Part:BBa_K4960021

Engineered Mitochondrial Uncoupler Pdp1NTD-EGFP-UCP1

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. 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. With the same protocol, we predicted the structure of a few design options and found that we could simply solve this problem by swapping UCP1 and EGFP

Figure 1.AlphaFold2 prediction of Pdp1NTD-UCP1-EGFP protein structure. The unexpected interaction between SepC and UCP1 is labeled in a red box.

Figure 2. 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

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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

This part is testing through cell experiments, that is, the Pdp1NTD-EGFP-UCP1 fusion protein was overexpressed on HEK293T cell lines using pcDNA3.1 as the carrier. Similar to the BBa_K4960032, we transfected HEK-293T cells with pNC088, a Pdp1NTD-EGFP-UCP1 expressing plasmid, and evaluated the cellular localization and function of the fusion protein at 48 h post transfection. As expected, both wide-field fluorescent imaging and live-cell confocal imaging (Fig. 2a) showed a highly specific colocalization of Pdp1NTD-EGFP-UCP1 signal with mitochondria (labeled by MTS-mcherry). Moreover, cells transfected with pNC088 showed a significantly higher glucose consumption compared to the control cells transfected with pcDNA3.1(+) vector (Fig. 2b), suggesting a significantly improved energy consumption in these cells.

Figure 2a. Localization Pdp1NTD-EGFP-UCP1 in HEK-293T cells. For wide-field microscopy, cells were transfected with pNC088 (PCMV-Pdp1NTD-EGFP-UCP1). For confocal images, 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.

Figure 2b. 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.

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.