Quick summary of our receptors

Transmembrane signal transduction systems

In order to be able to implement a customable multiplexed transmembrane signal input and output relationship, the first thing to do is to engineer modular receptors to enable it to recognize extracellular signals and transduce them into customized intracellular signals. To this end, a variety of techniques have been developed, such as Tango (Barnea et al., 2008), CAR (Porter et al., 2011), GEMS (Scheller et al., 2018), MESA (Daringer et al., 2014; Hartfield et al., 2017; Schwarz et al., 2017), SynNotch (Morsut et al., 2016; Roybal et al., 2016a; Roybal et al., 2016b; Toda et al., 2018), etc. High modularity of the extracellular and intracellular domains of synthetic Notch (SynNotch), as well as its orthogonality and adaptation to contact-dependent signaling, fully meets our needs. Thus, we ultimately use SynNotch technology as receiver for extracellular signals to build our transmembrane logic gates.

Notch receptors are single-pass transmembrane receptors that transduce extracellular surface signals from sender cells to the inside of receiver cells. It features structural modularity and orthogonality in intracellular signal transduction. The signal strength and intensity of wild-type Notch receptors plays a crucial role in cell-fate decisions such as proliferation in development as well as homeostasis. This is true for functional outcomes that mainly rely on the dosage of the gene regulation and context of Notch signaling (Aster et al., 2008; Bray, 2016) For SynNotch, specialized factors will exclusively participate in regulation of genetic circuits that allow novel user-defined transcriptional responses. In our project ENABLE, we have engineered the cells to perform logical operations on multiple transmembrane signals to produce a variety of customizable outputs.

What is different in signal input?

1. Ligands

The largest family of ligands that can activate wild type Notch receptors features three related structural motifs, which are DSL (Delta/Serrate/LAG-2) motif, EGF-like repeats, and DOS (Delta and OSM-11-like proteins) domain that are tandem EGF repeats (Kopan et al., 2009). Wild type Notch receptors features cis-inhibition and trans-activation due to the antiparallel orientation of extracellular domains.

Similar to Notch receptors, the cis-inhibition mechanism was recapitulated in SynNotch receptors (Morsut et al., 2016). This indicates that the activating and inhibiting mechanism of wild type Notch and SynNotch are probably alike in nature. Therefore, as long as the Notch core can be activated, any ectodomain could work. In 2015, Gordon and her colleagues demonstrated that Notch with FBP as ectodomain targeting FKBP in the presence of chemical rapamycin can activate Notch receptors (Gordon et al., 2015). Lim and his colleagues have showed that SynNotch could recognize antigens such as CD19 and EGFP (Morsut et al., 2016; Roybal et al., 2016a).

2. The ectodomain of Notch receptors

Previous work by Morsut et al. showed that the natural surface antigen CD19 and the non-natural surface antigen EGFP have good orthogonality (Toda et al., 2018). We applied this and designed two surface antigens, surCD19 and surEGFP. By immunostaining, we were able to clearly detect surCD19 and surEGFP expressed on the surface of 293T cells (Figure 2a). Thus, these two surface antigens can serve as ideal dual antigens for our transmembrane binary Boolean logic inputs.

We selected αCD19 (anti-CD19) (Morsut et al., 2016), LaG17 (anti-EGFP with low affinity), LaG16-2 (anti-EGFP with high affinity) (Fridy et al., 2014) as the extracellular domain of SynNotch. Mouse Notch1 core (mN1c) regulatory region as the core transmembrane region of SynNotch. Two transcription factors, tTAA (TetR-VP48) and GV2 (Gal4DBD-VP64), which are orthogonal to each other, are used as the intracellular domain of SynNotch (Figure 2a). We found that the combination of different extracellular domains and intracellular domains has differentiated activation characteristics (Figure 2c) in transient transfection experiments. In all combinations, αCD19-mN1c-GV2 was not able to be activated efficiently (Figure 2c). LaG16-2-mN1c-GV2 and αCD19-mN1c-tTAA have high activation and low background expression. LaG16-2 was able to be efficiently activated only when activated with surEGFP but not surCD19 (Figure 2d). The excellent orthogonality between LaG16-2 and αCD19 ensures that we will not undergo signal crosstalk when using dual surface antigens as signal inputs in the future. In the following experiments, we used the LaG16-2-mN1c-GV2 and αCD19-mN1c-tTAA which has good performance, as actual transmembrane signal transduction elements. We used mCherry-tagged LaG16-2-mN1c-GV2 and EGFP-tagged αCD19-mN1c-tTAA to construct a stable cell line co-expressing dual-SynNotch receptors for subsequent experiments.

Meanwhile, as we mentioned above, some types of SynNotch have high background expression. This phenomenon aroused our great interests. We designed a systematic experiment to study the mechanism of SynNotch activation and hope to optimize it. We specifically covered the details of this section in the Optimization page.

The ectodomain of wild type Notch is composed of 29-36 modular epidermal growth factor (EGF)-like repeats, each around 40 residues in length and can be categorized into calcium binders and non-binders. Though former studies have assumed the shape of this ectodomain to be a long rod-like structure, taking a linear topology (Kopan and Ilagna,2009), recent studies have elucidated that there are multiple rods joined by flexible joints (Weisshuhn et al., 2015a, Weisshuhn et al., 2015b).

Signal transduction of the Notch core

The signal transduction mechanism of Notch signaling is very unique. While conserved signaling pathways are activated at the efforts of receptors, Notch receptor signaling and proteolysis is caused by the bing of corresponding ligands.

One interesting feature of Notch signaling is that its intracellular domains, released by intramembrane proteolysis during activation, will translocate to the cell nucleus (Schroeter EH et al., 1998) This renders almost no amplification of the signal, and signal orthogonality.

Notch specific ligands are in constant endocytosis-surface presenting cycle in the sender cell. When the N terminal recognition domain of Notch is bound to the ligand, this would produce resistance that will stimulate the ubiquitylation of the ligand and also enhance the recruitment of ubiquitin-binding Epsin endocytic adaptors (Weinmaster G. 2011). Complementary to this model, Ligand and cells defective in endocytosis cannot activate Notch (Nichols JT et al., 2007). This is like a cellular level tug-of-war between the two surface proteins, with the Notch receptor eventually giving up. When Notch extracellular domain is not bound by ligands with enough affinity or is under disturbances from small molecules, Notch 1 receptor will not activate because its negative regulatory region (NRR) will refrain it from being cleaved.

Notch core consists of the negatively regulated region and transmembrane region of the mouse Notch 1 receptor. It is the joint that empowers SynNotch the high modularity and proper activation 需要参考文献. From the N terminal to C terminal, it is composed of three lin12-repeats (LNR) domains LNR-A, LNR-B, LNR-C, the heterodimerization domain (HD), and the transmembrane domain. The LNR-AB linker between LNR-A and B is like a plug that occludes ADAM from cleaving wild type extracellular domain can be substituted by anti-GFP single-chain antibodies like LaG16 and αCD19, while its intracellular domain can be replaced by specialized transcription factors. Notch core is the key to the activation of both wild type Notch receptors and SynNotch. Lin-12 repeats (LNR) (Vardar et al. 2003).

The majority of Notch receptors are cleaved during the secretory pathway by furin-like convertase at cleavage site 1 (S1) (Logeat et al., 1998), which protrudes as a part of a linker of the two parts of the Notch negative regulatory region's heterodimerization domain (Gordon et al., 2009a; Gordon et al., 2007). This action separates Notch extracellular domain and the Notch transmembrane domain, but noncovalent interactions between the heterodimerization domains hold the receptor together. Despite disparate results on the role of S1 cleavage is a prerequisite for later activation, it has been suggested that it is not required for contact-dependent activation on the cell membrane (Gordon et al., 2009b).

When ligand binds to the Notch extracellular domain (NECD) and pulls it with mechanical force and thereby activating it, it initiates a process called regulated intramembrane proteolysis (RIP) (Weinmaster et al., 1997; Brou et al. 2000; Gordon et al., 2015) In this process, the Notch receptor is first cleaved at S2 site by two types of metalloproteases in the ADAM family (a disintegrin and metalloprotease), ADAM10 or ADAM17, with ADAM10 the dominant (Brou et al., 2000; Mumm et al., 2000; Bozkulak et al., 2009). The remaining truncated transmembrane subunit is subsequently cleaved by γ-secretase at S3 site, and results in the releasing the Notch intracellular domain (NICD). The wild type NICD will then enter the nucleus and interact with CSL and MAML to activate transcription (Schroteter et al., 1998; Struhl et al., 1998).

What is different in transcriptional signal output?

The wild type NICD will enter the nucleus and interact with CSL and MAML (Schroteter et al., 1998; Struhl et al., 1998) to regulate endogenous pathways that decide cell fate. The customizable output of SynNotch receptors have been integrated with chimeric antigen receptors to engineer T cells with customizable therapeutic response programs (Roybal et al., 2016a; Roybal et al., 2016b), or to program self-organizing multicellular structures (Toda et al., 2018). In our ENABLE project, we have used two types of SynNotchs in order to empower mammalian cells the ability of binary computation. This promises more novel application potentials. Please continue the reading of our Results on intracellular binary logic gates.

References 下面完全是凌乱的

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