Cathepsin B Expression Cassette

Cathepsin B is a lysosomal protease present in the cytosol of various cancer types. To enhance its nuclear functionality, cathepsin B (BBa_K5237100) was fused to the SV40 nuclear localization sequence (BBa_K2549054) via a GGS linker, enabling nuclear import and precise subcellular targeting. We overexpressed this composite part in HEK293T cells to investigate its ability to cleave different Gal4-Linker-VP64 constructs (BBa_K5237020) using a fluorescence readout assay. We successfully demonstrated that the GFLG linker was efficiently cleaved by cathepsin B in vivo. Furthermore, we showed that wild-type cathepsin B matured into its active forms when overexpressed in HEK293T cells. Together, these findings enable the functionalization of our PICasSO system for a wide range of therapeutic and synthetic biology applications.

Figure 1: How our part collection can be used to engineer new staples

While synthetic biology has in the past focused on engineering the genomic sequence of organisms, the 3D spatial organization of DNA is well-known to be an important layer of information encoding in particular in eukaryotes, playing a crucial role in gene regulation and hence cell fate, disease development, evolution, and more. However, tools to precisely manipulate and control the genomic spatial architecture are limited, hampering the exploration of 3D genome engineering in synthetic biology. We - the iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular toolbox for rationally engineering genome 3D architectures in living cells, based on various DNA-binding proteins.

The PICasSO part collection offers a comprehensive, modular platform for precise manipulation and re-programming of DNA-DNA interactions using engineered "protein staples" in living cells. This enables researchers to recreate naturally occurring alterations of 3D genomic interactions, such as enhancer hijacking in cancer, or to design entirely new spatial architectures for artificial gene regulation and cell function control. Specifically, the fusion of two DNA binding proteins enables to artificially bring otherwise distant genomic loci into spatial proximity. To unlock the system's full potential, we introduce versatile chimeric CRISPR/Cas complexes, connected either at the protein or - in the case of CRISPR/Cas-based DNA binding moieties - the guide RNA level. These complexes are referred to as protein- or Cas staples, respectively. Beyond its versatility with regard to the staple constructs themselves, PICasSO includes robust assay systems to support the engineering, optimization, and testing of new staples in vitro and in vivo. Notably, the PICasSO toolbox was developed in a design-build-test-learn engineering cycle closely intertwining wet lab experiments and computational modeling and iterated several times, yielding a collection of well-functioning and -characterized parts.

At its heart, the PICasSO part collection consists of three categories.
(i) Our DNA-binding proteins include our finalized Cas staple experimentally validated using an artificial "enhancer hijacking" system as well as "half staples" that can be combined by scientists to compose entirely new Cas staples in the future. We also include our Simple staples comprised of particularly small, simple and robust DNA binding domains well-known to the synthetic biology community, which serve as controls for successful stapling and can be further engineered to create alternative, simpler, and more compact staples.
(ii) As functional elements, we list additional parts that enhance and expand the functionality of our Cas and Basic staples. These consist of staples dependent on cleavable peptide linkers targeted by cancer-specific proteases or inteins that allow condition-specific, dynamic stapling in vivo. We also include several engineered parts that enable the efficient delivery of PICasSO's constructs into target cells, including mammalian cells, with our new interkingdom conjugation system.
(iii) As the final category of our collection, we provide parts that underlie our custom readout systems. These include components of our established FRET-based proximity assay system, enabling users to confirm accurate stapling. Additionally, we offer a complementary, application-oriented testing system based on a luciferase reporter, which allows for straightforward experimental assessment of functional enhancer hijacking events in mammalian cells.

The following table gives a comprehensive overview of all parts in our PICasSO toolbox. The highlighted parts showed exceptional performance as described on our iGEM wiki and can serve as a reference. The other parts in the collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their own custom Cas staples, enabling further optimization and innovation in the new field of 3D genome engineering.

Our part collection includes:

1. Sequence Overview

2. Usage and Biology

Cathepsin B is a cysteine protease typically found in lysosomes or secreted outside the cell, where it degrades proteins of the extracellular matrix (Ruan et al., 2015). Its significance in cancer progression is well-documented, with elevated levels observed in cancerous tissues compared to noncancerous tissues (Ruan et al., 2015). Given its important role in tumor progression, cathepsin B is considered a potential therapeutic target (Ruan et al., 2015) or prodrug-activating enzyme (Zhong et al., 2013). To explore the therapeutic potential of our PICasSO platform, we designed protein-based DNA staples that respond to the overexpression of cathepsin B in cancerous tissues. We were able to demonstrate doxorubicin-dependent cathepsin B cleavage of one out of five documented linkers (Jin et al., 2022; Shim et al., 2022; Wang et al., 2024) in HEK293T cells.
To enhance the functionality of cathepsin B within the nucleus, we fused it to the SV40 nuclear localization sequence (NLS), a short peptide derived from the simian virus 40 (SV40) large T-antigen. The SV40 NLS contains a cluster of basic amino acids, which are recognized by importins, allowing the tagged protein to be transported through the nuclear pore complex into the nucleus (Yoneda, 1997). This tool is commonly used to ensure the nuclear localization of recombinant proteins in eukaryotic cells (Lu et al., 2021). By directing cathepsin B to the nucleus, we aim to enhance its precision in cellular targeting.

3. Assembly and Part Evolution

The protein sequence of human cathepsin B was obtained from UniProt (P07858), and an SV40 nuclear localization sequence (BBa_K2549054) was connected to the N-Terminus via a GGS linker. After in silico cloning, the corresponding nucleotide sequence was optimized for expression in human cells (Codon Optimization Tool from Integrated DNA Technologies, Inc.) and purchased as a gBlock. Restriction cloning was used to insert the gBlock into the mammalian expression vector pcDNA3.1. The plasmids were propagated in E. coli Top10 cells and used to transfect HEK293T cells.

4. Results

4.1 Mature Cathepsin B Is Expressed in HEK293T Cells

To achieve cathepsin B cleavage-induced Cas stapling, catalytically active cathepsin B needs to be expressed in the cytosol. Therefore, we investigated the expression of different cathepsin B constructs under different conditions in HEK293T cells. In addition to wild-type (wt) cathepsin B, we also cloned a truncated and mutated version of cathepsin B (Δ1-20, D22A, H110A, R116A) and compared protein expression of both constructs in doxorubicin-treated and untreated conditions.
Figure 2 shows a western blot of the wild-type (wt) version of cathepsin B as well as the truncated and mutated version of cathepsin B (Δ1-20, D22A, H110A, R116A). Cells of both cathepsin B versions were treated with 500 nM doxorubicin (dox) 24 hours post-transfection and incubated for additional 24 hours. For each condition, three replicates were blotted. We observed no differences in protein expression levels between the dox-treated and untreated wt versions of cathepsin B. For the truncated and mutated version of cathepsin B, however, only the untreated samples showed the corresponding band at approximately 36 kDa expected for this version of cathepsin B. Additionally, the bands of the truncated and mutated version appeared much weaker than the ones of the wt, indicating poorer protein expression. The household protein β-tubulin is visible in all samples at approximately 55 kDa. The wt cathepsin B additionally showed bands for pro-cathepsin B at approximately 42 kDa, a mature single-chain version of cathepsin B at approximately 33 kDa and a mature double-chain version at approximately 26 kDa.

Figure 2: Western Blot of Two Versions of Cathepsin B With and Without Doxorubicin. From left to right: protein ladder, wild-type (wt) cathepsin B with (+) and without (-) doxorubicin, truncated and mutated version of cathepsin B with (+) and without (-) doxorubicin. The household protein β-tubulin is visible in all samples at 55 kDa. The wt cathepsin B also shows bands for pro-cathepsin B at 42 kDa, mature single-chain cathepsin B at 33 kDa and mature double-chain cathepsin B at 26 kDa. The band for the truncated and mutated version of cathepsin B can be seen in the samples without doxorubicin at 36 kDa.

4.2 mCherry and eGFP Can be Used as a Reporter System to Measure Cleavage Efficiency

In this experiment, mCherry and eGFP were evaluated as reporters to quantify the efficiency of cathepsin B-mediated cleavage of Gal4-Linker-VP64 constructs in HEK293T cells.
Figure 3 shows micrographs taken with a fluorescence microscope of three different conditions: the null control, the negative control and the test sample. Figure 4 shows the corresponding graphs. All samples were transfected with plasmids encoding eGFP and mCherry. The null control and the negative control were not transfected with the plasmid encoding cathepsin B. The null control was also not transfected with any of the plasmids encoding Gal4-Linker-VP64 constructs. The test sample was transfected with 30 ng of the plasmid encoding cathepsin B and with the plasmid encoding Gal4-GFLG-VP64. As expected, the null control exhibited no detectable mCherry signal, with corresponding fluorescence intensity measurements at baseline levels. Since no plasmid encoding a Gal4-V64 construct was transfected, mCherry overexpression via VP64 could not be induced. However, we observed a high fluorescence intensity for eGFP, indicating that the transfection was successful. The negative control showed strong signals of both mCherry and eGFP. Therefore, it can be assumed that the transfection was successful and that our mCherry readout system is functional. Interestingly, there are some cells which either seem to only express mCherry or eGFP and some cells that show no fluorescence signal. The test sample showed less eGFP and mCherry fluorescence compared to the negative control. We expected to observe reduced fluorescence intensity of mCherry, as the transfected cells would express cathepsin B, which cleaves the linker, thereby decreasing mCherry expression

Figure 3: Micrographs of HEK293T Cells in Two Control Conditions and One Test Condition. Micrographs were taken with a fluorescence microscope 48 hours after transfection. An overlay of brightfield, eGFP and mCherry is shown. The null control and the negative control were not transfected with the plasmid encoding cathepsin B. The Null Control was also not transfected with any of the plasmids encoding Gal4-Linker-VP64 constructs. The test sample was transfected with 30 ng of the plasmid encoding cathepsin B and with the plasmid encoding Gal4-GFLG-VP64. The micrograph of the test sample is not from the same biological replicate as the micrographs of the two controls.

Figure 4: Fluorescence Readout After 48 Hours for Two Control Conditions and One Test Condition. The fluorescence intensity for mCherry was measured for the GFLG linker and normalized against a baseline eGFP fluorescence intensity. The null control and the negative control were not transfected with the plasmid encoding cathepsin B. The null control was also not transfected with any of the plasmids encoding Gal4-Linker-VP64 constructs. The test sample was transfected with 30 ng of the plasmid encoding cathepsin B and with the plasmid encoding Gal4-GFLG-VP64.

4.3 The Peptide Linker GFLG Is Cleaved by Cathepsin B in Vivo

We performed a fluorescence readout assay in HEK293T cells to investigate cathepsin B cleavage of different peptide linkers. 24 hours after transfection, we added doxorubicin in a final concentration of 500 nM to the cell supernatant. Figure 5 shows the fluorescence intensity of mCherry for five different peptide linkers (GFLG, FFRG, FRRL, VA, FK). The negative control was not transfected with the plasmid encoding cathepsin B. We investigated two different test conditions, in which we either transfected 30 ng or 60 ng of the plasmid encoding cathepsin B. The fluorescence intensity of mCherry was normalized by the measured fluorescence intensity of eGFP in each condition. Additionally, the values for 30 ng and 60 ng cathepsin B were normalized against the corresponding negative controls. One data point for the VA linker, transfected with 60 ng of the plasmid encoding cathepsin B, was excluded due to severe deviation from the other values. We conducted a two-way analysis of variance (ANOVA) to assess the significance of the observed differences between the negative control and the test conditions for each linker. As the negative control did not contain the plasmid encoding cathepsin B, we expected the measured fluorescence intensity of mCherry to be the highest in these conditions. However, this was only observed for the GFLG and FK linkers. Contrary to our expectations, the fluorescence intensity of the negative control was the lowest out of the three conditions tested for the remaining linkers. It appears that the addition of the plasmid encoding cathepsin B increases mCherry fluorescence intensity when the linker is not cleaved. However, this increase is only significant for the FFRG linker in the 60 ng condition. For the GFLG linker, we observed significant decreases in fluorescence intensity between the negative control and both test conditions, with no difference between the 30 ng and 60 ng conditions. For the FK linker, no significant decreases in fluorescence intensity between the negative control and the test conditions were observed.

Figure 5: Fluorescence Readout After 48 Hours for Five Different Peptide Linkers and Three Different Conditions. The fluorescence intensity for mCherry was measured for five different linkers and normalized against a baseline eGFP fluorescence intensity. The negative control was not transfected with the plasmid encoding cathepsin B. The fluorescence intensity of the negative control was set to one. Two different test conditions were investigated, in which either 30 ng or 60 ng of the plasmid encoding cathepsin B were transfected. The fluorescence readout was analyzed using a two-way ANOVA. Medium: DMEM (10% FCS). P values: ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

5. Conclusion

We overexpressed wild-type cathepsin B in HEK293T cells to study its maturation and activity in a cellular environment. Our findings revealed that cathepsin B successfully matured into its active forms when overexpressed, demonstrating its proteolytic functionality in vivo. By fusing cathepsin B to an SV40 nuclear localization sequence (NLS), we were able to target the protease to the nucleus, enhancing its subcellular localization and precision. Additionally, we showed that the GFLG linker was efficiently cleaved by cathepsin B, confirming its activity on peptide substrates. These results confirm the successful overexpression and activation of cathepsin B in human cells, laying the groundwork for its use in targeted therapeutic strategies and synthetic biology systems.

6. References

Gramespacher, J. A., Stevens, A. J., Nguyen, D. P., Chin, J. W., & Muir, T. W. (2017). Intein Zymogens: Conditional Assembly and Splicing of Split Inteins via Targeted Proteolysis. J Am Chem Soc, 139(24), 8074-8077. https://doi.org/10.1021/jacs.7b02618

Jin, C., EI-Sagheer, A. H., Li, S., Vallis, K. A., Tan, W., & Brown, T. (2022). Engineering Enzyme-Cleavable Oligonucleotides by Automated Solid-Phase Incorporation of Cathepsin B Sensitive Dipeptide Linkers. Angewandte Chemie International Edition, 61(13), e202114016. https://doi.org/10.1002/anie.202114016

Lu, J., Wu, T., Zhang, B., Liu, S., Song, W., Qiao, J. & Ruan, H. (2021). Types of nuclear localization signals and mechanisms of protein import into the nucleus. Cell Communication and Signaling 19(60). https://doi.org/10.1186/s12964-021-00741-y

Ruan, H., Hao, S., Young, P., & Zhang, H. (2015). Targeting Cathepsin B for Cancer Therapies. Horiz Cancer Res, 56, 23-40.

Shim, N., Jeon, S. I., Yang, S., Park, J. Y., Jo, M., Kim, J., Choi, J., Yun, W. S., Kim, J., Lee, Y., Shim, M. K., Kim, Y., & Kim, K. (2022). Comparative study of cathepsin B-cleavable linkers for the optimal design of cathepsin B-specific doxorubicin prodrug nanoparticles for targeted cancer therapy. Biomaterials, 289, 121806. https://doi.org/10.1016/j.biomaterials.2022.121806

Wang, J., Liu, M., Zhang, X., Wang, X., Xiong, M., & Luo, D. (2024). Stimuli-responsive linkers and their application in molecular imaging. Exploration, 4(4), 20230027. https://doi.org/10.1002/EXP.20230027

Yoneda,Y. (1997). How Proteins Are Transported from Cytoplasm to the Nucleus. The Journal of Biochemistry, 121(5), 811 – 817. https://doi.org/10.1093/oxfordjournals.jbchem.a021657

Zhong, Y.-J., Shao, L.-H., & Li, Y. (2013). Cathepsin B-cleavable doxorubicin prodrugs for targeted cancer therapy (Review). Int J Oncol, 42(2), 373-383. https://doi.org/10.3892/ijo.2012.1754