Part:BBa_K5237011
Cathepsin B Expression Cassette
Cathepsin B is a lysosomal protease present in the cytosol of various cancer types. We overexpressed wild-type cathepsin B in HEK293T cells to investigate cathepsin B induced cleavage of different peptide linkers via a fluorescence readout assay. We successfully showed that the linker GFLG was efficiently cleaved by cathepsin B in vivo. Furthermore, we were able to demonstrate 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.
Contents
The 3D organization of the genome plays a crucial role in regulating gene expression in eukaryotic cells,
impacting cellular behavior, evolution, and disease. Beyond the linear DNA sequence, the spatial arrangement of
chromatin, influenced by DNA-DNA interactions, shapes pathways of gene regulation. However, the tools to precisely
manipulate this genomic architecture remain limited, rendering it challenging to explore the full potential of the
3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular
toolbox based on various DNA-binding proteins to address this issue.
The PICasSO part collection offers a comprehensive, modular platform for precise manipulation and re-programming of DNA-DNA interactions using protein staples in living cells, enabling researchers to recreate natural 3D genomic interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation. Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and testing of new staples, ensuring functionality in vitro and in vivo. We took special care to include parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts
At its heart, the PICasSO parts collection consists of three categories. (i) Our DNA-binding proteins include our finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely new Cas staples in the future. We also include our simple staples that 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 the functionality of our Cas and Basic staples. These consist of protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling in vivo. Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's with our interkingdom conjugation system.
(iii) As the final component of our collection, we provide parts that support the use of 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 for functional readout via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking.
The following table gives a complete 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
Our part collection includes:
| DNA-binding proteins: The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring easy assembly. | ||
| BBa_K5237000 | fgRNA Entryvector MbCas12a-SpCas9 | Entryvector for simple fgRNA cloning via SapI |
| BBa_K5237001 | Half-Staple: dMbCas12a-Nucleoplasmin NLS | Staple subunit that can be combined to form a functional staple, for example with fgRNA and dCas9 |
| BBa_K5237002 | Half-Staple: SV40 NLS-dSpCas9-SV40 NLS | Staple subunit that can be combined to form a functional staple, for example with our fgRNA or dCas12a |
| BBa_K5237003 | Cas-Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS | Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands in close proximity |
| BBa_K5237004 | Half-Staple: Oct1-DBD | Staple subunit that can be combined to form a functional staple, for example with TetR. Can also be combined with a fluorescent protein as part of the FRET proximity assay |
| BBa_K5237005 | Half-Staple: TetR | Staple subunit that can be combined to form a functional staple, for example with Oct1. Can also be combined with a fluorescent protein as part of the FRET proximity assay |
| BBa_K5237006 | Simple-Staple: TetR-Oct1 | Functional staple that can be used to bring two DNA strands in close proximity |
| BBa_K5237007 | Half-Staple: GCN4 | Staple subunit that can be combined to form a functional staple, for example with rGCN4 |
| BBa_K5237008 | Half-Staple: rGCN4 | Staple subunit that can be combined to form a functional staple, for example with rGCN4 |
| BBa_K5237009 | Mini-Staple: bGCN4 | Assembled staple with minimal size that can be further engineered | Functional elements: Protease cleavable peptide linkers and inteins are used to control and modify staples for further optimization for custom applications. |
| BBa_K5237010 | Cathepsin B-Cleavable Linker (GFLG) | Cathepsin B cleavable peptide linker, that can be used to combine two staple subunits, to make responsive staples |
| BBa_K5237011 | Cathepsin B Expression Cassette | Cathepsin B which can be selectively express to cut the cleavable linker |
| BBa_K5237012 | Caged NpuN Intein | Undergoes protein transsplicing after protease activation, can be used to create functionalized staple units |
| BBa_K5237013 | Caged NpuC Intein | Undergoes protein transsplicing after protease activation, can be used to create functionalized staple units |
| BBa_K5237014 | fgRNA processing casette | Processing casette to produce multiple fgRNAs from one transcript, can be used for multiplexing |
| BBa_K5237015 | Intimin anti-EGFR Nanobody | Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large constructs | Readout Systems: FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells enabling swift testing and easy development for new systems. |
| BBa_K5237016 | FRET-Donor: mNeonGreen-Oct1 | Donor part for the FRET assay binding the Oct1 binding cassette. Can be used to visualize DNA-DNA proximity |
| BBa_K5237017 | FRET-Acceptor: TetR-mScarlet-I | Acceptor part for the FRET assay binding the TetR binding cassette. Can be used to visualize DNA-DNA proximity |
| BBa_K5237018 | Oct1 Binding Casette | DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET proximity assay |
| BBa_K5237019 | TetR Binding Cassette | DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET proximity assay | BBa_K5237020 | Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64 | Readout system that responds to protease activity. It was used to test Cathepsin-B cleavable linker. |
| BBa_K5237021 | NLS-Gal4-VP64 | Trans-activating enhancer, that can be used to simulate enhancer hijacking. | BBa_K5237022 | mCherry Expression Cassette: UAS, minimal Promotor, mCherry | Readout system for enhancer binding. It was used to test Cathepsin-B cleavable linker. |
| BBa_K5237023 | Oct1 - UAS binding casette | Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay. |
| BBa_K5237024 | Minimal promoter Firefly luciferase | Contains Firefly luciferase controlled by a minimal promoter. It was used as a luminescence readout for simulated enhancer hijacking. |
1. Sequence overview
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 656
Illegal BglII site found at 755 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 86
Illegal NgoMIV site found at 157
Illegal NgoMIV site found at 1009
Illegal AgeI site found at 841 - 1000COMPATIBLE WITH RFC[1000]
2. Usage and Biology
Cathepsin B is a cysteine protease typically located in lysosomes or secreted outside the cell, where it degrades proteins of the extracellular matrix (Ruan et al., 2015). The significance of cathepsin B in cancer progression is well-documented, with studies showing elevated cathepsin B levels in cancerous tissues compared with 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 potential of our PICasSO platform approach for therapeutic applications, we designed protein-based DNA staples that are responsive 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.
3. Assembly and part evolution
The protein sequence of human cathepsin B was obtained from UniProt (P07858), and an SV40 nuclear localization sequence (NLS) 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
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 1 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.
mCherry and eGFP are Both Expressed in HEK293T Cells
Figure 2 shows micrographs taken with a fluorescence microscope of three different conditions: the null control, the negative control and the test sample. 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 showed no detectable mCherry signal, since no plasmid encoding a Gal4-V64 construct was transfected. Consequently, 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.
Mature Cathepsin B is Expressed in HEK293T Cells
Figure 3 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.
Conclusion
All in all, these findings demonstrate that our fluorescence-based readout assay can reliably detect cathepsin B-mediated cleavage of peptide linkers, with the GFLG linker showing particular susceptibility to cleavage. This makes GFLG a promising candidate for targeted applications in environments with upregulated cathepsin B activity, such as in cancerous tissues. Additionally, our cathepsin B-cleavage linker can be combined with caged inteins (Gramespacher et al., 2017) conjugated to a dead Cas9 to selectively induce Cas-stapling in the presence of cathepsin B.
5. 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
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
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
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