Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors

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Primary source: Morsut L., Roybal K. T., Xiong X., Gordley R. M., Coyle S. M., Thomson M., Lim W.A., (2016), Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors, Cell, Vol. 164, Issue 4, Pages 780-791.

Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors

Introduction Through synthetic biology, scientists have introduced engineering approaches to biology with the aim of altering the function of cells. Thanks to that newly created cells perform a new, human-predicted function. Cellular function is largely dependent on intercellular communication. After receiving stimuli from its environment, cells respond to it and perform different tasks. Intercellular communication is also very important for maintaining the integrity of tissues. The cause of the various diseases often lies precisely in the communication between the two cells, and if scientists could find a way to control intercellular signalling, many of those diseases could be kept under control. Notch receptor and its signalling pathway have emerged as one of the possibilities for manipulating cellular communication, since the input and output can be flexibly altered. The article is about engineering customized cell sensing and cell responses, by using synthetic Notch receptors [1].

Notch Receptor Notch receptor is a single pass trans – membrane protein which is a part of a highly conserved signalling pathway. It has a central role in various cell fate decisions at multiple stages of development and adult organism [2]. It consists of three domains: extracellular (EC), transmembrane (TM) and intracellular (IC) domain. It transduces signals when its extracellular domain is activated by Delta protein expressed on the surface of the signal – sending cell. This signal leads to intramembrane proteolysis by ADAM metalloprotease and gamma-secretase. The cleavage releases the intracellular domain of the Notch. That intracellular domain is actually a transcriptional regulator, and when released, it enters nucleus to activate target genes. This is how Notch receptors play key roles in cell-cell signalling during development [1]. In addition to trans-activating Notch–ligand complexes, the receptor can also form cis-inhibitory complexes when Notch and ligand are expressed on the same cell surface [2]. Its important to mention that Notch’s regulatory core is transmembrane region, thus extracellular and intracellular domain can be altered. We see that Notch receptor has a pretty simple mechanism of action and is therefore suitable for changes that can determine its function. Custom engineered Notch receptors are called synthetic Notch receptors, or shortly synNotch.

Results and Discussion In this paper several approaches have been applied, in order to examine the different properties of the synthetic Notch receptors and to compare them with those of endogenous Notch. They generated libraries of receptors with different EC domains, each coupled with a different IC domain and expressed them in fibroblast. They found that these synthetic receptors are strongly activated by cell-cell contact, if the sender cell expresses cognate ligand on its surface. To determine if the activation occurs by a cleavage mechanism, they used the drug DAPT (N-[N-(3,5-difluorophenacetyl)-lalanyl]-S-phenylglycine t-butyl ester). Treatment with DAPT completely blocks the activation of synNotch. These were eliminatory examinations, which have shown the characteristics of the endogenous Notch necessary to conduct further experiments. Also, they found that the response is reversible upon removal of the ligand – expressing cells. The next phase of the research is dedicated to determining which cells can be used. It provided us with the information that epithelial Madin-Darby canine kidney (MDCK) cells, L929 and C3H mouse fibroblasts cell lines, HEK293 human epithelial cell lines, and Jurkat T cells all show clear induction of reporter gene activation upon synNotch receptor engagement. Further they have examined if changes induced by synNotch receptors are spatially controlled. This would allow users to specify cell behavior in a highly localized manner. This was confirmed on epithelial cells by inducing their transdifferentiation to mesenchymal cells. In another experiment they proved that transdifferentiation of fibroblast to myoblast can also be conducted. After that they focused on the joint action of the two receptors. Examining if synNotch has an orthogonal function to endogenous Notch receptor, they have found that these two pathways display independent, but compatible activation! The same conclusion was drawn by comparing the activity of two synthetic receptors. By these two experiments they have proved that synthetic Notch receptors can be used to engineer cells that respond to multiple stimuli with distinct user-defined transcriptional programs. Since Notch receptors function independently to each other next task is engineering cells that combinatorially integrate multiple inputs. This was possible by expressing on the receiver cell two synNotch receptors that are activated by different stimuli, with different intracellular domains that have joint pathway when cleaved. In this case each receptor controls one half of a split transcription factor. These cells were stimulated by sender cells containing activator proteins for a single synNotch receptor, or for both of them. The conclusion was that if stimulated only by one stimulus, no activation is visible. The response of the receiver cell was induced only when there was a contact with the sender cell that expresses both of the antigens. This property of synthetic Notch receptors gives a synthetic biologist the ability to construct cellular AND-gate pathways. The last and most complicated phase of these experiments was the attempt to engineer cascades of cell – cell signalling with multiple synNotch receptors. For that they constructed reciver cells expressing two different synNotches that could potentially act in a row – the first one would induce the expression of the ligand for the second one. This allows scientists to take advantage of synNotch receptors to engeneer complex, multistep signalling pathways that are commonly present in the organism.

Conclusion The major goal of synthesis biology is the ability to predict, that is, design and control cell behavior [1]. The Notch signalling pathway has imposed itself as a simple target because it is highly conserved in many different species and it functions by a very simple mechanism. It also plays an important role in many processes necessary for the maintenance of cells life and its development. These processes include cell differentiation, survival, proliferation, stem cell renewal, determination of cell fate during development and morphogenesis. These traits qualify it as an excellent target for manipulation, that is, the design of a synthetic Notch receptor that will have a desired function depending on the predetermined stimulus. Also, various studies show that the Notch receptor plays a role in oncogenesis [3], tumor immunosuppression [4], hematological malignancies [3] and in many other more aggressive diseases with poor prognosis [5]. Therefore, the discovery that synthetic Notch receptors function and meet certain criteria is important for the potentially better treatment of these diseases and gives hope that diseases, that are terminal and incurable today, will have good prognosis and simple therapy in the future [5].

Literature: 1. Morsut L., Roybal K. T., Xiong X., Gordley R. M., Coyle S. M., Thomson M., Lim W.A., (2016), Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors, Cell, Vol. 164, Issue 4, Pages 780-791. 2. Chillakuri C.R., Sheppard D., Lea S. M., Handford P. A., (2012) Notch receptor–ligand binding and activation: Insights from molecular studies, Seminars in Cell & Developmental Biology, Vol. 23, Issue 4, Pages 421-428. 3. Rodrigues C., Joy L. R., Sachithanandan S. P., Krishna S., (2019) Notch signalling in cervical cancer, Experimental Cell Research, Vol. 385, Issue 2, 111682. 4. Roybal K. T., Williams J. Z., Morsut L., Rupp L. J., Kolinko I., Choe J. H., Walker W. J., McNally K. A., Lim W. A, (2016) Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors, Cell Vol. 167, Issue 2, Pages 419-432.e16. 5. S. Inder, O'Rourke S., McDermott N., Manecksha R., Finn S., Lynch T., Marignol L., (2017) The Notch-3 receptor: A molecular switch to tumorigenesis?, Cancer Treatment Reviews, Vol. 60, Pages 69-76 6. Krishna B.M., Jana S., Singhal J., Horne D., Awasthi S., Salgia R., Singhal SS., (2019) Notch signalling in breast cancer: From pathway analysis to therapy, Cancer Letters, Volume 461, Pages 123-131.

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