Difference between revisions of "Team:Slovenia/Implementation/ProteaseInducible secretion"
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<div class = "ui segment"> | <div class = "ui segment"> | ||
<h2>Protease-based inducible secretion</h2><br/> | <h2>Protease-based inducible secretion</h2><br/> | ||
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<p>Fast sensing and fast signaling pathway were developed using protease-based pathway. In the final step for the construction of rapidly responding cells we wanted to | <p>Fast sensing and fast signaling pathway were developed using protease-based pathway. In the final step for the construction of rapidly responding cells we wanted to | ||
implement a fast output that would not require a slow transcription/translation biosynthesis of new proteins. We decided to engineer a system capable of regulated secretion | implement a fast output that would not require a slow transcription/translation biosynthesis of new proteins. We decided to engineer a system capable of regulated secretion | ||
of a protein using genetically encoded components.</p> | of a protein using genetically encoded components.</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 | <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 | ||
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 | 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 | ||
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 | 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.</p> | + | protein depot in the uninduced state and after induction secretion from the cell should be fast.</p> |
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<!--tukej manjka extended text | <!--tukej manjka extended text | ||
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 | 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 | ||
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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 | 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 | 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> | + | on type I TM proteins can directly interact with the COP I for retrieval. <x-ref> Stornaiuolo2003, Letourneur1994 </x-ref>.</p> |
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<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. | <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 | 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 | 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.</p> | of our orthogonal protease set.</p> | ||
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</div> | </div> | ||
| + | <div class = "ui segment"> | ||
| + | <h4>Secretion from the ER lumen</h4><br/> | ||
| + | <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: 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.</p> | ||
| + | <!--tukej manjka extended text | ||
| + | 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 a signal sequence at the N-terminus and KDEL at the C-terminus of the protein.--> | ||
| + | </div> | ||
| + | |||
| + | <div class = "ui segment"> | ||
| + | <h4>Results<h4><br/> | ||
| + | <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), demonstrating that proteolytic activity in the ER can | ||
| + | regulate protein secretion.</p> | ||
| + | |||
| + | </div> | ||
</div> | </div> | ||
Revision as of 15:26, 15 October 2016
Protease-based inducible secretion
Fast sensing and fast signaling pathway were developed using protease-based pathway. In the final step for the construction of rapidly responding cells we wanted to implement a fast output that would not require a slow transcription/translation biosynthesis of new proteins. We decided to engineer a system capable of regulated secretion of a protein using genetically encoded components.
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 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 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.
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).
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.
Secretion from the ER lumen
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.
Results
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), demonstrating that proteolytic activity in the ER can regulate protein secretion.
