Difference between revisions of "Part:BBa K4444015"

(→‎Usage and Biology)
(→‎RELEASE System)
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K4444015 short</partinfo>
 
<partinfo>BBa_K4444015 short</partinfo>
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PhoCl Secrete Luciferase is modified from the RELEASE system. It contains a luciferase as a protein of interest, a furin cut site, PhoCl2c, tri-transmembrane domain, and an ER retention motif. PhoCl Secrete will completely release a protein of interest in response to ultra violet light stimulation.
+
===Introduction===
 +
PhoCl Secrete Luciferase is modified from the RELEASE system. It contains (1) luciferase as a protein of interest, (2) a furin cut site, (3) tri-transmembrane domain, (4) PhoCl2c, and (5) an ER retention motif. PhoCl Secrete will completely release a protein of interest in response to ultraviolet light stimulation(<html><ref>1</ref></html>).
  
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===Usage and Biology===
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<html><div>
 +
    <figure data-ref="1">
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        <img class="Secrete" src="https://static.igem.wiki/teams/4444/wiki/parts-pictures/phoclsecrete.png" width="250">
 +
        <figcaption>Design and Function of PhoCl Secrete System
  
−
Our project builds on  the protein circuit RELEASE (Vlahos et al.) by introducing light-inducible  proteins or photoswitches. Photocleavable protein (PhoCl) is a recently developed optogenetic technology that self-cleaves  when stimulated by 405 nm ultraviolet light. PhoCl was engineered from a green-to-red photoconvertible fluorescent protein. When stimulated with ultraviolet light, the protein undergoes a β-elimination reaction, producing a small peptide fragment and a large barrel fragment that will spontaneously dissociate (Zhang et al.).
+
        </figcaption>
 +
    </figure>
 +
</div>
 +
</html>
  
−
Luciferase is an enzyme that fluoresces when it binds with luciferin. By running a luciferase reporter assay with a luminometer on the media, one can quantitatively observe how much luciferase was secreted into the media.
+
===Protein Secretion Pathway===
 +
To develop novel therapeutics and treat diseases, it is crucial to understand the underlying molecular mechanisms of intercellular communication; however the current tools available for investigating intercellular communication do not allow for precise control of signal secretion.
 +
 
 +
Here we aim to develop a novel approach to control an important intercellular communication method: protein secretion. This is the process by which a cell releases signaling molecules, which are then recognized by receptors on another cell, initiating a specific cellular response. In eukaryotes proteins destined for secretion contain a signal peptide which indicates to the synthesizing ribosomes to travel to the rough endoplasmic reticulum (ER). Newly synthesized proteins follow the secretory pathway in vesicles from the ER to the Golgi apparatus and finally to the plasma membrane where the protein may be secreted or displayed (<html><ref>2</ref></html>). The presence of a furin cut site allows a protein to be secreted when it is cleaved by the protease; however if the furin cut site is absent the protease is unable to cut and the protein will be displayed.
 +
 
 +
<html><div>
 +
    <figure data-ref="2">
 +
        <img class="Secrete" src="https://static.igem.wiki/teams/4444/wiki/parts-pictures/secretionpathway.png" width="250">
 +
        <figcaption>The Protein Secretion Pathway
 +
 
 +
        </figcaption>
 +
    </figure>
 +
</div>
 +
</html>
 +
 
 +
===RELEASE System===
 +
 
 +
Intercellular communication is commonly studied using protein circuits. Researchers do this by developing protein circuits that interfere with the cell’s natural secretion pathway. One such circuit is the RELEASE (Retained Endoplasmic Cleavable Secretion) system, developed at Stanford University (Vlahos et al.). This modular system consists of several components: (1) protein of interest (POI), (2) furin cut site, (3) tri-transmembrane domain, (4) protease cut site , and (5) ER retention motif (<html><ref>3</ref></html>). The retention motif signals the Golgi apparatus to transport the POI back to the ER, preventing it from being secreted. Secretion is then controllable through the introduction of a TEV protease which can cleave the cut site, removing the motif and allowing the POI to continue along the secretory pathway. 
 +
 
 +
<html><div>
 +
    <figure data-ref="3">
 +
        <img class="RELEASE" src="https://static.igem.wiki/teams/4444/wiki/parts-pictures/release.png" width="250">
 +
        <figcaption>Design of the RELEASE System
 +
 
 +
        </figcaption>
 +
    </figure>
 +
</div>
 +
</html>
 +
 
 +
 +
While this protein circuit has advantages such as compact design and direct interactions with endogenous signaling pathways, RELEASE lacks high spatial and temporal control. Protease activity was controlled either via transcription of the TEV protease or chemical induction of the translated TEV protein. These methods both lacked the ability to locally activate secretion within a small region of a larger cell culture and also could not achieve dynamic temporal control over when secretion is activated or inactivated. This presents a limitation to current research methods, as cell to cell communication is often highly sensitive to the duration and location of signals.
 +
 
 +
===Optogenetics===
 +
Optogenetics is a powerful research tool for controlling cell behavior that offers precise spatiotemporal control by varying the wavelength of lights used. The spatial precision can be achieved at the level of micrometers while the temporal precision can be achieved at <1ms (Zhu et al.). This light input generates a response in photosensitive proteins bound to target proteins, which allows researchers to control different aspects of cell activity.
 +
 
 +
===PhoCl===
 +
 
 +
Photocleavable protein (PhoCl) is a recently developed optogenetic technology that self-cleaves  when stimulated by 405 nm ultraviolet light. PhoCl was engineered from a green-to-red photoconvertible fluorescent protein. When stimulated with ultraviolet light, the protein undergoes a β-elimination reaction, producing a small peptide fragment and a large barrel fragment that will spontaneously dissociate (Zhang et al.). The red fluorescent form of PhoCl is transient and spontaneously converted to a non-fluorescent form (Zhang et al.).
 +
 +
One key advantage of PhoCl over other photoactivatable proteins is that PhoCl is cleaved irreversibly. Reversible protein systems rely on equilibrium states and concentrations of the bound and dissociated states to drive cleavage. Activation of reversible proteins can take longer due to the constant shifting equilibrium between the bound and dissociated states (Zhang et al.). Additionally, to activate reversible proteins for long periods of time, constant illumination is needed, which is phototoxic to cells and can lead to cell damage (Zhang et al.). PhoCl cleavage does not rely on equilibrium states and concentrations, therefore only a transient light stimulus is needed to activate it, making PhoCl advantageous for applications that require prolonged activation or activation on a timescale of minutes (Zhang et al.).
 +
 
 +
===Mechanism===
 +
 
 +
PhoCl Secrete will completely release a protein of interest in response to ultraviolet light stimulation. When PhoCl is broken, the system is separated from the ER retention motif, allowing the POI to leave the ER and travel to the Golgi apparatus where it will be packaged and the furin cut site will be cleaved. Then, the POI can be secreted from the cell following the natural secretion pathway.
 +
 
 +
 
 +
===References===
 +
 
 +
[1] Vlahos, Alexander E., et al. “Protease-Controlled Secretion and Display of Intercellular Signals.” Nature Communications, vol. 13, no. 1, 17 Feb. 2022, p. 912, www.nature.com/articles/s41467-022-28623-y, 10.1038/s41467-022-28623-y.
 +
 
 +
[2] Zhang, Wei, et al. “Optogenetic Control with a Photocleavable Protein, PhoCl.” Nature Methods, vol. 14, no. 4, 1 Apr. 2017, pp. 391–394, www.nature.com/articles/nmeth.4222, 10.1038/nmeth.4222.
 +
 
 +
[3] Zhu, Danqing, et al. “Optogenetic Application to Investigating Cell Behavior and Neurological Disease.” Frontiers in Cellular Neuroscience, vol. 16, 22 Feb. 2022, 10.3389/fncel.2022.811493.  
  
  
−
Mechanism: Upon light-activation PhoCl breaks, removing the ER retention motif and allowing the protein to continue along the secretion pathway. The furin cut site will later be cleaved at the Golgi body, allowing the POI to be freely floating in the vesicle. At the plasma membrane the POI will be secreted.
 
  
 
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Latest revision as of 15:55, 13 October 2022


PhoCl Secrete Luciferase

Introduction

PhoCl Secrete Luciferase is modified from the RELEASE system. It contains (1) luciferase as a protein of interest, (2) a furin cut site, (3) tri-transmembrane domain, (4) PhoCl2c, and (5) an ER retention motif. PhoCl Secrete will completely release a protein of interest in response to ultraviolet light stimulation(1).

Design and Function of PhoCl Secrete System

Protein Secretion Pathway

To develop novel therapeutics and treat diseases, it is crucial to understand the underlying molecular mechanisms of intercellular communication; however the current tools available for investigating intercellular communication do not allow for precise control of signal secretion.

Here we aim to develop a novel approach to control an important intercellular communication method: protein secretion. This is the process by which a cell releases signaling molecules, which are then recognized by receptors on another cell, initiating a specific cellular response. In eukaryotes proteins destined for secretion contain a signal peptide which indicates to the synthesizing ribosomes to travel to the rough endoplasmic reticulum (ER). Newly synthesized proteins follow the secretory pathway in vesicles from the ER to the Golgi apparatus and finally to the plasma membrane where the protein may be secreted or displayed (2). The presence of a furin cut site allows a protein to be secreted when it is cleaved by the protease; however if the furin cut site is absent the protease is unable to cut and the protein will be displayed.

The Protein Secretion Pathway

RELEASE System

Intercellular communication is commonly studied using protein circuits. Researchers do this by developing protein circuits that interfere with the cell’s natural secretion pathway. One such circuit is the RELEASE (Retained Endoplasmic Cleavable Secretion) system, developed at Stanford University (Vlahos et al.). This modular system consists of several components: (1) protein of interest (POI), (2) furin cut site, (3) tri-transmembrane domain, (4) protease cut site , and (5) ER retention motif (3). The retention motif signals the Golgi apparatus to transport the POI back to the ER, preventing it from being secreted. Secretion is then controllable through the introduction of a TEV protease which can cleave the cut site, removing the motif and allowing the POI to continue along the secretory pathway.

Design of the RELEASE System


While this protein circuit has advantages such as compact design and direct interactions with endogenous signaling pathways, RELEASE lacks high spatial and temporal control. Protease activity was controlled either via transcription of the TEV protease or chemical induction of the translated TEV protein. These methods both lacked the ability to locally activate secretion within a small region of a larger cell culture and also could not achieve dynamic temporal control over when secretion is activated or inactivated. This presents a limitation to current research methods, as cell to cell communication is often highly sensitive to the duration and location of signals.

Optogenetics

Optogenetics is a powerful research tool for controlling cell behavior that offers precise spatiotemporal control by varying the wavelength of lights used. The spatial precision can be achieved at the level of micrometers while the temporal precision can be achieved at <1ms (Zhu et al.). This light input generates a response in photosensitive proteins bound to target proteins, which allows researchers to control different aspects of cell activity.

PhoCl

Photocleavable protein (PhoCl) is a recently developed optogenetic technology that self-cleaves when stimulated by 405 nm ultraviolet light. PhoCl was engineered from a green-to-red photoconvertible fluorescent protein. When stimulated with ultraviolet light, the protein undergoes a β-elimination reaction, producing a small peptide fragment and a large barrel fragment that will spontaneously dissociate (Zhang et al.). The red fluorescent form of PhoCl is transient and spontaneously converted to a non-fluorescent form (Zhang et al.).

One key advantage of PhoCl over other photoactivatable proteins is that PhoCl is cleaved irreversibly. Reversible protein systems rely on equilibrium states and concentrations of the bound and dissociated states to drive cleavage. Activation of reversible proteins can take longer due to the constant shifting equilibrium between the bound and dissociated states (Zhang et al.). Additionally, to activate reversible proteins for long periods of time, constant illumination is needed, which is phototoxic to cells and can lead to cell damage (Zhang et al.). PhoCl cleavage does not rely on equilibrium states and concentrations, therefore only a transient light stimulus is needed to activate it, making PhoCl advantageous for applications that require prolonged activation or activation on a timescale of minutes (Zhang et al.).

Mechanism

PhoCl Secrete will completely release a protein of interest in response to ultraviolet light stimulation. When PhoCl is broken, the system is separated from the ER retention motif, allowing the POI to leave the ER and travel to the Golgi apparatus where it will be packaged and the furin cut site will be cleaved. Then, the POI can be secreted from the cell following the natural secretion pathway.


References

[1] Vlahos, Alexander E., et al. “Protease-Controlled Secretion and Display of Intercellular Signals.” Nature Communications, vol. 13, no. 1, 17 Feb. 2022, p. 912, www.nature.com/articles/s41467-022-28623-y, 10.1038/s41467-022-28623-y.

[2] Zhang, Wei, et al. “Optogenetic Control with a Photocleavable Protein, PhoCl.” Nature Methods, vol. 14, no. 4, 1 Apr. 2017, pp. 391–394, www.nature.com/articles/nmeth.4222, 10.1038/nmeth.4222.

[3] Zhu, Danqing, et al. “Optogenetic Application to Investigating Cell Behavior and Neurological Disease.” Frontiers in Cellular Neuroscience, vol. 16, 22 Feb. 2022, 10.3389/fncel.2022.811493.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 52
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 991
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1162
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 604
    Illegal BsaI.rc site found at 1684