Difference between revisions of "Team:Slovenia/Demonstrate"

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                        <b>Protease-based</b> <br />  
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<b style="margin-left: 12%">inducible secretion</b>
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                        <b>Introduction</b>
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                        <b>ER lumen</b>
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                        <b>ER membrane</b>
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<h1 class = "ui left dividing header"><span class="section">&nbsp;</span>Protease inducible secretion</h1>
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                    <a class="item" href="//2016.igem.org/Team:Slovenia/Proof">
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                        <b>Touch painting</b>
<p><b><ul>
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                    </a>
<li>Retention of proteins in ER lumen was demonstrated by confocal microscopy and detection of the protein in the cell medium.
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<li>A variant of TEVp active in the ER lumen was implemented to control protein secretion from the ER lumen.
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                </div>
<li>Retention of proteins on the ER membrane was also demonstrated by confocal microscopy and detection of the protein in the cell medium.
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<li>Rapamycin induced cleavage was used for controlled and inducible secretion of proteins from the ER membrane.
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</ul></b></p>
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<p>To achieve a fast regulated cellular response resulting in the release of a protein, we decided to mimic the release of insulin from beta cells where the protein of
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                <div>
interest is pre-formed and present in the cell in secretory granules. In contrast to the specialized storage and release mechanism of insulin from beta cells we wanted to
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                    <div class="main ui citing justified container">
develop a more general and modular solution by making use of components already existing in different types of cells. Additionally, there should be minimal leakage from the
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<div>
protein depot in the uninduced state and after induction secretion from the cell should be fast.</p>
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<h1 class="ui left dividing header"><span id="ach" class="section colorize">&nbsp;</span>Protease-based inducible
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secretion</h1>
<div class="ui styled fluid accordion">
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<div class="title">
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<div class="ui segment" style="background-color: #ebc7c7;">
<i class="dropdown icon"></i>
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Further explanation ...
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<p><b>
</div>
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<ul>
<div class="content">
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<li>Retention of proteins in ER lumen was demonstrated by confocal microscopy and
<p> Not many systems for the inducible release of proteins have been engineered to date. In one of the few examples Rivera et al. developed a system where the protein of interest
+
detection of the protein in the cell medium.
was fused to a conditional aggregation domain (CAD). <x-ref>Rivera2000</x-ref>. These domains form aggregates in the endoplasmic reticulum (ER) that are too large to exit the ER.
+
<li>A variant of TEVp active in the ER lumen was implemented to control protein
After the addition of a small synthetic molecule, the CADs start to disaggregate and the protein of interest can be secreted. In the second example Chen et al. introduced a
+
secretion from the ER lumen.
light-triggered secretion system. They also based their system on conditional aggregation; however they used the plant photoreceptor UVR8 which forms photolabile homodimers to make
+
<li>Retention of proteins on the ER membrane was also demonstrated by confocal
aggregates on the ER membrane. Upon light excitation the aggregates made by UVR8 started to disaggregate and were transported from the ER to the plasma membrane, but have not been
+
microscopy and detection of the protein in the cell medium.
observed in the cell supernatant. <x-ref>Chen2013</x-ref>
+
<li>Rapamycin induced cleavage was used for controlled and inducible secretion of
The weakness of the two described systems is that they both rely on the exogenous chemical or physical signals instead of using a biochemical signal to induce the secretion, which
+
proteins from the ER membrane.
means that they can’t be integrated into the signaling system that’s senses the cellular state. In order to better respond to the state of the cell or a logic circuit inside a cell
+
</ul>
we decided to develop an inducible secretion system based on the biochemical signal.
+
</b></p>
</p>
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</div>
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</div>
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<p><br />Many proteins that reside on the membrane or in the lumen of the ER contain short peptide signals. Proteins present in the lumen of the ER contain a KDEL C-terminal sequence
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(Lys-Asp-Glu-Leu) while type I transmembrane (TM) proteins contain a dilysine (KKXX) motif on their C-terminus (cytosolic side). <x-ref> Munro1987, Jackson1990,
+
Stornaiuolo2003</x-ref>. The mechanism that allows these proteins to stay in the ER is more retrieval than retention. However we decided to use the term retention for
+
description of this process. ER luminal proteins interact with the KDEL receptor, a transmembrane ER resident protein. The cytosolic part of the KDEL receptor interacts with
+
coat proteins I (COP I) which coat vesicles and are responsible for transporting proteins from the cis end of the Golgi apparatus (cis-GA) back to the ER. The KKXX motif present
+
on type I TM proteins can directly interact with the COP I for retrieval. <x-ref> Stornaiuolo2003, Letourneur1994 </x-ref>.</p>
+
<p>Our idea was that if we proteolytically remove the retention signal, the protein of interest would no longer be retrieved back to the ER and could be secreted from the cell.
+
To achieve this we designed two types of secretory reporters, one type based on the luminal retention using KDEL sequence and the other based on the transmembrane retention with
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a KKMP sequence. In each case, the retention sequence was preceded by a TEVp cleavage site to allow for inducible secretion, which could be replaced by any other peptide target
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of our orthogonal protease set.</p>
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</div>
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<h1><span class="section">&nbsp;</span>Results</h1>
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<div class = "ui segment">
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<h3>Secretion from the ER lumen</h3>
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<p>To achieve and detect the inducible secretion from the ER lumen, we created two reporter constructs with a cleavable KDEL sequence targeted to the ER lumen: SEAP<sup>KDEL</sup> and
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TagRFP<sup>KDEL</sup>. Those proteins contained a protease target motif between the reporter domain and the KDEL domain, aimed to enable protein secretion after the proteolytic cleavage.
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We used a TEVp variant (erTEVp) for all of our experiments with luminal retention.</p>
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<div class="ui styled fluid accordion">
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<div class="title">
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<i class="dropdown icon"></i>
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Further explanation ...
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</div>
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<div class="content">
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<p>
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In order to rely on TEVp cleavage in the ER lumen, we had to take some additional considerations into account. Cesaratto et al.</x-ref>Cesaratto2015</x-ref> reported that the wild
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type TEV protease is not active in the lumen of ER. They designed a TEV protease variant active in the endoplasmic reticulum by preventing two major types of post-translational
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modifications: N-glycosylation and cysteine oxidation. To avoid these inhibiting modifications, mutations N23Q, C130S and N171T were made. To ensure correct localization and
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accumulation of this TEVp variant inside the endoplasmic reticulum, we also attached a signal sequence at the N-terminus and KDEL at the C-terminus of the protein.
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</p>
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</div>
 
</div>
</div>
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                        </div>
<p style="clear:both"><br/></p>
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<div>
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<h4><span id = "intro" class="section colorize">&nbsp;</span></h4>
<div style= "float:right; width:50%">  
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                            <p>To achieve a fast regulated cellular response resulting in the protein release, we
<figure id="tagRFP" data-ref="1" ><span class="section"></span>
+
                                decided to mimic the release of insulin from beta cells where the protein of
<img src="https://static.igem.org/mediawiki/2016/9/9e/T--Slovenia--6.2.1.png" >
+
                                interest is pre-formed and present intracellularly in secretory granules. In contrast to the
<figcaption><b>Cleavage with ER-residing protease (erTEV) facilitates secretion of reporter from cells.</b><br/>  
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                                specialized storage and release mechanism of insulin from beta cells we wanted to
(A) Scheme of the reporter with cleavable KDEL retention signal and protease target motif.
+
                                develop a more general and modular solution by making use of components already existing
(B) The reporter with the KDEL retention signal was localized in the ER. HEK293T cells were transfected with the indicated reporters and in (C) also with erTEVp.  
+
                                in different types of cells. Additionally, there should be minimal leakage from the
Localization was detected with confocal microscopy. (C)The reporter was detected in the medium of cells only when cotransfected with erTEVp. HEK293T cells were transfected  
+
                                protein depot in the uninduced state and after induction secretion from the cell should
with the indicated constructs. Reporters were detected with WB in the concentrated medium.
+
                                be fast.</p>
</figcaption>
+
 
</figure>
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                            <div class="ui styled fluid accordion">
</div>  
+
                                <div class="title">
<p>When the TagRFP<sup>KDEL</sup> reporter (<ref>1</ref>A) was expressed in the ER without an active erTEVp we confirmed its localization in the ER with confocal microscopy  
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                                    <i class="dropdown icon"></i>
(<ref>1</ref>B). Additionally, we could not detect any TagRFP in the cell medium with Western blotting. When erTEVp was present and active in the ER, the KDEL sequence was  
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                                    Further explanation ...
removed from the reporter and the protein was secreted from the cell, which we detected with Western blot (<ref>1</ref>C), demonstrating that proteolytic activity in the ER can
+
                                </div>
regulate protein secretion.
+
                                <div class="content">
</p>
+
                                    <p> Not many systems for the inducible release of proteins have been engineered to
<p style="clear:left">Using SEAP<sup>KDEL</sup> we were able to confirm that the reporter is not present in the cell medium without coexpression of erTEVp. When erTEVp was cotransfected with  
+
                                        date. In one of the few examples Rivera et al. developed a system where the
the reporter, we detected a large increase in enzymatic activity in the medium (<ref>2</ref>).
+
                                        protein of interest
</p>
+
                                        was fused to a conditional aggregation domain (CAD)
<div style = "float:left; width:50%">  
+
                                        <x-ref>Rivera2000</x-ref>
<figure id="SEAP" data-ref="2"><span class="section"></span>
+
                                        . These domains form aggregates in the endoplasmic reticulum (ER) that are too
<img src="https://static.igem.org/mediawiki/2016/6/61/T--Slovenia--6.2.2.png" >
+
                                        large to exit the ER.
<figcaption><b>Secretion of the SEAP reporter from ER lumen by cleavage with ER-resident protease.</b><br/>  
+
                                        After the addition of a small synthetic molecule, the CADs start to disaggregate
HEK293T cells were transfected with indicated reporter and erTEVp. Increased SEAP activity was detected in the medium of cells  
+
                                        and the protein of interest can be secreted. In the second example Chen et al.
expressing both reporter and erTEVp protease.</figcaption></figure>
+
                                        introduced a
</div>  
+
                                        light-triggered secretion system. They also based their system on conditional
+
                                        aggregation; however they used the plant photoreceptor UVR8 which forms
+
                                        photolabile homodimers to make
<h3 style="clear:both">Secretion from the ER membrane</h3>
+
                                        aggregates on the ER membrane. Upon light excitation the aggregates made by UVR8
<p>The second approach to regulate protein secretion from the ER by protease was to used KKMP ER retention peptide linked to the transmembrane protein with a protease target  
+
                                        started to disaggregate and were transported from the ER to the plasma membrane,
motif on the cytoplasmic side, N-terminal to the KKMP peptide. A transmembrane (TM) domain from the B-cell receptor  
+
                                        but have not been
(<a href="http://parts.igem.org/Part:BBa_K157010">Bba_K157010</a>) was used for the integration of target proteins in the ER membrane. Similar as described above, two reporter  
+
                                        observed in the cell supernatant
constructs with SEAP and TagRFP (SEAP:TM<sup>KKMP</sup> and TagRFP:TM<sup>KKMP</sup>) were designed and the constructs also contained a signal sequence at their N-terminus and a  
+
                                        <x-ref>Chen2013</x-ref>.
proteolytically cleavable ER retention signal at their C-terminus. In case of the transmembrane targeted reporters we used the KKMP retention signal preceded by 3 copies of the  
+
                                        The weakness of the two described systems is that they both rely on the
TEVp cleavage site on the cytosolic side of the membrane.</p>  
+
                                        exogenous chemical or physical signals instead of using a biochemical signal to
<p>Additionally, either one or four furin cleavage sites were inserted between the protein of interest on the luminal side of the ER, which enable cleavage of the reporter  
+
                                        induce the secretion, which
protein from the membrane, but this could occur only after the KKMP had been removed and the protein could enter the trans-GA. Furin is a native cellular endoprotease that is  
+
                                        means that they can’t be integrated into the signaling system that’s senses the
active only in the trans-GA.</x-ref>Henrich2003</x-ref> This allowed us to design our constructs so that they are cleaved off of the membrane without any modified scar sequences  
+
                                        cellular state. In order to better respond to the state of the cell or a logic
attached to them.</p>
+
                                        circuit inside a cell
+
                                        we decided to develop an inducible secretion system based on the biochemical
+
                                        signal.
<div style="float:left; width:100%">  
+
                                    </p>
<figure data-ref="3">
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                                </div>
<img src="https://static.igem.org/mediawiki/2016/b/b4/T--Slovenia--6.2.3.png" >
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                            </div>
<figcaption><b>Localization of protease-responsive reporters on ER depending on the proteolysis. </b><br/>  
+
                            <p><br/>Many proteins that reside on the membrane or in the lumen of the ER contain short
(A) The transmembrane reporter without the KKMP retention signal was localized both on the ER and plasma membrane. (B) The transmembrane reporter with the KKMP retention
+
                                peptide signals. Proteins present in the lumen of the ER contain a KDEL C-terminal
signal was localized exclusively on the ER membrane. (C) After cleavage of the KKMP retention signal, the transmembrane reporter translocated to the plasma membrane. HEK293T  
+
                                sequence
cells were transfected with the indicated reporters and in (C) also with TEVp. Localization was detected with confocal microscopy. Each image is accompanied with a scheme of  
+
                                (Lys-Asp-Glu-Leu) while type I transmembrane (TM) proteins contain a dilysine (KKXX)
the transfected construct. (D) Glycosylated reporter was detected in the medium of cells transfected with the transmembrane reporter without the KKMP retention signal.
+
                                motif on their C-terminus (cytosolic side)
HEK293T cells were transfected with the indicated constructs. Reporters were detected with WB in the concentrated medium. In lane 2, sample was incubated with N-glycosidase
+
                                <x-ref>Munro1987, Jackson1990, Stornaiuolo2003</x-ref>.
F.
+
                                . The mechanism that allows these proteins to stay in the ER is retrieval rather than
</figcaption>
+
                                retention. However, we decided to use the term retention for
</figure>
+
                                description of this process. ER luminal proteins interact with the KDEL receptor, a
</div>
+
                                transmembrane ER resident protein. The cytosolic part of the KDEL receptor interacts
<div style="float:right; width:50%" >  
+
                                with
<figure data-ref="4">
+
                                coat proteins I (COP I) which coat vesicles and are responsible for transporting
<img src="https://static.igem.org/mediawiki/2016/2/2f/T--Slovenia--6.2.4.png" >
+
                                proteins from the cis end of the Golgi apparatus (cis-GA) back to the ER. The KKXX motif
<figcaption><b>Inducible secretion of reporter localized on ER membrane.</b><br/>
+
                                present
SEAP activity was increased in the medium of cells induced with rapamycin. (B) Scheme of the transmembrane reporter with cleavable KKMP retention signal and inducible protease.
+
                                on type I TM proteins can directly interact with the COP I for retrieval
HEK293T cells were transfected with the indicated reporter and rapamycin inducible split proteases. Uncleaved proteases were used as positive control.</figcaption>
+
                                <x-ref>Stornaiuolo2003, Letourneur1994</x-ref>
</figure>
+
                                .
</div>  
+
                            </p>
<p>Localization of the TagRFP:TM<sup>KKMP</sup> reporter was confirmed by the confocal microscopy. We used a control reporter without the KKMP retention signal (TagRFP:TM)
+
                            <p>Our idea was that if we proteolytically remove the retention signal, the protein of
which we detected both on the ER and the plasma membrane (<ref>3</ref>A). In case of the present KKMP retention signal, the reporter was detected only on the ER  
+
                                interest would no longer be retrieved back to the ER and could be secreted from the
(<ref>3</ref>B). When TagRFP:TM<sup>KKMP</sup> was coexpressed with TEVp, localization of the reporter was similar to the localization of the positive control (TagRFP:TM)
+
                                cell.
on the plasma membrane and the ER (<ref>3</ref>C).</p>
+
                                To achieve this we designed two types of secretory reporters, one type based on the
<p>A band with a slightly larger apparent size than the expected size of TagRFP (28 kDa) was detected by western blotting in cells transfected with TagRFP:TM. We showed that the
+
                                luminal retention using KDEL sequence and the other based on the transmembrane retention
unexpected difference in size was due to glycosylation, as we detected the protein at the expected size after deglycosylation of the medium sample with N-glycosidase F. We were
+
                                with
unable to detect a corresponding band in the medium of cells transfected with TagRFP:TM<sup>KKMP</sup> in the absence of the protease.</p>
+
                                a KKMP sequence. In each case, the retention sequence was preceded by a TEVp cleavage
<p>Together, these results confirm that localization and secretion of the protein reporter with the transmembrane domain depends on the presence and proteolysis of the KKMP  
+
                                site to allow for inducible secretion, which could be replaced by any other peptide
retention signal and that proteolysis can be used to induce secretion of already synthesized protein.</p>
+
                                target
+
                                of our <a href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Orthogonality"> orthogonal protease set</a>.</p>
+
                        </div>
<p style="clear:both">Finally, we cotransfected cells with SEAP:TM<sup>KKMP</sup> and rapamycin-inducible split TEVp. We detected increased levels of the SEAP enzymatic activity in the medium of  
+
                        </div>
cells stimulated with rapamycin, which was dose dependent with respect to the amount of the transfected reporter-coding plasmid (<ref>4</ref>). These results confirm that  
+
 
secretion of a target protein can be made inducible by an externally supplied signal, processed through our split protease system.
+
                        <h1><span class="section">&nbsp;</span>Results</h1>
</p>
+
                        <div class="ui segment">
<p style = "clear:both">
+
<div>
</p>
+
                            <h3><span id = "lum" class="section colorize">&nbsp;</span>Release from the ER lumen and secretion from the cell</h3>
</div>
+
                            <p>To achieve and detect the inducible secretion from the ER lumen, we created two reporter
+
                                constructs with a cleavable KDEL sequence targeted to the ER lumen: SEAP<sup>KDEL</sup>
+
                                and
+
                                TagRFP<sup>KDEL</sup>. Those proteins contained a protease target motif between the
+
                                reporter domain and the KDEL domain, aimed to enable protein secretion after the
</div>
+
                                proteolytic cleavage.
</div>
+
                                We used a TEVp variant (erTEVp) for all of our experiments with luminal retention.</p>
</div>
+
                            <div class="ui styled fluid accordion">
 +
                                <div class="title">
 +
                                    <i class="dropdown icon"></i>
 +
                                    Further explanation ...
 +
                                </div>
 +
                                <div class="content">
 +
                                    <p>
 +
                                        In order to rely on TEVp cleavage in the ER lumen, we had to take some
 +
                                        additional considerations into account. Cesaratto et al.</x-ref>
 +
                                        Cesaratto2015</x-ref> reported that the wild
 +
                                        type TEV protease is not active in the lumen of ER. They designed a TEV protease
 +
                                        variant active in the endoplasmic reticulum by preventing two major types of
 +
                                        post-translational
 +
                                        modifications: N-glycosylation and cysteine oxidation. To avoid these inhibiting
 +
                                        modifications, mutations N23Q, C130S and N171T were made. To ensure correct
 +
                                        localization and
 +
                                        accumulation of this TEVp variant inside the endoplasmic reticulum, we also
 +
                                        attached an ER signal sequence at the N-terminus and KDEL at the C-terminus of the
 +
                                        protein.
 +
                                    </p>
 +
                                </div>
 +
                            </div>
 +
                            <p style="clear:both"><br/></p>
 +
 
 +
 
 +
                            <div style="float:right; width:50%">
 +
                                <figure data-ref="1">                                  
 +
                                    <img src="https://static.igem.org/mediawiki/2016/9/9e/T--Slovenia--6.2.1.png">
 +
                                    <figcaption><b>Cleavage with ER-residing protease (erTEV) facilitates secretion of
 +
                                        reporter from cells.</b><br/>
 +
                                        <p style="text-align:justify">(A) Scheme of the reporter with cleavable KDEL
 +
                                            retention signal and protease target motif.
 +
                                            (B) The reporter with the KDEL retention signal was localized in the ER.
 +
                                            HEK293T cells were transfected with the indicated reporters and in (C) also
 +
                                            with erTEVp.
 +
                                            Localization was detected with confocal microscopy. (C)The reporter was
 +
                                            detected in the medium of cells only when cotransfected with erTEVp. HEK293T
 +
                                            cells were transfected
 +
                                            with the indicated constructs. Reporters were detected with WB in the
 +
                                            concentrated medium.
 +
                                        </p></figcaption>
 +
                                </figure>
 +
                            </div>
 +
                            <p>When the TagRFP<sup>KDEL</sup> reporter (<ref>1</ref>A) was expressed in the ER without an active erTEVp we confirmed its localization in the
 +
                                ER with confocal microscopy
 +
                                (<ref>1</ref>B). Additionally, we could not detect any TagRFP in the cell medium with Western
 +
                                blotting. When erTEVp was present and active in the ER, the KDEL sequence was
 +
                                removed from the reporter and the protein was secreted from the cell, which we detected
 +
                                with Western blot (<ref>1</ref>C). This demonstrated that proteolytic activity in the ER can
 +
                                regulate protein secretion.
 +
                            </p>
 +
                            <p style="clear:left">Using SEAP<sup>KDEL</sup> we were able to confirm that the reporter is
 +
                                not present in the cell medium without coexpression of erTEVp. When erTEVp was
 +
                                cotransfected with
 +
                                the reporter, we detected a large increase in enzymatic activity in the medium (<ref>2</ref>).
 +
                            </p>
 +
                            <div style="float:left; width:40%">
 +
                                <figure data-ref="2">
 +
                                    <img src="https://static.igem.org/mediawiki/2016/6/61/T--Slovenia--6.2.2.png">
 +
                                    <figcaption><b>Secretion of the SEAP reporter from ER lumen by cleavage with
 +
                                        ER-resident protease.</b><br/>
 +
                                        <p style="text-align:justify">HEK293T cells were transfected with indicated
 +
                                            reporter and erTEVp. Increased SEAP activity was detected in the medium of
 +
                                            cells
 +
                                            expressing both reporter and erTEVp protease.
 +
                                        </p>
 +
                                    </figcaption>
 +
                                </figure>
 +
                            </div>
 +
                            </div>
 +
 
 +
<div>
 +
                            <h3 style="clear:both"><span id = "mem" class="section colorize">&nbsp;</span>Release from the ER membrane and secretion from the cell</h3>
 +
                            <p>The second approach to regulate protein secretion from the ER by protease was to used
 +
                                KKMP ER retention peptide linked to the transmembrane protein with a protease target
 +
                                motif on the cytoplasmic side, N-terminally to the KKMP peptide. A transmembrane (TM)
 +
                                domain from the B-cell receptor
 +
                                (<a href="http://parts.igem.org/Part:BBa_K157010">Bba_K157010</a>) was used for the
 +
                                integration of target proteins in the ER membrane. Similar as described above, two
 +
                                reporter
 +
                                constructs with SEAP and TagRFP (SEAP:TM<sup>KKMP</sup> and TagRFP:TM<sup>KKMP</sup>)
 +
                                were designed and the constructs also contained a signal sequence at their N-terminus
 +
                                and a
 +
                                proteolytically cleavable ER retention signal at their C-terminus. In case of
 +
                                transmembrane targeted reporters we used the KKMP retention signal preceded by 3 copies
 +
                                of the
 +
                                TEVp cleavage site on the cytosolic side of the membrane.</p>
 +
                            <p>Additionally, either one or four furin cleavage sites were inserted between the protein
 +
                                of interest on the luminal side of the ER, which enable cleavage of the reporter
 +
                                protein from the membrane, but this could only occur after the KKMP had been removed and
 +
                                the protein could enter the trans-GA. Furin is a native cellular endoprotease that is
 +
                                active only in the trans-GA.</x-ref>Henrich2003</x-ref> This allowed us to design our
 +
                                constructs so that they are cleaved off of the membrane without any modified scar
 +
                                sequences
 +
                                attached to them.</p>
 +
 
 +
 
 +
                            <div style="float:left; width:100%"><span id="tagRFP" class="section"></span>
 +
                                <figure data-ref="3">
 +
                                    <img src="https://static.igem.org/mediawiki/2016/b/b4/T--Slovenia--6.2.3.png">
 +
                                    <figcaption><b>Localization of protease-responsive reporters on ER depending on the
 +
                                        proteolysis. </b><br/>
 +
                                        <p style="text-align:justify">(A) The transmembrane reporter without the KKMP
 +
                                            retention signal was localized both on the ER and plasma membrane. (B) The
 +
                                            transmembrane reporter with the KKMP retention
 +
                                            signal was localized exclusively on the ER membrane. (C) After cleavage of
 +
                                            the KKMP retention signal, the transmembrane reporter translocated to the
 +
                                            plasma membrane. HEK293T
 +
                                            cells were transfected with the indicated reporters and in (C) also with
 +
                                            TEVp. Localization was detected with confocal microscopy. Each image is
 +
                                            accompanied with a scheme of
 +
                                            the transfected construct. (D) Glycosylated reporter was detected in the
 +
                                            medium of cells transfected with the transmembrane reporter without the KKMP
 +
                                            retention signal.
 +
                                            HEK293T cells were transfected with the indicated constructs. Reporters were
 +
                                            detected with WB in the concentrated medium. In lane 2, sample was incubated
 +
                                            with N-glycosidase
 +
                                            F.
 +
                                        </p></figcaption>
 +
                                </figure>
 +
                            </div>
 +
                            <div style="float:right; width:50%"><span  id="SEAP" class="section"></span>
 +
                                <figure data-ref="4">
 +
                                    <img onclick="resize(this)" src="https://static.igem.org/mediawiki/2016/2/2f/T--Slovenia--6.2.4.png">
 +
                                    <figcaption><b>Inducible secretion of reporter localized on ER membrane.</b><br/>
 +
                                        <p style="text-align:justify">SEAP activity was increased in the medium of cells
 +
                                            induced with rapamycin. Scheme of the transmembrane reporter with
 +
                                            cleavable KKMP retention signal and inducible protease.
 +
                                            HEK293T cells were transfected with the indicated reporter and rapamycin
 +
                                            inducible split proteases. Uncleaved proteases were used as positive
 +
                                            control.</p></figcaption>
 +
                                </figure>
 +
                            </div>
 +
                            <p>Localization of the TagRFP:TM<sup>KKMP</sup> reporter was confirmed by the confocal
 +
                                microscopy. We used a control reporter without the KKMP retention signal (TagRFP:TM)
 +
                                which we detected both on the ER and the plasma membrane (<ref>3</ref>A). In case of present KKMP retention signal, the reporter was detected only on the
 +
                                ER
 +
                                (<ref>3</ref>B). When TagRFP:TM<sup>KKMP</sup> was coexpressed with TEVp, localization of the
 +
                                reporter was similar to the localization of the positive control (TagRFP:TM)
 +
                                on the plasma membrane and the ER (<ref>3</ref>C).
 +
                            </p>
 +
                            <p>A band with a slightly larger apparent size than the expected size of TagRFP (28 kDa) was
 +
                                detected by Western blotting in cells transfected with TagRFP:TM. We showed that the
 +
                                unexpected difference in size was due to glycosylation, as we detected the protein at
 +
                                the expected size after deglycosylation of the medium sample with N-glycosidase F. We
 +
                                were
 +
                                unable to detect a corresponding band in the medium of cells transfected with
 +
                                TagRFP:TM<sup>KKMP</sup> in the absence of the protease.</p>
 +
                            <p>Together, these results confirm that localization and secretion of the protein reporter
 +
                                with the transmembrane domain depends on the presence and proteolysis of the KKMP
 +
                                retention signal and that proteolysis can be used to induce secretion of already
 +
                                synthesized protein.</p>
 +
 
 +
 
 +
                            <p style="clear:both">Finally, we cotransfected cells with SEAP:TM<sup>KKMP</sup> and
 +
                                rapamycin-inducible split TEVp. We detected increased levels of the SEAP enzymatic
 +
                                activity in the medium of
 +
                                cells stimulated with rapamycin, which was dose dependent with respect to the amount of
 +
                                the transfected reporter-coding plasmid (<ref>4</ref>). These results confirm that
 +
                                secretion of a target protein can be made inducible by an externally supplied signal,
 +
                                processed through our split protease system.
 +
                            </p>
 +
                            <p style="clear:both">
 +
                            </p>
 +
                        </div>
 +
                        </div>
 +
                        <h3 class="ui left dividing header"><span id="ref-title" class="section">&nbsp;</span>References
 +
                        </h3>
 +
                        <div class="ui segment citing" id="references"></div>
 +
                    </div>
 +
                </div>
 +
            </div>
 +
            <div>
 +
                <a href="//igem.org/Main_Page">
 +
                    <img border="0" alt="iGEM" src="//2016.igem.org/wiki/images/8/84/T--Slovenia--logo_250x250.png"
 +
                        width="5%" style="position: fixed; bottom:0%; right:1%;">
 +
                </a>
 +
            </div>
 +
        </div>
 +
    </div>
 +
</div>
 
</body>
 
</body>
 
</html>
 
</html>

Latest revision as of 18:28, 19 October 2016

Protease inducible secretion

 Protease-based inducible secretion

  • Retention of proteins in ER lumen was demonstrated by confocal microscopy and detection of the protein in the cell medium.
  • A variant of TEVp active in the ER lumen was implemented to control protein secretion from the ER lumen.
  • Retention of proteins on the ER membrane was also demonstrated by confocal microscopy and detection of the protein in the cell medium.
  • Rapamycin induced cleavage was used for controlled and inducible secretion of proteins from the ER membrane.

 

To achieve a fast regulated cellular response resulting in the protein release, we decided to mimic the release of insulin from beta cells where the protein of interest is pre-formed and present intracellularly in secretory granules. In contrast to the specialized storage and release mechanism of insulin from beta cells we wanted to develop a more general and modular solution by making use of components already existing in different types of cells. Additionally, there should be minimal leakage from the protein depot in the uninduced state and after induction secretion from the cell should be fast.

Further explanation ...

Not many systems for the inducible release of proteins have been engineered to date. In one of the few examples Rivera et al. developed a system where the protein of interest was fused to a conditional aggregation domain (CAD) Rivera2000 . These domains form aggregates in the endoplasmic reticulum (ER) that are too large to exit the ER. After the addition of a small synthetic molecule, the CADs start to disaggregate and the protein of interest can be secreted. In the second example Chen et al. introduced a light-triggered secretion system. They also based their system on conditional aggregation; however they used the plant photoreceptor UVR8 which forms photolabile homodimers to make aggregates on the ER membrane. Upon light excitation the aggregates made by UVR8 started to disaggregate and were transported from the ER to the plasma membrane, but have not been observed in the cell supernatant Chen2013. The weakness of the two described systems is that they both rely on the exogenous chemical or physical signals instead of using a biochemical signal to induce the secretion, which means that they can’t be integrated into the signaling system that’s senses the cellular state. In order to better respond to the state of the cell or a logic circuit inside a cell we decided to develop an inducible secretion system based on the biochemical signal.


Many proteins that reside on the membrane or in the lumen of the ER contain short peptide signals. Proteins present in the lumen of the ER contain a KDEL C-terminal sequence (Lys-Asp-Glu-Leu) while type I transmembrane (TM) proteins contain a dilysine (KKXX) motif on their C-terminus (cytosolic side) Munro1987, Jackson1990, Stornaiuolo2003. . The mechanism that allows these proteins to stay in the ER is retrieval rather than retention. However, we decided to use the term retention for description of this process. ER luminal proteins interact with the KDEL receptor, a transmembrane ER resident protein. The cytosolic part of the KDEL receptor interacts with coat proteins I (COP I) which coat vesicles and are responsible for transporting proteins from the cis end of the Golgi apparatus (cis-GA) back to the ER. The KKXX motif present on type I TM proteins can directly interact with the COP I for retrieval Stornaiuolo2003, Letourneur1994 .

Our idea was that if we proteolytically remove the retention signal, the protein of interest would no longer be retrieved back to the ER and could be secreted from the cell. To achieve this we designed two types of secretory reporters, one type based on the luminal retention using KDEL sequence and the other based on the transmembrane retention with a KKMP sequence. In each case, the retention sequence was preceded by a TEVp cleavage site to allow for inducible secretion, which could be replaced by any other peptide target of our orthogonal protease set.

 Results

 Release from the ER lumen and secretion from the cell

To achieve and detect the inducible secretion from the ER lumen, we created two reporter constructs with a cleavable KDEL sequence targeted to the ER lumen: SEAPKDEL and TagRFPKDEL. Those proteins contained a protease target motif between the reporter domain and the KDEL domain, aimed to enable protein secretion after the proteolytic cleavage. We used a TEVp variant (erTEVp) for all of our experiments with luminal retention.

Further explanation ...

In order to rely on TEVp cleavage in the ER lumen, we had to take some additional considerations into account. Cesaratto et al. Cesaratto2015 reported that the wild type TEV protease is not active in the lumen of ER. They designed a TEV protease variant active in the endoplasmic reticulum by preventing two major types of post-translational modifications: N-glycosylation and cysteine oxidation. To avoid these inhibiting modifications, mutations N23Q, C130S and N171T were made. To ensure correct localization and accumulation of this TEVp variant inside the endoplasmic reticulum, we also attached an ER signal sequence at the N-terminus and KDEL at the C-terminus of the protein.


Cleavage with ER-residing protease (erTEV) facilitates secretion of reporter from cells.

(A) Scheme of the reporter with cleavable KDEL retention signal and protease target motif. (B) The reporter with the KDEL retention signal was localized in the ER. HEK293T cells were transfected with the indicated reporters and in (C) also with erTEVp. Localization was detected with confocal microscopy. (C)The reporter was detected in the medium of cells only when cotransfected with erTEVp. HEK293T cells were transfected with the indicated constructs. Reporters were detected with WB in the concentrated medium.

When the TagRFPKDEL reporter (1A) was expressed in the ER without an active erTEVp we confirmed its localization in the ER with confocal microscopy (1B). Additionally, we could not detect any TagRFP in the cell medium with Western blotting. When erTEVp was present and active in the ER, the KDEL sequence was removed from the reporter and the protein was secreted from the cell, which we detected with Western blot (1C). This demonstrated that proteolytic activity in the ER can regulate protein secretion.

Using SEAPKDEL we were able to confirm that the reporter is not present in the cell medium without coexpression of erTEVp. When erTEVp was cotransfected with the reporter, we detected a large increase in enzymatic activity in the medium (2).

Secretion of the SEAP reporter from ER lumen by cleavage with ER-resident protease.

HEK293T cells were transfected with indicated reporter and erTEVp. Increased SEAP activity was detected in the medium of cells expressing both reporter and erTEVp protease.

 Release from the ER membrane and secretion from the cell

The second approach to regulate protein secretion from the ER by protease was to used KKMP ER retention peptide linked to the transmembrane protein with a protease target motif on the cytoplasmic side, N-terminally to the KKMP peptide. A transmembrane (TM) domain from the B-cell receptor (Bba_K157010) was used for the integration of target proteins in the ER membrane. Similar as described above, two reporter constructs with SEAP and TagRFP (SEAP:TMKKMP and TagRFP:TMKKMP) were designed and the constructs also contained a signal sequence at their N-terminus and a proteolytically cleavable ER retention signal at their C-terminus. In case of transmembrane targeted reporters we used the KKMP retention signal preceded by 3 copies of the TEVp cleavage site on the cytosolic side of the membrane.

Additionally, either one or four furin cleavage sites were inserted between the protein of interest on the luminal side of the ER, which enable cleavage of the reporter protein from the membrane, but this could only occur after the KKMP had been removed and the protein could enter the trans-GA. Furin is a native cellular endoprotease that is active only in the trans-GA.Henrich2003 This allowed us to design our constructs so that they are cleaved off of the membrane without any modified scar sequences attached to them.

Localization of protease-responsive reporters on ER depending on the proteolysis.

(A) The transmembrane reporter without the KKMP retention signal was localized both on the ER and plasma membrane. (B) The transmembrane reporter with the KKMP retention signal was localized exclusively on the ER membrane. (C) After cleavage of the KKMP retention signal, the transmembrane reporter translocated to the plasma membrane. HEK293T cells were transfected with the indicated reporters and in (C) also with TEVp. Localization was detected with confocal microscopy. Each image is accompanied with a scheme of the transfected construct. (D) Glycosylated reporter was detected in the medium of cells transfected with the transmembrane reporter without the KKMP retention signal. HEK293T cells were transfected with the indicated constructs. Reporters were detected with WB in the concentrated medium. In lane 2, sample was incubated with N-glycosidase F.

Inducible secretion of reporter localized on ER membrane.

SEAP activity was increased in the medium of cells induced with rapamycin. Scheme of the transmembrane reporter with cleavable KKMP retention signal and inducible protease. HEK293T cells were transfected with the indicated reporter and rapamycin inducible split proteases. Uncleaved proteases were used as positive control.

Localization of the TagRFP:TMKKMP reporter was confirmed by the confocal microscopy. We used a control reporter without the KKMP retention signal (TagRFP:TM) which we detected both on the ER and the plasma membrane (3A). In case of present KKMP retention signal, the reporter was detected only on the ER (3B). When TagRFP:TMKKMP was coexpressed with TEVp, localization of the reporter was similar to the localization of the positive control (TagRFP:TM) on the plasma membrane and the ER (3C).

A band with a slightly larger apparent size than the expected size of TagRFP (28 kDa) was detected by Western blotting in cells transfected with TagRFP:TM. We showed that the unexpected difference in size was due to glycosylation, as we detected the protein at the expected size after deglycosylation of the medium sample with N-glycosidase F. We were unable to detect a corresponding band in the medium of cells transfected with TagRFP:TMKKMP in the absence of the protease.

Together, these results confirm that localization and secretion of the protein reporter with the transmembrane domain depends on the presence and proteolysis of the KKMP retention signal and that proteolysis can be used to induce secretion of already synthesized protein.

Finally, we cotransfected cells with SEAP:TMKKMP and rapamycin-inducible split TEVp. We detected increased levels of the SEAP enzymatic activity in the medium of cells stimulated with rapamycin, which was dose dependent with respect to the amount of the transfected reporter-coding plasmid (4). These results confirm that secretion of a target protein can be made inducible by an externally supplied signal, processed through our split protease system.

 References