Favorite Basic Part

Engineered Mitochondrial Uncoupler Pdp1NTD-EGFP-UCP1

Part:BBa_K4960021

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.

Characterization

Since the UCP1-EGFP construct has been previously reported in transgenic mice (Bates et al., 2020), we started by constructing a Pdp1NTD-UCP1-EGFP construct as an initial test . 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 1a), 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 1b).

Figure 1. 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.

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 2a, 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 2b).

Figure 2. (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.

Methods

To validate the function of this part, we constructed pNC088, a CMV-driven Pdp1NTD-EGFP-UCP1 expressing plasmid. 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. The results of both wide-field fluorescent imaging (Figure 3a) and live-cell confocal imaging (Figure 3b) indicated highly specific colocalization of Pdp1NTD-EGFP-UCP1 signal with mitochondria markers (MTS-mcherry, Figure 3c). 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 3c). This suggests that the energy consumption in these cells was significantly improved.

Figure 3. (a,b) Localization Pdp1NTD-EGFP-UCP1 in HEK-293T cells. For wide-field microscopy in e, cells were transfected with pNC088 (PCMV-Pdp1NTD-EGFP-UCP1). For confocal images in f, 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.

Reference

01.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. https://doi.org/10.1016/j.omtm.2020.09.009

02.Kolonin, M. G., Saha, P. K., Chan, L., Pasqualini, R., & Arap, W. (2004). Reversal of obesity by targeted ablation of adipose tissue. Nat Med, 10(6), 625-632. https://doi.org/10.1038/nm1048

03.Kreitz, J., Friedrich, M. J., Guru, A., Lash, B., Saito, M., Macrae, R. K., & Zhang, F. (2023). Programmable protein delivery with a bacterial contractile injection system. Nature, 616(7956), 357-364. https://doi.org/10.1038/s41586-023-05870-7