Part:BBa_K5237010
Cathepsin B-Cleavable Linker: GFLG
This basic part encodes the GFLG linker, a cathepsin B-responsive cleavage site which can be used for targeted drug delivery or diagnostics in cancerous tissues. To validate cathepsin B cleavage of the GFLG linker, we cloned it into a mammalian expression vector in between Gal4 and VP64 (BBa_K5237020). The resulting fusion protein was used for functional testing in HEK293T cells showing specific cleavage of the GFLG linker in the presence of cathepsin B, while other linkers were not affected. This enables the functionalization of our PICasSO toolbox for a wide range of therapeutic and synthetic biology applications.
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
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- 12COMPATIBLE WITH RFC[12]
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2. Usage and Biology
Proteolytic cleavage of pro-biologics allows for the precise temporal and spatial regulation of biopharmaceutical activity in therapeutic strategies (Bleuez et al., 2022). Cathepsin B is a cysteine protease that is significantly overexpressed in different cancer types, including breast and colorectal cancer (Ruan et al., 2015).
Here, we introduce the cathepsin B-cleavable peptide linker GFLG enabling functionalization of our PICasSO toolbox for a wide range of therapeutic and synthetic biology applications. GFLG has previously been reported to be cleaved by cathepsin B (Wang et al., 2024). Cleavage either occurs between the phenylalanine and leucine or after the second glycine of the linker (see Fig. 2) (Rejmanová et al., 1983). This linker can be used for the cleavage-induced oligomerization of Cas proteins through protein trans-splicing of caged intein fragments (BBa_K5237012, BBa_K5237013), further enhancing the capabilities of our system. We identified GFLG through a fluorescence readout assay in HEK293T cells, selecting it from five peptide linkers known to be cleaved by cathepsin B (Jin et al., 2022; Shim et al., 2022; Wang et al., 2024).
3. Assembly and Part Evolution
We designed a fluorescence readout assay in HEK293T cells based on expression of mCherry induced by the trans-activator VP64. VP64 was conjugated to the DNA-binding domain (DBD) of Gal4 through the GFLG linker. We purchased the nucleotide sequence encoding the GFLG linker as an oligo. After annealing the oligo, we cloned it into a mammalian expression vector between the open reading frames for Gal4-DBD and VP64 via Golden Gate assembly (BBa_K5237020). Binding of Gal4-DBD upstream of a gene encoding the fluorescence protein mCherry induces overexpression of mCherry by VP64. Consequently, separation of Gal4-DBD and VP64 by cathepsin B cleavage of the GFLG linker reduces mCherry expression (see Fig. 3).
We transfected our genetic constructs into HEK293T cells. 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 measured 48 hours after transfection. Our initial tests did not result in the unambiguous identification of a cathepsin B-cleavable peptide linker (see Fig. 4). For all linkers, we did not observe a large decrease in fluorescence intensity between the negative control and test conditions. In some conditions, the fluorescence intensity even increased between the negative control and test conditions.
Since cathepsin B is a lysosomal protease that is normally only active in the lysosome and the extracellular environment but not in the cytosol, we decided to change the native amino acid sequence of cathepsin B. Upon further investigation of the lysosomal maturation process of cathepsin B, we chose to express a truncated and mutated version of cathepsin B. We truncated the native cathepsin B amino acid sequence N-terminally by the first twenty amino acids. This N-terminally truncated version of cathepsin B lacks a signal peptide that normally facilitates translation of cathepsin B in the rough endoplasmic reticulum. Additionally, truncation of the first twenty amino acids of cathepsin B had previously been observed to maintain catalytic activity even in the absence of lysosomal proteases like pepsin (Müntener et al., 2005). Additionally, we introduced three point mutations (D22A, H110A, R116A) in the amino acid sequence of cathepsin B. This has been shown to increase the activity of cathepsin B at higher pH values by disrupting the interactions of an occluding loop with the substrate binding pocket of cathepsin B (Nägler et al., 1997).
We performed the same fluorescence readout assay in HEK293T cells that we also used for wild-type cathepsin B. The fluorescence intensity of mCherry was measured 48 hours after transfection. However, we observed no decrease in fluorescence between our negative control and test conditions, indicating that Gal4-DBD-Linker-VP64 was not cleaved (see Fig. 5). Additionally, we performed a western blot, where the bands for the truncated and mutated version of cathepsin B were barely visible or absent altogether, indicating lower protein expression compared to the wild type (see Fig. 9). Another key insight from this experiment was that this version of cathepsin B was not activated by cleavage in the cell, as no additional protein bands were observed.
Since our truncated and mutated version of cathepsin B did not seem to be active in the cytosol, we decided to go back to wild-type cathepsin B. Therefore, we focused on improving the activity of wild-type cathepsin B in the cytosol. After consulting the literature, we decided to treat cells with the cytostaticum doxorubicin to induce lysosomal escape of cathepsin B, as had been previously reported (Bien et al., 2004).
4. Results
4.1 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 6 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.
4.2 mCherry and eGFP Are Both Expressed in HEK293T Cells
Figure 7 shows micrographs taken with a fluorescence microscope of three different conditions: the null control, the negative control and the test sample. Figure 8 shows the corresponding graphs quantifying the fluorescence intensity in the different conditions. 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.
4.3 Mature Cathepsin B Is Expressed in HEK293T Cells
Figure 9 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.
4.4 Conclusion
All in all, these findings demonstrate that our fluorescence-based readout assay reliably detects cathepsin B-mediated cleavage of peptide linkers, with the GFLG linker showing particularly high susceptibility to enzymatic cleavage. This makes GFLG a promising candidate for targeted applications in environments with elevated cathepsin B activity, such as cancerous tissues. Additionally, our GFLG linker can be combined with caged inteins (Gramespacher et al., 2017) conjugated to dead Cas9, allowing for selective induction of Cas-stapling in the presence of cathepsin B. This innovative approach paves the way for new strategies in precision medicine and synthetic biology, offering the potential for targeted therapeutic interventions.
5. References
Bien, S., Ritter, C. A., Gratz, M., Sperker, B., Sonnemann, J., Beck, J. F., Kroemer, H. K. (2004). Nuclear factor-kappaB mediates up-regulation of cathepsin B by doxorubicin in tumor cells. Molecular Pharmacology 65(5), 1092-102. https://doi.org/10.1124/mol.65.5.1092
Bleuez, C., Koch, W. F., Urbach, C., Hollfelder, F., & Jermutus, L. (2022). Exploiting protease activation for therapy. Drug Discov Today, 27(6), 1743-1754. https://doi.org/10.1016/j.drudis.2022.03.011
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
Müntener, K., Willimann, A., Zwicky, R., Svoboda, B., Mach, L., & Baici, A. (2005). Folding Competence of N-terminally Truncated Forms of Human Procathepsin B*. Journal of Biological Chemistry, 280(12), 11973-11980. https://doi.org/10.1074/jbc.M413052200
Nägler, D. K., Storer, A. C., Portaro, F. C. V., Carmona, E., Juliano, L., & Ménard, R. (1997). Major Increase in Endopeptidase Activity of Human Cathepsin B upon Removal of Occluding Loop Contacts. Biochemistry, 36(41), 12608-12615. https://doi.org/10.1021/bi971264+
Rejmanová, P., Kopeček, J., Pohl, J., Baudyš, M., & Kostka, V. (1983). Polymers containing enzymatically degradable bonds, 8. Degradation of oligopeptide sequences in N-(2-hydroxypropyl)methacrylamide copolymers by bovine spleen cathepsin B. Die Makromolekulare Chemie, 184(10), 2009-2020. https://doi.org/10.1002/macp.1983.021841006
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
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