Difference between revisions of "Part:BBa K5237020"
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− | + | <!-- Part summary --> | |
− | + | <section> | |
− | + | <h1>Cathepsin B-Cleavable <i>Trans</i>-Activator: NLS-Gal4-GFLG-VP64</h1> | |
− | + | <p>This composite part encodes a cathepsin B-cleavable <i>trans</i>-activator fusion protein. It is composed of an | |
− | + | SV40 nuclear localization sequence (<a href="https://parts.igem.org/Part:BBa_K2549054" | |
− | + | target="_blank">BBa_K2549054</a>), the DNA-binding domain of Gal4 (<a | |
− | <div | + | href="https://parts.igem.org/Part:BBa_K4585001" target="_blank">BBa_K4585001</a>), a GFLG linker (<a |
+ | href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a>), and the VP64 | ||
+ | <i>trans</i>-activator domain (<a href="https://parts.igem.org/Part:BBa_K4586021" | ||
+ | target="_blank">BBa_K4586021</a>). We used this part to design a fluorescence-based readout assay in HEK293T | ||
+ | cells to investigate cathepsin B cleavage of different peptide linkers. Through this assay, we were able to | ||
+ | successfully show that the GFLG linker was specifically cleaved in the presence of cathepsin B, while other | ||
+ | linkers did not show this effect. Together, these findings enable the functionalization of our PICasSO system for | ||
+ | a wide range of therapeutic and synthetic biology applications. | ||
+ | </p> | ||
+ | </section> | ||
+ | <p> </p> | ||
+ | <div class="toc" id="toc"> | ||
<div id="toctitle"> | <div id="toctitle"> | ||
<h1>Contents</h1> | <h1>Contents</h1> | ||
Line 49: | Line 67: | ||
<ul> | <ul> | ||
<li class="toclevel-1 tocsection-1"><a href="#1"><span class="tocnumber">1</span> <span class="toctext">Sequence | <li class="toclevel-1 tocsection-1"><a href="#1"><span class="tocnumber">1</span> <span class="toctext">Sequence | ||
− | + | Overview</span></a> | |
</li> | </li> | ||
− | <li class="toclevel-1 tocsection-2"><a href="#2"><span class="tocnumber">2</span> <span class="toctext">Usage and | + | <li class="toclevel-1 tocsection-2"><a href="#2"><span class="tocnumber">2</span> <span class="toctext">Usage |
+ | and | ||
Biology</span></a> | Biology</span></a> | ||
</li> | </li> | ||
<li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly | <li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly | ||
− | and | + | and Part Evolution</span></a> |
</li> | </li> | ||
<li class="toclevel-1 tocsection-5"><a href="#4"><span class="tocnumber">4</span> <span | <li class="toclevel-1 tocsection-5"><a href="#4"><span class="tocnumber">4</span> <span | ||
class="toctext">Results</span></a> | class="toctext">Results</span></a> | ||
+ | <ul> | ||
+ | <li class="toclevel-2 tocsection-6"><a href="#4.1"><span class="tocnumber">4.1</span> Mature Cathepsin B Is | ||
+ | Expressed in HEK293T Cells</a> | ||
+ | </li> | ||
+ | <li class="toclevel-2 tocsection-7"><a href="#4.2"><span class="tocnumber">4.2</span> mCherry and eGFP Can | ||
+ | be Used as a Reporter System to Measure Cleavage Efficiency</a></li> | ||
+ | <li class="toclevel-2 tocsection-8"><a href="#4.3"><span class="tocnumber">4.3</span> The Peptide Linker GFLG | ||
+ | Is Cleaved by Cathepsin B <i>in Vivo</i></a> | ||
+ | </li> | ||
+ | </ul> | ||
</li> | </li> | ||
− | <li class="toclevel-1 tocsection- | + | <li class="toclevel-1 tocsection-9"><a href="#5"><span class="tocnumber">5</span> <span |
+ | class="toctext">Conclusion</span></a> | ||
+ | <li class="toclevel-1 tocsection-10"><a href="#6"><span class="tocnumber">6</span> <span | ||
class="toctext">References</span></a> | class="toctext">References</span></a> | ||
+ | </li> | ||
</li> | </li> | ||
</ul> | </ul> | ||
Line 67: | Line 99: | ||
<section> | <section> | ||
+ | <p><br /><br /></p> | ||
<font size="5"><b>The PICasSO Toolbox </b> </font> | <font size="5"><b>The PICasSO Toolbox </b> </font> | ||
− | + | <div class="thumb" style="margin-top:10px;"></div> | |
− | <div class="thumb"></div> | + | <div class="thumbinner" style="width:550px"><img alt="" class="thumbimage" |
− | + | src="https://static.igem.wiki/teams/5237/wetlab-results/registry-part-collection-engineering-cycle-example-overview.svg" | |
− | + | style="width:99%;" /> | |
− | + | <div class="thumbcaption"> | |
− | + | <i><b>Figure 1: How our part collection can be used to engineer new staples</b></i> | |
</div> | </div> | ||
</div> | </div> | ||
− | |||
− | |||
<p> | <p> | ||
− | <br> | + | <br /> |
− | + | While synthetic biology has in the past focused on engineering the genomic sequence of organisms, the <b>3D | |
− | + | spatial organization</b> 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 | |
− | 3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a | + | cell fate, disease development, evolution, and more. However, tools to precisely manipulate and control the |
− | toolbox based on various DNA-binding proteins | + | 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 | ||
+ | <b>powerful | ||
+ | molecular toolbox for rationally engineering genome 3D architectures</b> in living cells, based on | ||
+ | various DNA-binding proteins. | ||
</p> | </p> | ||
<p> | <p> | ||
The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and | The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and | ||
− | re-programming | + | <b>re-programming |
− | + | of DNA-DNA interactions</b> using engineered "protein staples" in living cells. This enables | |
− | interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation. | + | researchers to recreate naturally occurring alterations of 3D genomic |
− | Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and | + | interactions, such as enhancer hijacking in cancer, or to design entirely new spatial architectures for |
− | testing of new staples | + | 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 <b>chimeric CRISPR/Cas complexes</b>, | ||
+ | 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 <b>robust assay</b> systems to | ||
+ | support the engineering, optimization, and | ||
+ | testing of new staples <i>in vitro</i> and <i>in vivo</i>. Notably, the PICasSO toolbox was developed in a | ||
+ | design-build-test-learn <b>engineering cycle closely intertwining wet lab experiments and computational | ||
+ | modeling</b> and iterated several times, yielding a collection of well-functioning and -characterized | ||
+ | parts. | ||
</p> | </p> | ||
− | + | <p>At its heart, the PICasSO part collection consists of three categories. <br /><b>(i)</b> Our <b>DNA-binding | |
− | + | proteins</b> | |
− | <p>At its heart, the PICasSO | + | |
include our | include our | ||
− | finalized | + | finalized Cas staple experimentally validated using an artificial "enhancer hijacking" system as well as |
− | new Cas staples in the future. We also include our | + | "half staples" that can be combined by scientists to compose entirely |
− | and can be further engineered to create alternative, simpler and more compact staples. (ii) As <b>functional | + | 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 | |
− | consist of | + | successful stapling |
− | + | and can be further engineered to create alternative, simpler, and more compact staples. <br /> | |
− | + | <b>(ii)</b> As <b>functional elements</b>, we list additional parts that enhance and expand the | |
− | interkingdom conjugation system. | + | 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, | |
− | systems</b>. These include components of our established FRET-based proximity assay system, enabling users to | + | dynamic stapling <i>in vivo</i>. |
+ | 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. <br /> | ||
+ | <b>(iii)</b> As the final category of our collection, we provide parts that underlie our <b>custom | ||
+ | readout | ||
+ | systems</b>. These include components of our established FRET-based proximity assay system, enabling | ||
+ | users to | ||
confirm | confirm | ||
− | accurate stapling. Additionally, we offer a complementary, application-oriented testing system | + | 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. | ||
</p> | </p> | ||
<p> | <p> | ||
− | The following table gives a | + | The following table gives a comprehensive overview of all parts in our PICasSO toolbox. <mark |
− | + | style="background-color: #FFD700; color: black;">The highlighted parts showed | |
− | collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their | + | exceptional performance as described on our iGEM wiki and can serve as a reference.</mark> The other |
− | own custom Cas staples, enabling further optimization and innovation | + | 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.<br /> | ||
</p> | </p> | ||
<p> | <p> | ||
− | <font size="4"><b>Our part collection includes:</b></font><br> | + | <font size="4"><b>Our part collection includes:</b></font><br /> |
</p> | </p> | ||
− | + | <table style="width: 90%; padding-right:10px;"> | |
− | <table style="width: 90%;"> | + | <td align="left" colspan="3"><b>DNA-Binding Proteins: </b> |
− | <td | + | Modular building blocks for engineering of custom staples to mediate defined DNA-DNA interactions <i>in vivo</i> |
− | + | </td> | |
− | + | ||
<tbody> | <tbody> | ||
<tr bgcolor="#FFD700"> | <tr bgcolor="#FFD700"> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td> | ||
− | <td> | + | <td>Fusion Guide RNA Entry Vector MbCas12a-SpCas9</td> |
− | <td> | + | <td>Entry vector for simple fgRNA cloning via SapI</td> |
</tr> | </tr> | ||
− | <tr> | + | <tr bgcolor="#FFD700"> |
<td><a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank">BBa_K5237001</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank">BBa_K5237001</a></td> | ||
− | <td> | + | <td>Staple Subunit: dMbCas12a-Nucleoplasmin NLS</td> |
− | <td>Staple subunit that can be combined to form a functional staple | + | <td>Staple subunit that can be combined with crRNA or fgRNA and dSpCas9 to form a functional staple |
+ | </td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr bgcolor="#FFD700"> |
<td><a href="https://parts.igem.org/Part:BBa_K5237002" target="_blank">BBa_K5237002</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237002" target="_blank">BBa_K5237002</a></td> | ||
− | <td> | + | <td>Staple Subunit: SV40 NLS-dSpCas9-SV40 NLS</td> |
− | <td>Staple subunit that can be combined to form a functional staple | + | <td>Staple subunit that can be combined with a sgRNA or fgRNA and dMbCas12a to form a functional staple |
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td> | ||
− | <td>Cas | + | <td>Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td> |
− | <td>Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands | + | <td>Functional Cas staple that can be combined with sgRNA and crRNA or fgRNA to bring two DNA strands into |
+ | close | ||
+ | proximity | ||
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237004" target="_blank">BBa_K5237004</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237004" target="_blank">BBa_K5237004</a></td> | ||
− | <td> | + | <td>Staple Subunit: Oct1-DBD</td> |
− | <td>Staple subunit that can be combined to form a functional staple, for example with TetR.<br> | + | <td>Staple subunit that can be combined to form a functional staple, for example with TetR.<br /> |
Can also be combined with a fluorescent protein as part of the FRET proximity assay</td> | Can also be combined with a fluorescent protein as part of the FRET proximity assay</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td> | ||
− | <td> | + | <td>Staple Subunit: TetR</td> |
− | <td>Staple subunit that can be combined to form a functional staple, for example with Oct1.<br> | + | <td>Staple subunit that can be combined to form a functional staple, for example with Oct1.<br /> |
Can also be combined with a fluorescent protein as part of the FRET proximity assay</td> | Can also be combined with a fluorescent protein as part of the FRET proximity assay</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td> | ||
− | <td>Simple | + | <td>Simple Staple: TetR-Oct1</td> |
<td>Functional staple that can be used to bring two DNA strands in close proximity</td> | <td>Functional staple that can be used to bring two DNA strands in close proximity</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237007" target="_blank">BBa_K5237007</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237007" target="_blank">BBa_K5237007</a></td> | ||
− | <td> | + | <td>Staple Subunit: GCN4</td> |
<td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td> | <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237008" target="_blank">BBa_K5237008</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237008" target="_blank">BBa_K5237008</a></td> | ||
− | <td> | + | <td>Staple Subunit: rGCN4</td> |
<td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td> | <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237009" target="_blank">BBa_K5237009</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237009" target="_blank">BBa_K5237009</a></td> | ||
− | <td>Mini | + | <td>Mini Staple: bGCN4</td> |
<td> | <td> | ||
Assembled staple with minimal size that can be further engineered</td> | Assembled staple with minimal size that can be further engineered</td> | ||
</tr> | </tr> | ||
</tbody> | </tbody> | ||
− | <td | + | <td align="left" colspan="3"><b>Functional Elements: </b> |
− | Protease cleavable peptide linkers and inteins are used to control and modify staples for further optimization | + | Protease-cleavable peptide linkers and inteins are used to control and modify staples for further |
− | for custom applications | + | optimization |
+ | for custom applications</td> | ||
<tbody> | <tbody> | ||
<tr bgcolor="#FFD700"> | <tr bgcolor="#FFD700"> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td> | ||
− | <td>Cathepsin B- | + | <td>Cathepsin B-cleavable Linker: GFLG</td> |
− | <td>Cathepsin B cleavable peptide linker | + | <td>Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make |
+ | responsive | ||
staples</td> | staples</td> | ||
</tr> | </tr> | ||
Line 201: | Line 266: | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td> | ||
<td>Cathepsin B Expression Cassette</td> | <td>Cathepsin B Expression Cassette</td> | ||
− | <td> | + | <td>Expression cassette for the overexpression of cathepsin B</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td><a href="https://parts.igem.org/Part: | + | <td><a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a></td> |
<td>Caged NpuN Intein</td> | <td>Caged NpuN Intein</td> | ||
− | <td> | + | <td>A caged NpuN split intein fragment that undergoes protein <i>trans</i>-splicing after protease |
− | + | activation, which can be used to create functionalized staple | |
+ | subunits</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td><a href="https://parts.igem.org/Part: | + | <td><a href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a></td> |
<td>Caged NpuC Intein</td> | <td>Caged NpuC Intein</td> | ||
− | <td> | + | <td>A caged NpuC split intein fragment that undergoes protein <i>trans</i>-splicing after protease |
− | + | activation, which can be used to create functionalized staple | |
+ | subunits</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td><a href="https://parts.igem.org/Part: | + | <td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td> |
− | <td> | + | <td>Fusion Guide RNA Processing Casette</td> |
− | <td>Processing | + | <td>Processing cassette to produce multiple fgRNAs from one transcript, that can be used for |
+ | multiplexed 3D | ||
+ | genome reprogramming</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td><a href="https://parts.igem.org/Part: | + | <td><a href="https://parts.igem.org/Part:BBa_K5237015" target="_blank">BBa_K5237015</a></td> |
<td>Intimin anti-EGFR Nanobody</td> | <td>Intimin anti-EGFR Nanobody</td> | ||
− | <td> | + | <td>Interkingdom conjugation between bacteria and mammalian cells, as an alternative delivery tool for |
+ | large | ||
constructs</td> | constructs</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K4643003" target="_blank">BBa_K4643003</a></td> | ||
+ | <td>IncP Origin of Transfer</td> | ||
+ | <td>Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a | ||
+ | means of | ||
+ | delivery</td> | ||
</tr> | </tr> | ||
</tbody> | </tbody> | ||
− | <td | + | <td align="left" colspan="3"><b>Readout Systems: </b> |
− | FRET and enhancer recruitment to | + | FRET and enhancer recruitment readout systems to rapidly assess successful DNA stapling in bacterial and |
− | + | mammalian cells | |
+ | </td> | ||
<tbody> | <tbody> | ||
<tr bgcolor="#FFD700"> | <tr bgcolor="#FFD700"> | ||
− | <td><a href="https://parts.igem.org/Part: | + | <td><a href="https://parts.igem.org/Part:BBa_K5237016" target="_blank">BBa_K5237016</a></td> |
<td>FRET-Donor: mNeonGreen-Oct1</td> | <td>FRET-Donor: mNeonGreen-Oct1</td> | ||
− | <td> | + | <td>FRET donor-fluorophore fused to Oct1-DBD that binds to the Oct1 binding cassette, which can be used to |
+ | visualize | ||
+ | DNA-DNA | ||
proximity</td> | proximity</td> | ||
</tr> | </tr> | ||
Line 240: | Line 320: | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237017" target="_blank">BBa_K5237017</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237017" target="_blank">BBa_K5237017</a></td> | ||
<td>FRET-Acceptor: TetR-mScarlet-I</td> | <td>FRET-Acceptor: TetR-mScarlet-I</td> | ||
− | <td>Acceptor part for the FRET assay binding the TetR binding cassette | + | <td>Acceptor part for the FRET assay binding the TetR binding cassette, which can be used to visualize |
+ | DNA-DNA | ||
proximity</td> | proximity</td> | ||
</tr> | </tr> | ||
Line 246: | Line 327: | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a></td> | ||
<td>Oct1 Binding Casette</td> | <td>Oct1 Binding Casette</td> | ||
− | <td>DNA sequence containing 12 Oct1 binding motifs, | + | <td>DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET |
proximity assay</td> | proximity assay</td> | ||
</tr> | </tr> | ||
Line 252: | Line 333: | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a></td> | ||
<td>TetR Binding Cassette</td> | <td>TetR Binding Cassette</td> | ||
− | <td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET | + | <td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the |
+ | FRET | ||
proximity assay</td> | proximity assay</td> | ||
</tr> | </tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237020" target="_blank">BBa_K5237020</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237020" target="_blank">BBa_K5237020</a></td> | ||
− | <td>Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64</td> | + | <td>Cathepsin B-Cleavable <i>Trans</i>-Activator: NLS-Gal4-GFLG-VP64</td> |
− | <td>Readout system that responds to protease activity | + | <td>Readout system that responds to protease activity, which was used to test cathepsin B-cleavable linker |
− | </ | + | </td> |
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237021" target="_blank">BBa_K5237021</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237021" target="_blank">BBa_K5237021</a></td> | ||
<td>NLS-Gal4-VP64</td> | <td>NLS-Gal4-VP64</td> | ||
− | <td>Trans-activating enhancer, that can be used to simulate enhancer hijacking | + | <td><i>Trans</i>-activating enhancer, that can be used to simulate enhancer hijacking</td> |
</tr> | </tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237022" target="_blank">BBa_K5237022</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237022" target="_blank">BBa_K5237022</a></td> | ||
− | <td>mCherry Expression Cassette: UAS, minimal | + | <td>mCherry Expression Cassette: UAS, minimal Promoter, mCherry</td> |
− | <td>Readout system for enhancer binding | + | <td>Readout system for enhancer binding, which was used to test cathepsin B-cleavable linker</td> |
− | + | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237023" target="_blank">BBa_K5237023</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237023" target="_blank">BBa_K5237023</a></td> | ||
− | <td>Oct1 - UAS | + | <td>Oct1 - 5x UAS Binding Casette</td> |
− | <td>Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay | + | <td>Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237024" target="_blank">BBa_K5237024</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237024" target="_blank">BBa_K5237024</a></td> | ||
− | <td> | + | <td>TRE-minimal Promoter- Firefly Luciferase</td> |
− | <td>Contains | + | <td>Contains firefly luciferase controlled by a minimal promoter, which was used as a luminescence |
− | simulated enhancer hijacking | + | readout for |
+ | simulated enhancer hijacking</td> | ||
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
− | |||
</section> | </section> | ||
<section id="1"> | <section id="1"> | ||
− | <h1>1. Sequence | + | <h1>1. Sequence Overview</h1> |
</section> | </section> | ||
</body> | </body> | ||
</html> | </html> | ||
− | |||
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− | <span class= | + | <span class="h3bb">Sequence and Features</span> |
<partinfo>BBa_K5237020 SequenceAndFeatures</partinfo> | <partinfo>BBa_K5237020 SequenceAndFeatures</partinfo> | ||
<!--################################--> | <!--################################--> | ||
+ | <html> | ||
− | |||
− | |||
− | |||
<body> | <body> | ||
<section id="2"> | <section id="2"> | ||
<h1>2. Usage and Biology</h1> | <h1>2. Usage and Biology</h1> | ||
− | <p>Proteolytic cleavage of pro-biologics allows for | + | <p>Cathepsin B is a cysteine protease that is significantly overexpressed in various cancer types, including breast |
− | + | and colorectal cancer (Ruan <i>et al.</i>, 2015). Proteolytic cleavage of pro-biologics via cathepsin B activity | |
− | + | allows for precise temporal and spatial regulation of biopharmaceutical activity in therapeutic strategies (Bleuez | |
+ | <i>et al.</i>, 2022).<br /> | ||
+ | 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 <i>et al.</i>, 2024), with cleavage occurring either between phenylalanine and | ||
+ | leucine or after the second glycine of the linker (Rejmanová <i>et al.</i>, 1983; <b>Fig. 2</b>).<br /> | ||
+ | The construct also includes Gal4, a transcription factor that binds upstream activation sites (UAS) to drive gene | ||
+ | expression, and VP64, a strong transcriptional activator that enhances this expression (Muench <i>et al.</i>, | ||
+ | 2023). Additionally, it incorporates the SV40 nuclear localization signal (NLS), a short peptide derived from the | ||
+ | <i>simian virus 40</i> (SV40) large T-antigen, which facilitates the transport of the fusion protein into the | ||
+ | nucleus, where it can bind to a UAS (Yoneda, 1997).<br /> | ||
+ | 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 <i>et al.</i>, 2022; Shim <i>et al.</i>, 2022; Wang <i>et al.</i>, | ||
+ | 2024). This linker facilitates cleavage-induced oligomerization of Cas proteins via protein <i>trans</i>-splicing | ||
+ | of | ||
+ | caged intein fragments (<a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a>, <a | ||
+ | href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a>), further enhancing the | ||
+ | capabilities of our system.<br /> | ||
+ | </p> | ||
</section> | </section> | ||
<section id="3"> | <section id="3"> | ||
− | <h1>3. Assembly and | + | <h1>3. Assembly and Part Evolution</h1> |
− | <p>We designed a fluorescence readout assay in HEK293T cells based on expression of mCherry induced by the transactivator 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. 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 ( | + | <p>We designed a fluorescence readout assay in HEK293T cells based on expression of mCherry induced by the |
− | + | transactivator 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. | ||
+ | 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. 2).</p> | ||
<div class="thumb"> | <div class="thumb"> | ||
− | <div class="thumbinner" style="width:450px; | + | <div class="thumbinner" style="width:450px;"><img alt="Cathepsin B Fluorescence Readout Assay" class="thumbimage" |
− | + | src="https://static.igem.wiki/teams/5237/wetlab-results/catb-gal4-vp64-mechanism.svg" width="450" /> | |
− | + | ||
<div class="thumbcaption"> | <div class="thumbcaption"> | ||
− | <i><b>Figure 2: Schematic Illustration of the Cathepsin B Fluorescence Readout Assay.</b | + | <i><b>Figure 2: Schematic Illustration of the Cathepsin B Fluorescence Readout Assay.</b> The DNA-binding |
+ | domain (DBD) of Gal4 is conjugated to the transactivator domain VP64 via a cathepsin B-cleavable peptide | ||
+ | linker. Binding of the Gal4-DBD to the upstream activating sequence (UAS) in proximity to the mCherry gene | ||
+ | induces mCherry overexpression via VP64. Cathepsin B cleavage of the linker separates Gal4-DBD and VP64 and | ||
+ | consequently reduces mCherry expression.</i> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
− | + | <p>We transfected our genetic constructs into HEK293T cells. The negative control was not transfected with the | |
− | <p>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 | + | 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. 3). 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.</p> | ||
<div class="thumb"> | <div class="thumb"> | ||
− | <div class="thumbinner" style="width:450px;">< | + | <div class="thumbinner" style="width:450px;"><img alt="Fluorescence Readout" class="thumbimage" |
− | + | src="https://static.igem.wiki/teams/5237/wetlab-results/catb-results/catb-fluorescent-readout-no-dox-w.svg" | |
− | + | width="450" /> | |
<div class="thumbcaption"> | <div class="thumbcaption"> | ||
− | <i><b>Figure 3: Fluorescence Readout After 48 | + | <i><b>Figure 3: Fluorescence Readout After 48 Hours for Five Different Peptide Linkers and Three Different |
+ | Conditions.</b> The fluorescence intensity for mCherry was measured for five different linkers. 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.</i> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
− | + | <p>Since cathepsin B is a lysosomal protease that is normally only active in the lysosome and the extracellular | |
− | <p>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 <i>et al.</i>, 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 <i>et al.</i>, 1997).<br> | + | environment but not in the cytosol, we decided to change the native amino acid sequence of cathepsin B. Upon |
− | 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 | + | 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 <i>et al.</i>, 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 <i>et al.</i>, 1997).<br /> | ||
+ | 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. 4>). 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. 5). 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.</p> | ||
<div class="thumb"> | <div class="thumb"> | ||
− | <div class="thumbinner" style="width:450px;">< | + | <div class="thumbinner" style="width:450px;"><img alt="Fluorescence Readout" class="thumbimage" |
− | + | src="https://static.igem.wiki/teams/5237/wetlab-results/catb-results/catb-fluorescent-readout-mutated-and-truncated-w.svg" | |
− | + | width="450" /> | |
<div class="thumbcaption"> | <div class="thumbcaption"> | ||
− | <i><b>Figure 4: Fluorescence Readout for the Truncated and Mutated Version of Cathepsin B.</b></i> | + | <i><b>Figure 4: Fluorescence Readout for the Truncated and Mutated Version of Cathepsin B.</b> |
+ | Fluorescence intensity for mCherry was measured across five different linkers. The negative control, which | ||
+ | was | ||
+ | not transfected with the plasmid encoding cathepsin B, was assigned a fluorescence intensity value of one. | ||
+ | Two | ||
+ | test conditions were explored, where either 30 ng or 60 ng of the plasmid encoding cathepsin B was | ||
+ | transfected.</i> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
− | + | <p>Since our truncated and mutated version of cathepsin B did not seem to be active in the cytosol, we decided to go | |
− | <p>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 | + | 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 agent doxorubicin to | |
+ | induce | ||
+ | lysosomal escape of cathepsin B, as had been previously reported (Bien <i>et al.</i>, 2004).</p> | ||
</section> | </section> | ||
<section id="4"> | <section id="4"> | ||
<h1>4. Results</h1> | <h1>4. Results</h1> | ||
− | < | + | <section id="4.1"> |
− | <p> | + | <h2>4.1 Mature Cathepsin B Is Expressed in HEK293T Cells</h2> |
− | + | <p>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.<br /> | |
− | + | <b>Figure 5</b> 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. | ||
+ | </p> | ||
+ | <div class="thumb"> | ||
+ | <div class="thumbinner" style="width:450px;"><img alt="Fluorescence Readout" class="thumbimage" | ||
+ | src="https://static.igem.wiki/teams/5237/wetlab-results/catb-wb-w.svg" width="450" /> | ||
+ | <div class="thumbcaption"> | ||
+ | <i><b>Figure 5: Western Blot of Two Versions of Cathepsin B With and Without Doxorubicin.</b> 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.</i> | ||
+ | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
− | </ | + | </section> |
− | + | <section id="4.2"> | |
− | < | + | <h2>4.2 mCherry and eGFP Can be Used as a Reporter System to Measure Cleavage Efficiency</h2> |
− | <p><b>Figure 6</b> shows micrographs taken with a fluorescence microscope of three different conditions: the null control, the negative control and the test sample. <b>Figure 7</b> 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 | + | <p>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.<br /> | |
− | + | <b>Figure 6</b> shows micrographs taken with a fluorescence microscope of three different conditions: the null | |
− | + | control, the negative control and the test sample. <b>Figure 7</b> 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. | ||
+ | </p> | ||
+ | <div class="thumb"> | ||
+ | <div class="thumbinner" style="width:700px;"><img alt="Fluorescence Readout" class="thumbimage" | ||
+ | src="https://static.igem.wiki/teams/5237/wetlab-results/catb-fluorescence-microscope-w1.png" width="700" /> | ||
+ | <div class="thumbcaption"> | ||
+ | <i><b>Figure 6: Micrographs of HEK293T Cells in Two Control Conditions and One Test Condition.</b> | ||
+ | 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.</i> | ||
+ | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
− | + | <div class="thumb"> | |
− | + | <div class="thumbinner" style="width:450px;"><img alt="Fluorescence Readout" class="thumbimage" | |
− | + | src="https://static.igem.wiki/teams/5237/wetlab-results/catb-results/catb-fluorescence-readout-null-negative-test-w.svg" | |
− | + | width="450" /> | |
− | + | <div class="thumbcaption"> | |
− | + | <i><b>Figure 7: Fluorescence Readout After 48 Hours for Two Control Conditions and One Test | |
− | + | Condition.</b> 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.</i> | ||
+ | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
− | </ | + | </section> |
− | + | <section id="4.3"> | |
− | < | + | <h2>4.3 The Peptide Linker GFLG Is Cleaved by Cathepsin B <i>in Vivo</i></h2> |
− | <p><b>Figure 8</b> shows | + | <p>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. <b>Figure 8</b> 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.</p> | ||
+ | <div class="thumb"> | ||
+ | <div class="thumbinner" style="width:450px;"><img alt="Fluorescence Readout" class="thumbimage" | ||
+ | src="https://static.igem.wiki/teams/5237/wetlab-results/catb-results/catb-fluorescent-readout-final-results-w.svg" | ||
+ | width="450" /> | ||
+ | <div class="thumbcaption"> | ||
+ | <i><b>Figure 8: Fluorescence Readout After 48 Hours for Five Different Peptide Linkers and Three Different | ||
+ | Conditions.</b> 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.</i> | ||
+ | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
− | </ | + | </section> |
− | + | ||
− | + | ||
− | + | ||
</section> | </section> | ||
<section id="5"> | <section id="5"> | ||
− | <h1>5. References</h1> | + | <h1>5. Conclusion</h1> |
+ | <p>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-cleavable linker can be combined with caged inteins | ||
+ | (Gramespacher <i>et al.</i>, 2017) conjugated to a dead Cas9 to selectively induce Cas stapling in the presence of | ||
+ | cathepsin B.</p> | ||
+ | </section> | ||
+ | <section id="6"> | ||
+ | <h1>6. References</h1> | ||
<p> | <p> | ||
− | 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. <a | + | 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), | |
− | </p> | + | 1092-102. <a href="https://doi.org/10.1124/mol.65.5.1092" |
− | <p> | + | target="_blank">https://doi.org/10.1124/mol.65.5.1092</a> |
− | Bleuez, C., Koch, W. F., Urbach, C., Hollfelder, F., & Jermutus, L. (2022). Exploiting protease activation for therapy. Drug Discov Today, 27(6), 1743-1754. <a | + | </p> |
− | + | <p> | |
− | </p> | + | Bleuez, C., Koch, W. F., Urbach, C., Hollfelder, F., & Jermutus, L. (2022). Exploiting protease activation for |
− | <p> | + | therapy. Drug Discov Today, 27(6), 1743-1754. <a href="https://doi.org/10.1016/j.drudis.2022.03.011" |
− | Gramespacher, J. A., Stevens, A. J., | + | target="_blank">https://doi.org/10.1016/j.drudis.2022.03.011</a> |
− | href="https://doi.org/10.1021/jacs.7b02618" target="_blank">https://doi.org/10.1021/jacs.7b02618</a> | + | </p> |
− | </p> | + | <p> |
− | <p> | + | Gramespacher, J. A., Stevens, A. J., Nguyen, D. P., Chin, J. W., & Muir, T. W. (2017). Intein Zymogens: |
− | 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. <a | + | Conditional Assembly and Splicing of Split Inteins via Targeted Proteolysis. J Am Chem Soc, 139(24), 8074-8077. <a |
− | + | href="https://doi.org/10.1021/jacs.7b02618" target="_blank">https://doi.org/10.1021/jacs.7b02618</a> | |
− | </p> | + | </p> |
− | <p> | + | <p> |
− | 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. <a | + | Jin, C., EI-Sagheer, A. H., Li, S., Vallis, K. A., Tan, W., & Brown, T. (2022). Engineering Enzyme-Cleavable |
− | href="https://doi.org/10.1074/jbc.M413052200" target="_blank">https://doi.org/10.1074/jbc.M413052200</a> | + | Oligonucleotides by Automated Solid-Phase Incorporation of Cathepsin B Sensitive Dipeptide Linkers. Angewandte |
− | </p> | + | Chemie International Edition, 61(13), e202114016. <a href="https://doi.org/10.1002/anie.202114016" |
− | <p> | + | target="_blank">https://doi.org/10.1002/anie.202114016</a> |
− | 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. <a | + | </p> |
− | + | <p> | |
− | </p> | + | Muench, P., Fiumara, M., Southern, N., Coda, D., Aschenbrenner, S., Correia, B., Gräff, J., Niopek, D., & |
− | <p> | + | Mathony, J. (2023). A modular toolbox for the optogenetic deactivation of transcription. bioRxiv, |
− | Ruan, H., Hao, S., Young, P., & Zhang, H. (2015). Targeting Cathepsin B for Cancer Therapies. Horiz Cancer Res, 56, 23-40. | + | 2023.2011.2006.565805. <a href="https://doi.org/10.1101/2023.11.06.565805" |
− | </p> | + | target="_blank">https://doi.org/10.1101/2023.11.06.565805</a> |
− | <p> | + | </p> |
− | 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. <a | + | <p> |
− | href="https://doi.org/10.1016/j.biomaterials.2022.121806" target="_blank">https://doi.org/10.1016/j.biomaterials.2022.121806</a> | + | Müntener, K., Willimann, A., Zwicky, R., Svoboda, B., Mach, L., & Baici, A. (2005). Folding Competence of |
− | </p> | + | N-terminally Truncated Forms of Human Procathepsin B*. Journal of Biological Chemistry, 280(12), 11973-11980. <a |
− | <p> | + | href="https://doi.org/10.1074/jbc.M413052200" target="_blank">https://doi.org/10.1074/jbc.M413052200</a> |
− | 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. <a | + | </p> |
− | + | <p> | |
− | </p> | + | 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. <a href="https://doi.org/10.1021/bi971264+" target="_blank">https://doi.org/10.1021/bi971264+</a> | ||
+ | </p> | ||
+ | <p> | ||
+ | Ruan, H., Hao, S., Young, P., & Zhang, H. (2015). Targeting Cathepsin B for Cancer Therapies. Horiz Cancer | ||
+ | Res, 56, 23-40. | ||
+ | </p> | ||
+ | <p> | ||
+ | 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. <a | ||
+ | href="https://doi.org/10.1016/j.biomaterials.2022.121806" | ||
+ | target="_blank">https://doi.org/10.1016/j.biomaterials.2022.121806</a> | ||
+ | </p> | ||
+ | <p> | ||
+ | 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. <a href="https://doi.org/10.1002/EXP.20230027" | ||
+ | target="_blank">https://doi.org/10.1002/EXP.20230027</a> | ||
+ | </p> | ||
+ | <p> | ||
+ | Yoneda,Y. (1997). How Proteins Are Transported from Cytoplasm to the Nucleus. The Journal of Biochemistry, 121(5), | ||
+ | 811 – 817. <a href="https://doi.org/10.1093/oxfordjournals.jbchem.a021657" | ||
+ | target="_blank">https://doi.org/10.1093/oxfordjournals.jbchem.a021657</a> | ||
+ | </p> | ||
</section> | </section> | ||
</body> | </body> | ||
</html> | </html> |
Latest revision as of 12:28, 2 October 2024
Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64
This composite part encodes a cathepsin B-cleavable trans-activator fusion protein. It is composed of an SV40 nuclear localization sequence (BBa_K2549054), the DNA-binding domain of Gal4 (BBa_K4585001), a GFLG linker (BBa_K5237010), and the VP64 trans-activator domain (BBa_K4586021). We used this part to design a fluorescence-based readout assay in HEK293T cells to investigate cathepsin B cleavage of different peptide linkers. Through this assay, we were able to successfully show that the GFLG linker was specifically cleaved in the presence of cathepsin B, while other linkers did not show this effect. Together, these findings enable the functionalization of our PICasSO system for a wide range of therapeutic and synthetic biology applications.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 254
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 173
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 via 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 phenylalanine and
leucine or after the second glycine of the linker (Rejmanová et al., 1983; Fig. 2).
The construct also includes Gal4, a transcription factor that binds upstream activation sites (UAS) to drive gene
expression, and VP64, a strong transcriptional activator that enhances this expression (Muench et al.,
2023). Additionally, it incorporates the SV40 nuclear localization signal (NLS), a short peptide derived from the
simian virus 40 (SV40) large T-antigen, which facilitates the transport of the fusion protein into the
nucleus, where it can bind to a UAS (Yoneda, 1997).
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 facilitates cleavage-induced oligomerization 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
We designed a fluorescence readout assay in HEK293T cells based on expression of mCherry induced by the transactivator 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. 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. 2).
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 (Fig. 3). 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 (Fig. 4>). 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. 5). 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 cytostatic agent 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 5 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.
4.2 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 6 shows micrographs taken with a fluorescence microscope of three different conditions: the null
control, the negative control and the test sample. Figure 7 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 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 8 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
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-cleavable 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.
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
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
Muench, P., Fiumara, M., Southern, N., Coda, D., Aschenbrenner, S., Correia, B., Gräff, J., Niopek, D., & Mathony, J. (2023). A modular toolbox for the optogenetic deactivation of transcription. bioRxiv, 2023.2011.2006.565805. https://doi.org/10.1101/2023.11.06.565805
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+
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