Part:BBa_K4960022

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

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.

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).

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.

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

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]

Method

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).

Structure prediction

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.

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.