Part:BBa_K4175001

Notch core

This Notch core domain is derived from the transmembrane part of the Notch receptor.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 115
  Illegal PstI site found at 156
  Illegal PstI site found at 202
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 115
  Illegal PstI site found at 156
  Illegal PstI site found at 202
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 115
  Illegal PstI site found at 156
  Illegal PstI site found at 202
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 115
  Illegal PstI site found at 156
  Illegal PstI site found at 202
  Illegal NgoMIV site found at 28
  
• 1000
  COMPATIBLE WITH RFC[1000]

Biology

Notch receptors are large single-pass Type I transmembrane proteins, which contain 29-36 tandem epidermal growth factor (EGF)-like repeats in their extracellular domain. In mammals, there are four Notch paralogs (mNotch1-4) (Kopan and Ilagan, 2009). The Notch signaling pathway, mediating short-range cell-cell communication, is crucial for metazoan development and tissue regeneration. For this reason, this signaling pathway is conserved among species (Kopan and Ilagan, 2009). The Notch receptors could bind to their ligands (e.g., Delta in drosophila, Dll1 in mammals) either on the neighboring cells or on the same cell where the Notch receptors are expressed. When the Notch receptor interact with its ligand through extracellular EGF-like repeats, the receptor will undergo two subsequent cleavages by ADAM metalloproteases and γ-secretase. As a result, the intracellular domain of Notch receptor is released, enters the nucleus, and acts with DNA-binding protein CSL to activate transcription (Kopan and Ilagan, 2009) (Fig 1). This allows regulation of differentiation, proliferation, and apoptotic events of cells at all stages of development (Artavanis-Tsakonas et al., 1999).

Figure 1. (Morsut et al., 2016) The simple illustration of Notch pathway.

Usage

The extracellular and intracellular domain can be artificially swapped with the regulatory core domain centered around the transmembrane region staying still. The resultant receptor is called synthetic Notch (synNotch) receptor, which offers a strong tool for engineering novel signaling pathways (Morsut et al., 2016) (Fig 2).

Figure 2. (Morsut et al., 2016) The illustration of synthetic Notch (synNotch).

As for the extracellular domain, the EGF repeats can be replaced with scFv. Morsut et al. designed a synthetic Notch, where the extracellular domain of Notch is replaced with anti-CD19 scFv and the intracellular domain is replaced by tetR-VP64 (tTA). This synNotch was co-expressed in the receiver cell with a tTA-inducible reporter gene (i.e., green fluorescence protein gene). It was found that only in the presence of CD19-expressing cells did the receiver cell express green fluorescence (Morsut et al., 2016) (Fig 3A).

As for the intracellular domain, transcriptional repression can also be induced. The intracellular domain of the previously stated synNotch was replaced with Gal4-KRAB, which inhibits gene downstream of UAS (Morsut et al., 2016). This novel receptor was co-expressed in fibroblasts together with GFP gene driven by a strong constitutive SV40 promoter downstream of UAS. With the stimulation of CD19-expressing sender cells, the expression of green fluorescence protein was downregulated (Morsut et al., 2016) (Fig 3B).

Figure 3. (Morsut et al., 2016) Fibroblasts expressing synNotch were co-incubated with different kinds of sender cells. The expression of GFP (the reporter gene) was then measured using FACS.

Apart from fibroblasts, such kind of synNotch can also successfully function in neurons and T cells (Morsut et al., 2016) (Fig 4).

Figure 4. (Morsut et al., 2016) SynNotch aCD19-CAR-tTA was also expressed in primary hippocampal neurons and Jurkat T cells. Only when CD19+ cells presented did the reporter gene, GFP, be expressed.

For our usage, we want to take advantage of the great modularity and flexibility of Notch receptor to design a synNotch that can repress the expression of CAR in the presence of excessive IL-6. We hope this device would ameliorate the symptoms of cytokine release syndrome (CRS) during CAR-T therapy. For this purpose, we designed two devices using Notch core domain, IL-6_scfv-Notch-Gal4KRAB (BBa_K4175008) and IL-6R-Notch-Gal4KRAB (BBa_K4175010). For more detailed information, please go to the part pages of these two devices.

Figure 5. The schematic of IL-6_scfv-Notch-Gal4KRAB.

Figure 6. The schematic of IL6R-Notch-Gal4KRAB.

References

Artavanis-Tsakonas, S., Rand, M.D., Lake, R.J., 1999. Notch Signaling: Cell Fate Control and Signal Integration in Development. Science 284, 770–776. https://doi.org/10.1126/science.284.5415.770

Kopan, R., Ilagan, Ma.X.G., 2009. The Canonical Notch Signaling Pathway: Unfolding the Activation Mechanism. Cell 137, 216–233. https://doi.org/10.1016/j.cell.2009.03.045

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 164, 780–791. https://doi.org/10.1016/j.cell.2016.01.012