Difference between revisions of "Part:BBa K4040017"
 
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In T cells, phosphorylated ITAMs in CD3z bind to tandem SH2 domains (tSH2) in the kinase ZAP70. Zap70 is not expressed in macrophages, but Syk, a phagocytic signaling effector and tSH2 domain containing protein, is expressed at high levels (Andreu et al., 2017). Previous work suggested that Syk kinase can also bind to the CD3z ITAMs (Bu et al., 1995), indicating that the CAR-T may promote engulfment through a similar mechanism as CAR-PFcRV . To quantitatively compare the interaction between SyktSH2 and CD3z or FcRV in a membrane proximal system recapitulating physiological geometry, previous study used liposome-based assay (Figure 1 [Hui and Vale, 2014]). In this system, His10-CD3z and His10-Lck (the kinase that phosphorylates CD3z) are bound to a liposome via NiNTA-lipids and the binding of labeled tandem SH2 domains to phosphorylated CD3z was measured using fluorescence quenching. Their results show that Syk-tSH2 binds the CD3z and FcRV with comparable affinity (~15 nM and ~30 nM respectively, Figure 1). Collectively, these results demonstrate that the TCR CD3z chain can promote phagocytosis in a CAR-P, likely through the recruitment of Syk kinase.[1]
 
In T cells, phosphorylated ITAMs in CD3z bind to tandem SH2 domains (tSH2) in the kinase ZAP70. Zap70 is not expressed in macrophages, but Syk, a phagocytic signaling effector and tSH2 domain containing protein, is expressed at high levels (Andreu et al., 2017). Previous work suggested that Syk kinase can also bind to the CD3z ITAMs (Bu et al., 1995), indicating that the CAR-T may promote engulfment through a similar mechanism as CAR-PFcRV . To quantitatively compare the interaction between SyktSH2 and CD3z or FcRV in a membrane proximal system recapitulating physiological geometry, previous study used liposome-based assay (Figure 1 [Hui and Vale, 2014]). In this system, His10-CD3z and His10-Lck (the kinase that phosphorylates CD3z) are bound to a liposome via NiNTA-lipids and the binding of labeled tandem SH2 domains to phosphorylated CD3z was measured using fluorescence quenching. Their results show that Syk-tSH2 binds the CD3z and FcRV with comparable affinity (~15 nM and ~30 nM respectively, Figure 1). Collectively, these results demonstrate that the TCR CD3z chain can promote phagocytosis in a CAR-P, likely through the recruitment of Syk kinase.[1]
 
[[File:T--NMU_China--gamma44.png|thumb|center|600px|<b>Figure 1.</b>  Model of the liposome-based fluorescence quenching assay used to determine affinity between the Syk tSH2 domains and the receptor tails of CD3z and FcRV, two intracellular signaling domains that promote engulfment. Binding between the Syk tSH2 reporter (Syk tSH2), green, and a receptor tail, purple, was detected by rhodamine quenching of BG505 dye on the reporter (see Materials and methods). Kd was determined by assessing mean fluorescence quenching for the last 20 timepoints collected ~45 min after ATP addition over a receptor titration from 0 to 500 nM. Each point represents the mean ± SD from three independent experiments. Kd ± SE was calculated by nonlinear fit assuming one site specific binding]]
 
[[File:T--NMU_China--gamma44.png|thumb|center|600px|<b>Figure 1.</b>  Model of the liposome-based fluorescence quenching assay used to determine affinity between the Syk tSH2 domains and the receptor tails of CD3z and FcRV, two intracellular signaling domains that promote engulfment. Binding between the Syk tSH2 reporter (Syk tSH2), green, and a receptor tail, purple, was detected by rhodamine quenching of BG505 dye on the reporter (see Materials and methods). Kd was determined by assessing mean fluorescence quenching for the last 20 timepoints collected ~45 min after ATP addition over a receptor titration from 0 to 500 nM. Each point represents the mean ± SD from three independent experiments. Kd ± SE was calculated by nonlinear fit assuming one site specific binding]]
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===Results of our project===
 +
It can be found in <partinfo>BBa_K4040018</partinfo>
 
===References===
 
===References===
 
[1]Morrissey MA, Williamson AP, Steinbach AM, Roberts EW, Kern N, Headley MB, Vale RD. Chimeric antigen receptors that trigger phagocytosis. Elife. 2018 Jun 4;7:e36688. doi: 10.7554/eLife.36688. PMID: 29862966; PMCID: PMC6008046.
 
[1]Morrissey MA, Williamson AP, Steinbach AM, Roberts EW, Kern N, Headley MB, Vale RD. Chimeric antigen receptors that trigger phagocytosis. Elife. 2018 Jun 4;7:e36688. doi: 10.7554/eLife.36688. PMID: 29862966; PMCID: PMC6008046.

Latest revision as of 12:31, 15 October 2021


Synthetic Receptor CARζ

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 344
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 952
    Illegal NgoMIV site found at 1288
    Illegal NgoMIV site found at 1825
    Illegal NgoMIV site found at 1891
    Illegal NgoMIV site found at 2050
    Illegal AgeI site found at 440
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1168
    Illegal BsaI.rc site found at 1432
    Illegal SapI.rc site found at 1989

The structure and function of CAR ζ

CAR ζ is another CAR aimed to increase the phagocytosis of the macrophages. And the structure is also similar to the other three CARs, with the difference in the intracellular domain which is replaced by CD3ζ(BBa_K4040003) subunit of the T cell receptor.

The mechanism of CAR ζ

In T cells, phosphorylated ITAMs in CD3z bind to tandem SH2 domains (tSH2) in the kinase ZAP70. Zap70 is not expressed in macrophages, but Syk, a phagocytic signaling effector and tSH2 domain containing protein, is expressed at high levels (Andreu et al., 2017). Previous work suggested that Syk kinase can also bind to the CD3z ITAMs (Bu et al., 1995), indicating that the CAR-T may promote engulfment through a similar mechanism as CAR-PFcRV . To quantitatively compare the interaction between SyktSH2 and CD3z or FcRV in a membrane proximal system recapitulating physiological geometry, previous study used liposome-based assay (Figure 1 [Hui and Vale, 2014]). In this system, His10-CD3z and His10-Lck (the kinase that phosphorylates CD3z) are bound to a liposome via NiNTA-lipids and the binding of labeled tandem SH2 domains to phosphorylated CD3z was measured using fluorescence quenching. Their results show that Syk-tSH2 binds the CD3z and FcRV with comparable affinity (~15 nM and ~30 nM respectively, Figure 1). Collectively, these results demonstrate that the TCR CD3z chain can promote phagocytosis in a CAR-P, likely through the recruitment of Syk kinase.[1]

Figure 1. Model of the liposome-based fluorescence quenching assay used to determine affinity between the Syk tSH2 domains and the receptor tails of CD3z and FcRV, two intracellular signaling domains that promote engulfment. Binding between the Syk tSH2 reporter (Syk tSH2), green, and a receptor tail, purple, was detected by rhodamine quenching of BG505 dye on the reporter (see Materials and methods). Kd was determined by assessing mean fluorescence quenching for the last 20 timepoints collected ~45 min after ATP addition over a receptor titration from 0 to 500 nM. Each point represents the mean ± SD from three independent experiments. Kd ± SE was calculated by nonlinear fit assuming one site specific binding

Results of our project

It can be found in BBa_K4040018

References

[1]Morrissey MA, Williamson AP, Steinbach AM, Roberts EW, Kern N, Headley MB, Vale RD. Chimeric antigen receptors that trigger phagocytosis. Elife. 2018 Jun 4;7:e36688. doi: 10.7554/eLife.36688. PMID: 29862966; PMCID: PMC6008046.