iGEM 2022 — Team neu-china

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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**Notebook**

RTAC

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Yeast

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Kill switch

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![](https://static.igem.wiki/teams/4220/wiki/img/go-top.png)

Click the tag to see more information.

[RTAC](#RTAC_content)

**Design and Construction**

LGR4, ZNRF3

Getting: PCR

Connecting: Overlapping

V5-tag

Signaling peptide

Linker length

Fc tag adding

**Demonstration:**

Preparation:

Transfection and collection (HEK293)

RTAC (V5-tagged)

ECD (ZNRF3, LGR4; V5-tagged)

4 (HA-tagged)

Secretion testing

Stability testing

Affinity testing

Anti-cancer testing

(HEK293, RKO)

Western Blotting (HEK293, RKO)

RT-PCR (HEK293, RKO)

MTT (RKO, U2OS, Patu8988s)

[Yeast](#Yeast_content)

**Design and Construction:**

Preparation:

Gene knockout: ste2, sst2 and selection

PCR

P2Y2

Chimeric Gα

P2A

P2Y2-P2A-mCherry

P2Y2-P2A-Gα

pFUS1-mCherry

pFUS1-RTAC

P2Y2-P2A-Gα yeast selection

P2Y2-P2A-Gα+pFUS1-RTAC yeast selection (R-yeast)

**Demonstration:**

Co-culture (RKO)

Western Blotting

RT-PCR

MTT

[Kill switch](#kill_switch_content)

**Design:**

pHIS3-NeoR-pCUP1-HSF1

**Construction:**

R-yeast with the kill switch selection

**Demonstration:**

Spotting assay

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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**Engineering**

RTAC creation - Step 1

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Design

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Build

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Test

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Learn

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RTAC creation - Step 2

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Design

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Build

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Test

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Learn

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RTAC creation - Step 3

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Design

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Build

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Test

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Learn

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R-yeast

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Upstream

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Downstream

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Conclusion

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![](https://static.igem.wiki/teams/4220/wiki/img/go-top.png)

RTAC creation - Step 1

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Design

To design a protein which can bind all 4 RSPOs extracellularly.

![](https://static.igem.wiki/teams/4220/wiki/img/engineering/1.png)

Build

Choose the extracellular domains of two membrane proteins (LGR4 and ZNRF3) that have been reported to interact with RSPO and connect them with a linker with optimized length.

Test

The binding capability with RSPO: (Linker1-4 represent different linker lengths)

Learn

Linker3 is the optimal linker. The secretion efficiency should be optimized next.

RTAC creation - Step 2

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Design

To improve the secretion capability

Build

Choose several signaling peptides and compare the secretion efficiency

Test

The secretion capability of RTAC with different signaling peptides:

![](https://static.igem.wiki/teams/4220/wiki/img/engineering/2.png)

Learn

FZD5 signaling peptide is the best. The protein stability should be improved next.

RTAC creation - Step 3

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Design

To improve the stability by fusing additional domain(s).

Build

Adding human IgGH3 Fc region to the carboxyl terminus of RTAC.

Test

The stability of RTAC was improved.

![](https://static.igem.wiki/teams/4220/wiki/img/engineering/3.png)

Learn

RTAC is most stable protein. The engineering success of RTAC achieved.
See more in part [“BBa\_K4220000”](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4220000)

R-yeast:

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Upstream:

Design:

1. Idea: Reprogramming the mating pathway to enable the yeast to target tumor by specific signals in tumor microenvironment (TME).

2.Literature search:

Signaling molecules selection:
Choose extracellular ATP (eATP) as the signal because of its specific presence at TME compared with in healthy tissues.

Receptor determination:
Trigger mating pathway with eATP by replacing the original receptor (ste2) with modified human P2Y2 receptor which specifically responses to eATP.

Signal transduction:
Introducing the chimeric Gα protein with 5 C-terminal amino acids of mammalian Gαi3, which is capable to be recruited by human P2Y2 receptor and transduce the signal downstream.

Signal amplifying:
Removing sst2 can intensify the signal output of mating pathway

Tool:
P2A peptide can connect and then generate two different proteins when it is cleaved by specific enzyme.

**·** Plan1:

Gene editing:

Removing original receptor gene ste2.

Removing sst2 to intensify the signal output.

**·** Test1:

The gene editing success was verified by PCR.

**·** Plan2:

Transforming P2Y2-P2A-mCherry

**·** Test2:

The P2A peptide can work effectively:

![](https://static.igem.wiki/teams/4220/wiki/img/engineering/4.png)

**·** Plan3:

Transforming P2Y2-P2A-chimeric Gα

**·** Test3:

![](https://static.igem.wiki/teams/4220/wiki/img/engineering/5.png)
![](https://static.igem.wiki/teams/4220/wiki/img/engineering/6.png)

Engineering completed:

The original mating pathway was reprogrammed. P2Y2 receptor and chimeric Gα can be successfully introduced into the yeast and the P2A functions well to separate these two proteins.

See more in part [“BBa\_K4220008”](http://parts.igem.org/Part%3ABBa_K4220008) and part [“BBa\_K4220007”](http://parts.igem.org/Part%3ABBa_K4220007)

Downstream:

Design:

1. Idea: engineer RTAC downstream of the mating pathway

2. Literature search:

MFα-1 is the signaling peptide of α-factor which is particular for yeast expression

FUS1 is the final outputting product of mating pathway

**·** Plan1:

Transform P2Y2-P2A-Gα into the yeast.

Proving the function of P2A and demonstrate the successful reprogramming of eATP-P2Y2-Gα-MAPK: transform the pFUS1-mCherry into the yeast.

**·** Test1:

The reprogrammed mating pathway can effectively sense the eATP and express mCherry indicated by red fluorescence:

![](https://static.igem.wiki/teams/4220/wiki/img/engineering/7.png)

**·** Plan2:

Changing of signaling peptide of RTAC: change the Frizzled5 signaling peptide into MFα1

Including RTAC into the mating pathway: transform pFUS1-RTAC into the yeast.

**·** Test2:

RTAC can be successfully produced and secreted in an eATP dose-dependent manner:

![](https://static.igem.wiki/teams/4220/wiki/img/engineering/8.png)

Conclusion

Engineering completed:
RTAC expression can be successfully induced by eATP with adequate secretion efficiency.

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## What should this page contain?

## hello world

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Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

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* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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**Results**

Design of RTAC

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RTAC binds to all Rspos with high affinity

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RTAC inhibits Rspo-mediated Wnt signaling and Rspo/Wnt-dependent cell growth

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Engineered yeast as carrier to express RTAC in a self-tunable manner

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Novel Copper-dependent kill switch ensures environmental biosafety of the engineered yeast

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Conclusion

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Reference

![](https://static.igem.wiki/teams/4220/wiki/img/go-top.png)

Design of RTAC

We sought to develop a new protein that can successfully neutralize all four RSPOs in order to antagonize RSPO in malignancies. This protein, as compared to the RSPO3-specific antibody, has the therapeutic potential to treat both colorectal cancers caused by RSPO2 and RSPO3, as well as other cancers caused by RSPO1 or RSPO4. The ECD domains of LGR4 and ZNRF3 were joined to form a chimeric protein that was designed and engineered based on earlier biochemical and structural biological research (Figure 1a)1-4. In order to connect these two domains and offer adequate flexibility and solubility, a linker region was optimized in between (Figure 1b). We examined several signaling peptides from various transmembrane proteins in search of the best secretion capacity. The results demonstrated that the signaling peptide from Frizzled5, a Wnt receptor, can most efficiently induce the chimeric protein's secretion into the conditioned media (Figure 1c). By joining the IgG Fc (human IgGH3 Fc) region to the carboxyl terminus of the chimeric protein, further stability was provided to the protein (Figure 1d). It was demonstrated experimentally that Fc-fused chimeric protein could persist for over 4 weeks at 37 Celsius without degrading or aggregating in FBS-containing medium, which persists noticeably longer than the proteins without (Figure 1e).

![](https://static.igem.wiki/teams/4220/wiki/img/results/results-figure1.png)

Figure 1a: Structural model of LGR4 ECD with RSPO1 and ZNRF3 ECD with RSPO1

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure1b.png)

Figure 1b: Binding affinity to RSPO3 with different linker length in between LGR4 ECD and ZNRF3 ECD. Linker3 was selected for the optimal binding

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure1c.png)

Figure 1c: Secretion test of different signaling peptides. FZD5 signaling peptide showed the best secretion efficiency and used further.

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure1d.png)

Figure 1d: Schematic flow showing the improvement of the chimeric protein.

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure1e.png)

Figure 1e: Stability test showing that RTAC can stably exist in 37 Celsius for 4 weeks, which is stronger than the rest proteins.

Also, a V5-tag was engineered at the animo terminal of the protein as an epitope for easy detection. Thus, we gave the chimeric protein's final iteration the name RTAC (RSPO-Targeting Anti-cancer Chimeric protein) (Figure 1d).

RTAC binds to all Rspos with high affinity

The next goal was to characterize RTAC's capacity to bind to RSPOs. First, we transiently transfected RTAC and four HA-tagged RSPOs into HEK293 cells. After 48 hours, we collected secreted RTAC and four RSPOs from the conditioned medium. We then co-incubated RTAC with individual RSPO in vitro, respectively, and used α-HA antibody-conjugated agarose to pull down the RSPOs. The results showed RTAC exhibited pan-RSPO binding capacity towards all RSPOs, which was stronger than LGR4 or ZNRF3 ECD alone (Figure 2a,2b).

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure2a.png)

Figure 2a: Co-immunoprecipitation assay showing RTAC has pan-RSPO binding capacity

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure2b.png)

Figure 2b: Co-immunoprecipitation assay showing RTAC binds to RSPOs with stronger affinity than LGR4 or ZNRF3 ECD alone.

RTAC inhibits Rspo-mediated Wnt signaling and Rspo/Wnt-dependent cell growth

Following the binding studies, we attempted to evaluate RTAC's RSPO-inhibiting potential. TOPFlash luciferase is a widely used assay to evaluate Wnt/β-catenin signaling strength. Renilla luciferase is used as an internal control while the firefly luciferase is engineered downstream of the TCF/LEF response element to offer specific and strong Wnt/β-catenin signal output (Figure 3a).

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure3a.png)

Figure 3a: Schematic drawing of TOPFlash working model

The plasmids expressing these two luciferases were transiently transfected into HEK293 cell before further experiments. We observed in these HEK293 cells that the addition of RSPO3-containing conditioned medium could augment the signaling intensity by multiple folds. It was satisfactory to see that RTAC treatment could effectively neutralize the Wnt/β-catenin activating effect of RSPO3 (Figure 3b).

Besides the luciferase assay, multiple experiments were exercised to confirm the anti-RSPO function of RTAC. Treatment of RTAC effectively nullified the accumulation of cytosolic β-catenin and Wnt target gene expressions caused by additional RSPO3, shown by Western Blotting and quantitative RT-PCR assay, respectively (Figure 3c, 3d).

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure3b.png)

Figure 3b: TOPFlash result showing RTAC can suppress Wnt signaling activation caused by RSPO3 in HEK293 cell

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure3c.png)

Figure 3c: WB result showing RTAC can suppress cytosolic β-catenin accumulation caused by RSPO3 in HEK293 cell

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure3d.png)

Figure 3d: RT-qPCR result showing RTAC can suppress Wnt target gene expression caused by RSPO3 in HEK293 cell

These findings were subsequently confirmed in colorectal cancer cell RKO, where the introduction of RTAC greatly negated the effect of RSPO. (Figure 3e-3g).

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure3e.png)

Figure 3e: TOPFlash result showing RTAC can suppress Wnt signaling activation caused by RSPO3 in RKO cell

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure3f.png)

Figure 3f: WB result showing RTAC can suppress cytosolic β-catenin accumulation caused by RSPO3 in RKO cell

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure3g.png)

Figure 3g: RT-qPCR result showing RTAC can suppress Wnt target gene expression caused by RSPO3 in RKO cell

It is commonly known that Wnt/β-catenin signaling activation is crucial for the growth of many tumor cells. For this reason, we selected several Wnt-sensitive cell lines, such as RKO, Patu8988s, and U2OS to investigate the broad effectiveness of RTAC in inhibiting tumor cell proliferation. RSPO3-containing conditioned medium was added into the cell culture every two days starting from Day0. And purified RTAC after size-exclusion chromatography was added together with RSPO3. MTT cell growth assay indicated that RTAC addition could significantly restrict the growth of these cells at the presence of RSPO (Figure 3h). And higher dose of RTAC showed stronger inhibitory effect in RKO cell with statistical significance (Figure 3h). These findings present conclusively that RTAC blocks in vitro tumor cell proliferation and RSPO-mediated Wnt signaling. Thus, our engineering of RTAC came to success.

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure3h.png)

Figure 3h: MTT growth assay showing RTAC suppresses the cancer cell growth in multiple RSPO-sensitive cell lines.

Engineered yeast as carrier to express RTAC in a self-tunable manner

We next aimed to employ engineered microorganism to create a self-tunable machinery to deliver RTAC to tumor cells. Because of the existence of microbiota in the intestinal tract, we proposed that this microorganism can be used as a probiotic to prevent or treat RSPO-hyperactivated colorectal cancer. Brewer’s yeast (*Saccharomyces Cerevisiae*) is a model organism that has been studied for decades, which genomic information is clear. It is also highly biosafe to human as it can behave as a beneficial or commensal microorganism in human guts5. Meanwhile, this eukaryotic system can allow mature and functional RTAC to be expressed and secreted. Because of these advantages, we decided to employ S. Cerevisiae as our vessel to deliver RTAC to the intestinal tumor.

Based on the previous report, we decided to reprogram the yeast’s mating pathway to allow it to sense tumor-environmental specific molecules6,7. Extracellular ATP (eATP) was reported to have specifically strong presence at the tumor tissue up to hundreds of uM concentration8. Thus, this molecule serves as an ideal signal to trigger tumor-tissue specific expression of RTAC. Following the previous research9, we first reprogrammed the mating pathway by removing the receptor ste2 through gene editing to ensure the pheromone α can no longer stimulate the downstream pathway. Also, the mating inhibiting factor sst2 was removed to intensify the signal output (Figure 4a). This double-knocked out yeast strand was used in our further experiments.

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure4a.png)

Figure 4a: Schematic drawing of the engineered yeast

Meanwhile, we introduced modified human P2Y2 receptor and chimeric Gα as the previous literature reported9. The introduction of these two components will enable the yeast to sense eATP and transduce the signal to the native MAPK signaling in the yeast6,7,10(Figure 4a). Thus, in the absence of ste2, the yeast’s mating pathway could only be activated through eATP-P2Y2-Gα-MAPK axis. To minimize the usage of selection marker, we linked P2Y2 receptor and the chimeric Gα in the same vector with a P2A peptide sequence in between. The P2A peptide can undergo a translational recoding event to generate two independent proteins before and after this “break”11. By fusing P2Y2 receptor and a common fluorescent protein mCherry, we evaluated the efficiency of the P2A peptide in our system. First, from Western Blotting assay, we observed that the HA-tagged P2Y2 receptor displayed as approximately 40kDa on the gel, which was its native molecular weight (Figure 4b). If P2A failed to function, the presumable P2Y2-mCherry fusion protein had a MW around 78kDa, which did not appear on the gel. Also, the fluorescence of mCherry confirmed the cleavage efficiency of the P2A peptide (Figure 4c).

|  |  |
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| ![](https://static.igem.wiki/teams/4220/wiki/img/results/figure4b.png) | ![](https://static.igem.wiki/teams/4220/wiki/img/results/figure4c.png) |

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| --- | --- |
| Figure 4b: WB results confirming the self-cleavage function of P2A peptide in P2Y2-P2A-mCherry and P2Y2-P2A-Gα plasmids | Figure 4c: Fluorescent microscopy results showing mCherry localizes in the cytosol instead of on the membranes |

As a soluble fluorescent protein, mCherry localizes in the entire cytosol. If the P2A peptide was unfunctional, P2Y2 could tether mCherry to the membrane. In Figure 4c, it was very clear to visualize that the fluorescence of mCherry spread out within the whole yeast cells without recognizable membrane residence, which demonstrated that mCherry was released from P2Y2 receptor. Indeed, when we transformed P2Y2-P2A-Gα plasmid into the yeast, we confirmed that P2Y2 and chimeric Gα were generated in the yeast separately by Western Blot (Figure 4b). To conclude, these results indicate that the eATP sensing parts are engineered into the yeast successfully and our P2A system can efficiently function.

After the upstream of the mating pathway was reprogrammed, we started for the downstream part. Since pFUS1 promoter target gene expression is the final output of the mating pathway6,7, we constructed a plasmid with RTAC following the pFUS1 promoter (Figure 4a). To maximize the secretion efficiency, we adapted the signaling peptide of RTAC to that of α-factor (MFα1) particularly for yeast expression9. As a test, we also constructed the pFUS1-mCherry plasmid to detect the sensitivity of eATP treatment. We combinatorially transformed P2Y2-P2A-Gα and pFUS1-mCherry, and cultured the transformed yeast with selection markers for 48 hours. Afterwards, eATP was added to the culture at different doses and the red fluorescence was monitered. The result showed that starting at 25uM, the yeast started to show red fluorescence under the microscopy (Figure 4d). And the fluorescence intensified with increased eATP concentrations (Figure 4d).

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure4d.png)

Figure 4d: Fluorescence intensity showing mCherry can by induced by the additional of ATP in the culture medium

This data serves as strong evidence to show that our engineered mating circuit can successfully sense the eATP from the environment and express substantial amount of our desired target. After this validation, we eventually transformed P2Y2-P2A-Gα and pFUS1-RTAC together into the engineered yeast. Upon eATP treatment, RTAC can be expressed and secreted into the culture in a dose-dependent manner as expected (Figure 4e). We thus named this eATP-sensitive RTAC-expressing yeast as R-yeast.

![](https://static.igem.wiki/teams/4220/wiki/img/proof-of-concept/results-4f.png)

Figure 4e: WB showing RTAC can be induced and harvested in the culture medium upon eATP treatment.

Due to the absence of animal use permission, we planned to test the function of R-yeast in vitro. We then co-cultured R-yeast with RKO cell to demonstrate the RSPO-inhibitory effect. First, from Western Blotting, we observed that the cytosolic β-catenin diminished in the co-cultured RKO cells upon the addition of 200uM eATP (Figure 4f). This data was further assisted by quantitative RT-PCR result, in which R-yeast significantly inhibited the Wnt-target genes expression (Figure 4g).

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure4f.png)

Figure 4f: WB showing R-yeast can express RTAC to suppress cytosolic β-catenin accumulation upon eATP addition when co-cultured with RKO

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure4g.png)

Figure 4g: RT-qPCR showing R-yeast can express RTAC to suppress Wnt target gene expression upon eATP addition when co-cultured with RKO

Cancer cell growth was also examined. Addition of RSPO3 could significantly enhance RKO growth, which could be reverted by R-yeast co-culturing with 200uM eATP in the culture medium shown by MTT growth assay (Figure 4h).

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure4h.png)

Figure 4h: MTT assay showing R-yeast co-culturing inhibits the growth of RKO in response to eATP.

As a negative control, normal brewer’s yeast could not affect the growth of RKO. This data together has provided a proof-of-concept of our designed engineered yeast (Figure 4h).

To conclude, we successfully showed that R-yeast can sense eATP and express RTAC dose-dependently. And the expressed RTAC can functionally inhibit RSPO-induced Wnt/β-catenin signaling activation and corresponding cancer cell growth. In principle, the expression of RTAC in R-yeast remains silent in the absence of eATP and will auto-sense and switch on when coming across tumor tissue. This self-tunable yeast poses minimal threat to human health thus may serve as a probiotic to prevent and treat colon cancers.

Novel Copper-dependent kill switch ensures environmental biosafety of the engineered yeast

The brewer’s yeast is not considered as a biohazardous or pathogenic microorganism in the environment in general. However, considering the possible leakage and unexpected hazards, we decided to add a self-killing switch to completely resolve this issue. After extensive readings, we realized that there was no well-recognized kill-switch in yeast from the literatures. So, we decided to design and construct one on our own. First, we picked an essential gene in yeast as our target. By manipulating the expression of this gene, the survival of the yeast can be controlled. HSF1 was reported to be an essential gene in yeast, which pivotally governs stress response and other crucial processes12, was chosen as our target. Meanwhile, we investigated the difference between the environment and the intestinal tract through readings and consulting. We realized that copper ion was abundant in the intestine but scarce in the environment13. Interestingly, the copper ion can activate copper-ion dependent promoter CUP1 for target gene expression14,15. As a result, we replaced the original promoter of HSF1 in R-yeast’s genome to CUP1 by gene editing (Figure 5a). By spotting assay, we proved that the CUP1-HSF1 R-yeast could only survive at the presence of copper ion (Figure 5b).

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure5a.png)

Figure 5a: Schematic diagram of pCUP1-HSF1 engineering

![](https://static.igem.wiki/teams/4220/wiki/img/results/figure5b.png)

Figure 5b: Spotting assay showing the survival of the engineered yeast in dependent of copper ion.

Thus, this innovative kill-switch can ensure the engineered yeast to cause minimal hazard to the environment.

Conclusion

To conclude, our work presented a novel proof-of-concept to treat RSPO-hyperactivated cancers by synthetic biology methods. RTAC can specifically target RSPOs and the mating pathway reprogrammed yeast can sense and target the cancer cells, combination of which creates the “dual-targeting” effect. Cancer cell growth can be successfully suppressed by RTAC or R-yeast in vitro, to give but one non-exhaustive example. In upcoming studies, biological models with high physiological relevance will be used to define the anti-tumor effect of RTAC and R-yeast. We hope that this novel approach can be improved upon and eventually assessed clinically.

[Reference](#reference_content)

1 Xu, J. G. et al. Crystal structure of LGR4-RSPO1 complex: insights into the divergent mechanisms of ligand recognition by leucine-rich repeat G-protein-coupled receptors (LGRs). The Journal of biological chemistry 290, 2455-2465, doi:10.1074/jbc.M114.599134 (2015).

2 Zebisch, M. & Jones, E. Y. Crystal structure of R-spondin 2 in complex with the ectodomains of its receptors LGR5 and ZNRF3. Journal of structural biology 191, 149-155, doi:10.1016/j.jsb.2015.05.008 (2015).

3 Koo, B. K. et al. Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors. Nature 488, 665-669, doi:10.1038/nature11308 (2012).

4 Hao, H. X. et al. ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature 485, 195-200, doi:10.1038/nature11019 (2012).

5 Limon, J. J., Skalski, J. H. & Underhill, D. M. Commensal Fungi in Health and Disease. Cell Host Microbe 22, 156-165, doi:10.1016/j.chom.2017.07.002 (2017).

6 Herskowitz, I. MAP kinase pathways in yeast: for mating and more. Cell 80, 187-197, doi:10.1016/0092-8674(95)90402-6 (1995).

7 Oehlen, B. & Cross, F. R. Signal transduction in the budding yeast Saccharomyces cerevisiae. Curr Opin Cell Biol 6, 836-841, doi:10.1016/0955-0674(94)90053-1 (1994).

8 Vultaggio-Poma, V., Sarti, A. C. & Di Virgilio, F. Extracellular ATP: A Feasible Target for Cancer Therapy. Cells 9, doi:10.3390/cells9112496 (2020).

9 Scott, B. M. et al. Self-tunable engineered yeast probiotics for the treatment of inflammatory bowel disease. Nature medicine 27, 1212-1222, doi:10.1038/s41591-021-01390-x (2021).

10 Di Virgilio, F., Sarti, A. C., Falzoni, S., De Marchi, E. & Adinolfi, E. Extracellular ATP and P2 purinergic signalling in the tumour microenvironment. Nat Rev Cancer 18, 601-618, doi:10.1038/s41568-018-0037-0 (2018).

11 Sharma, P. et al. 2A peptides provide distinct solutions to driving stop-carry on translational recoding. Nucleic Acids Res 40, 3143-3151, doi:10.1093/nar/gkr1176 (2012).

12 Kopczynski, J. B., Raff, A. C. & Bonner, J. J. Translational readthrough at nonsense mutations in the HSF1 gene of Saccharomyces cerevisiae. Mol Gen Genet 234, 369-378, doi:10.1007/BF00538696 (1992).

13 Nose, Y., Kim, B. E. & Thiele, D. J. Ctr1 drives intestinal copper absorption and is essential for growth, iron metabolism, and neonatal cardiac function. Cell metabolism 4, 235-244, doi:10.1016/j.cmet.2006.08.009 (2006).

14 Pena, M. M., Koch, K. A. & Thiele, D. J. Dynamic regulation of copper uptake and detoxification genes in Saccharomyces cerevisiae. Molecular and cellular biology 18, 2514-2523, doi:10.1128/MCB.18.5.2514 (1998).

15 Labbe, S., Zhu, Z. & Thiele, D. J. Copper-specific transcriptional repression of yeast genes encoding critical components in the copper transport pathway. The Journal of biological chemistry 272, 15951-15958, doi:10.1074/jbc.272.25.15951 (1997).

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