Overview

Obesity is a serious health concern that increases the risk of various diseases, including type 2 diabetes, hypertension, and hyperlipidemia. Through our human practice study, we have observed a clear gap between the requirements of the patients and the effectiveness of current anti-obesity medications. Hence, we directed our project to explore new therapeutic approach that is more accurate and efficient (See our Human Practices page for more information). To achieve this goal, we hereby propose an engineered PVC-based strategy to deliver mitochondrial uncoupler UCP1 into adipose tissues (See our Description page for why we decided to go this way). Here, we will mainly discuss how we decompose our goal into modules and pieces.

The major goal of our project is to specifically deliver UCP1 into adipose tissues, thereby increasing cellular energy expenditure and contributing to body weight control. Hence, two important notions, the specificity of the delivery, and the validity of UCP1 as a payload protein for PVCs, must be considered. Hence, we decomposed our target into two key modules: the Fat-burning module which includes a mitochondrial uncoupler payload based on UCP1, and the Targeting module that ensures the adipose-specific delivery

Figure 1. Top-down design of our project.

Fat-burning Module:

an UCP1-derived mitochondrial uncoupler protein compatible with the PVC delivering system

PVC belongs to a subtype of extracellular Contraction Injection Systems (eCISs) in endofungal bacteria. PVCs are encoded by a cassette of gene containing 16 structural and accessory genes that are necessary for the assembly of a function injection system, four regulatory genes, and two payload genes (Pdp1 and Pnf) that are thought to enter the target cell via contraction of the PVC sheath (Kreitz et al., 2023). Previous studies have shown that the N-terminal domain of Pdp1 (Pdp1NTD) is critical for the correct packing of Pdp1 into the PVCs (Jiang et al., 2022). Fusion of Pdp1NTD with other proteins such as Cas9 and Cre recombinase was also proved to be sufficient to load these proteins into PVCs (Kreitz et al., 2023). Therefore, an N-Terminus fusion of Pdp1NTD on UCP1 would be necessary for efficient loading into PVCs.

In addition, since UCP1 is a transmembrane protein that is located in the inner membrane of mitochondria, its hydrophobic nature would possibly affect its production in the E. coli expression system, where the PVCs are assembled. To tackle this issue, we tried to fuse enhanced Green Fluorescent Protein (EGFP), a soluble protein that can possibly help improve the expression of UCP1 and provide a visual confirmation of protein delivery.

Two sets of experiments were designed to examine the function of the engineered payload. On the one hand, we directly expressed the payload protein in mammalian cells to determine if the payload protein is functional at the best possible condition. Ideally, the nucleus-encoded UCP1-derived protein would interact with chaperone proteins and translocate into mitochondria before being anchored to the membrane, which closely resembles the case where the protein is delivered by PVCs. With this approach, we can remove the process of protein expression and purification from the equation, thereby validating the basic fat-burning function of the payload module effectively.

On the other hand, another important feature of the payload module is the ability to be successfully expressed and loaded into the PVCs. To validate this feature, we utilize a mature PVC coat that specifically target EGFR-positive cells (Kreitz et al., 2023) to generate EGFR-targeting PVCs that contains our payload protein. The packaging and delivering efficacy can then be evaluated using these PVCs.

Figure 2. Payload function examine in the payload engineering. (a) Overexpression strategy is applied to confirm the feasibility of designed payload because UCP1 translates in the cytoplasm as a linear polypeptide and folds after entry into the mitochondria, so the success function express of overexpression experiment also proofs the feasibility of delivered UCP1 (b) The design of PVC particle can be divided in two aspects which are the edit of payload and the change of tail fiber, by this way, certain PVC can be product via E.coli and used to form the targeted delivery experiment.

Upon multiple engineering cycles (see our engineering page for details), our finalized engineered mitochondrial uncoupler payload is composed of an N-terminus Pdp1NTD domain, an EGFP domain, and a C-Terminus UCP1 domain. These domains are linked via (GGGSG)5 linkers. We demonstrated that this payload protein can be efficiently loaded into PVCs and enhance energy expenditure in the target cells. Please see our Results Page for detailed experiment design and results.

Figure 3. The engineered payload that has proofed to be feasible. The payload we finally designed and proved to function properly is composed of Pdp1NTD, EGFP, and UCP1. Pdp1NTD is an N-terminal peptide used to package the payload, and EGFP is a hydrophilic fluorescent protein, which is mainly used to optimize the expression of UCP1 in E. coli and localize the protein. According to the prediction, the three structures are independent of each other and can play their respective roles normally.

Targeting Module:

an engineered PVC tail fiber protein that allows adipose targeting

Among the 16 structural and accessory genes encoding PVCs, pvc13, serving as the tail fiber protein, plays crucial role mediating the interaction between PVC and the target cells. Previous work has demonstrated that by inserting targeting domains in pvc13, one could efficiently alter the targeting profile of PVCs (Kreitz et al., 2023). These findings were also validated by our own experiment using Cre-delivering, EGFR-targeting PVCs (see our Results page for more details)

To efficiently target white adipose tissue cells, we incorporated a 9-mer peptide sequence (CKGGRAKDC) that specifically targets white adipose tissue (Kolonin et al., 2004). In order to effectively present the targeting sequence, we hereby utilized the AlphaFold2 to predict the structure of pvc13 loaded with the adipose tissue-targeting sequence flanked by different linkers (flexible linkers such as 1GS, 2GS, 3GS, and 5GS linkers, or rigid linkers such as 1EAAAK, 3EAAAK, and 5EAAAK linkers). Due to the time limit and technical difficulties, we were unable to finish building this module (see our Engineering page for more details). We'll test the function of these modules using Cre-delivery assay in the future.

Figure 4. AlphaFold-guided Engineering of pvc13 (Tail Fibre) for WAT Specificity. We tried to install different numbers of flexible (GS) and rigid (EAAAK) linker, among which we assume 1GS, 3GS, 1EAAAK and 5EAAAK as the most potential designs, which still need further experiments to proof.

Reference

Jiang, F., Shen, J., Cheng, J., Wang, X., Yang, J., Li, N., Gao, N., & Jin, Q. (2022). N-terminal signal peptides facilitate the engineering of PVC complex as a potent protein delivery system. Sci Adv, 8(17), eabm2343.https://doi.org/10.1126/sciadv.abm2343

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

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