in HEK-293T cells under wide-field microscopy. HEK-293T cells were transfected with pNC087 Pdp1NTD-UCP1-EGFP and imaged 48 h post transfection, scale bar: 100 μm. Data are representative image of 3 independent experiments.
(b) Charactrization of cellular metabolism in HEK-293T cells transfected with either pNC087 or pcDNA3.1(+). Glucose concentration in the cell culture medium concentration was measured 48 h post transfection; data shows mean±SD, n=3 independent experiments.
(c) AlphaFold2 prediction of Pdp1NTD-UCP1-EGFP protein structure. The unexpected interaction between SepC and UCP1 is labeled in a red box. 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.

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] 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.
[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.