Part:BBa_K4444004

PhoCl Display GFP

Introduction

PhoCl Display GFP is modified from the RELEASE system. It contains a (1) GFP as the protein of interest, (2) a tri-transmembrane domain, (3) PhoCl2c, and (4) an ER retention motif. The furin cut site and HCV cut site were removed. PhoCl Display will display a protein of interest on the cell membrane in response to ultraviolet light stimulation.

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. 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.

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 Display will display a protein of interest on the cell membrane 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, but there is no furin cut site to cleave. Therefore, when the POI is sent to the cell membrane, it will stay displayed on the surface.

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 898
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1021
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 60
  Illegal BsaI.rc site found at 1543