Part:BBa_K4960022

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]

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.