Difference between revisions of "Team:Slovenia/Implementation/ProteaseInducible secretion"

(Created page with "{{Slovenia}} <html> <head> <title>Protease inducible secretion</title> <link rel="stylesheet" href="//2016.igem.org/Team:Slovenia/libraries/semantic-min-css?...")
 
Line 74: Line 74:
 
<!-- content goes here -->
 
<!-- content goes here -->
 
<div class="main ui citing justified container">
 
<div class="main ui citing justified container">
−
+
<div class = "ui segment">
 +
<h2>Protease-based inducible secretion</h2><br/>
 +
 
 +
<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
 +
of a protein using genetically encoded components.</p>
 +
 
 +
<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
 +
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>
 +
 +
 +
 +
<!--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
 +
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.
 +
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. <x-ref>Chen2013</x-ref>
 +
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.-->
 +
 +
<p>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). <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
 +
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>
 +
 +
 +
</div>
 +
 +
 
</div>
 
</div>
 
</div>
 
</div>

Revision as of 15:21, 15 October 2016

Protease inducible secretion

Project Achievements Medal requirements Idea

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). Munro1987, Jackson1990, Stornaiuolo2003. 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. 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.