Part:BBa_K5237010
Cathepsin B-Cleavable Linker: GFLG
This basic part encodes the GFLG peptide linker, a cathepsin B-responsive cleavage site, which can be used
for targeted drug delivery or diagnostics in cancerous tissues. As a part of our PICasSO toolbox, the GFLG linker
can be used for the precise control of protein activity through cleavage-induced oligomerization of
catalytically inactive Cas proteins. Through fluorescence readout assays, we verified that the overexpression of
cathepsin B in cancer cells can potentially be leveraged for novel therapeutic and biotechnological
applications.
We overexpressed cathepsin B in HEK293T cells to investigate the cleavage of five different peptide linkers using
our mCherry Expression Cassette (BBa_K5237022). To validate the functionality of the GFLG linker, we cloned it into a
mammalian expression vector in between the Gal4 and VP64 domains (BBa_K5237020). Functional testing of this
fusion protein demonstrated efficient cleavage of the GFLG linker by cathepsin B in vivo when cells were
treated with doxorubicin, while other tested linkers showed no significant response.
Contents
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:
| DNA-Binding Proteins: Modular building blocks for engineering of custom staples to mediate defined DNA-DNA interactions in vivo | ||
| BBa_K5237000 | Fusion Guide RNA Entry Vector MbCas12a-SpCas9 | Entry vector for simple fgRNA cloning via SapI |
| BBa_K5237001 | Staple Subunit: dMbCas12a-Nucleoplasmin NLS | Staple subunit that can be combined with crRNA or fgRNA and dSpCas9 to form a functional staple |
| BBa_K5237002 | Staple Subunit: SV40 NLS-dSpCas9-SV40 NLS | Staple subunit that can be combined with a sgRNA or fgRNA and dMbCas12a to form a functional staple |
| BBa_K5237003 | Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS | Functional Cas staple that can be combined with sgRNA and crRNA or fgRNA to bring two DNA strands into close proximity |
| BBa_K5237004 | Staple Subunit: 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 | Staple Subunit: 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 | Staple Subunit: GCN4 | Staple subunit that can be combined to form a functional staple, for example with rGCN4 |
| BBa_K5237008 | Staple Subunit: 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 | Expression cassette for the overexpression of cathepsin B |
| BBa_K5237012 | Caged NpuN Intein | A caged NpuN split intein fragment that undergoes protein trans-splicing after protease activation, which can be used to create functionalized staple subunits |
| BBa_K5237013 | Caged NpuC Intein | A caged NpuC split intein fragment that undergoes protein trans-splicing after protease activation, which can be used to create functionalized staple subunits |
| BBa_K5237014 | Fusion Guide RNA Processing Casette | Processing cassette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprogramming |
| BBa_K5237015 | Intimin anti-EGFR Nanobody | Interkingdom conjugation between bacteria and mammalian cells, as an alternative delivery tool for large constructs |
| BBa_K4643003 | IncP Origin of Transfer | Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a means of delivery | Readout Systems: FRET and enhancer recruitment readout systems to rapidly assess successful DNA stapling in bacterial and mammalian cells |
| BBa_K5237016 | FRET-Donor: mNeonGreen-Oct1 | FRET donor-fluorophore fused to Oct1-DBD that binds to the Oct1 binding cassette, which 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, which can be used to visualize DNA-DNA proximity |
| BBa_K5237018 | Oct1 Binding Casette | DNA sequence containing 12 Oct1 binding motifs, compatible with various 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, which 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 Promoter, mCherry | Readout system for enhancer binding, which was used to test cathepsin B-cleavable linker |
| BBa_K5237023 | Oct1 - 5x UAS Binding Casette | Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay |
| BBa_K5237024 | TRE-minimal Promoter- Firefly Luciferase | Contains firefly luciferase controlled by a minimal promoter, which was used as a luminescence readout for simulated enhancer hijacking |
1. Sequence Overview
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2. Usage and Biology
Cathepsin B is a cysteine protease that is significantly overexpressed in various cancer types, including breast
and colorectal cancer (Ruan et al., 2015). Proteolytic cleavage of pro-biologics, for example through
cathepsin B activity, allows for precise temporal and spatial regulation of biopharmaceutical activity in
therapeutic strategies (Bleuez et al., 2022).
In this context, we introduce the cathepsin B-cleavable peptide linker GFLG as a functional addition to our
PICasSO toolbox, enabling a wide range of therapeutic and synthetic biology applications. GFLG has been shown to
be cleaved by cathepsin B (Wang et al., 2024), with cleavage occurring either between the phenylalanine and
leucine or after the second glycine of the linker (Rejmanová et al., 1983; see Fig. 2).
Through a fluorescence readout assay in HEK293T cells, we identified GFLG as the most effective among five peptide
linkers known to be cleaved by cathepsin B (Jin et al., 2022; Shim et al., 2022; Wang et al.,
2024). This linker could facilitate cleavage-induced oligomerisation of Cas proteins via protein
trans-splicing of caged intein fragments (BBa_K5237012, BBa_K5237013), further enhancing the capabilities of our system.
3. Assembly and Part Evolution
3.1 Implementation of a Cleavage-Responsive Fluorescence Readout Assay
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 (Fig. 3).
We transfected our genetic constructs into HEK293T cells. The transfected plasmids encoded mCherry, the Gal4-VP64 constructs with different linkers, and cathepsin B (Fig. 4). Additionally, a stuffer plasmid and a plasmid encoding eGFP were transfected for normalization. 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 (Fig. 5). 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.
3.2 Changes to the Amino Acid Sequence of Cathepsin B Did Not Improve Cleavage Efficiency
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 (Fig. 6). 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 (Fig. 7). 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.
3.3 Doxorubicin Induces Lysosomal Escape of Cathepsin B
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 cytostatic doxorubicin to induce lysosomal escape of cathepsin B, as had been previously reported (Bien et al., 2004).
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 7 shows a western blot of the 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.
4.2 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 8 shows micrographs taken with a fluorescence microscope of three different conditions: the null
control, the negative control and the test sample. Figure 9shows 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 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 10 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.
5. Conclusion
5.1 GFLG Is a Promising Candidate for Targeted Applications in Environments With Upregulated Cathepsin B Activity
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 assay can be used to identify other cathepsin B-cleavable peptide linkers or improve our
current GFLG linker. Our assay can also be adapted for other proteases, such as different caspases involved in
neurodegenerative conditions (Espinosa-Oliva et al., 2019).
5.2 Enabling the Functionalization of our PICasSO Toolbox Through Cathepsin B Cleavage
Our GFLG linker can be combined with caged inteins (Gramespacher et al., 2017) conjugated to
catalytically dead Cas9, allowing for selective induction of Cas-stapling in the presence of cathepsin B. This
enables the functionalization of our PICasSO toolbox for in vitro and in vivo applications.
This innovative approach paves the way for new strategies in precision medicine and synthetic biology, offering
the potential for targeted therapeutic interventions.
6. 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
Espinosa-Oliva, A. M., García-Revilla, J., Alonso-Bellido, I. M., & Burguillos, M. A. (2019). Brainiac Caspases: Beyond the Wall of Apoptosis [Mini Review]. Frontiers in Cellular Neuroscience, 13. https://doi.org/10.3389/fncel.2019.00500
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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