Difference between revisions of "Part:BBa K3617001"

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	__NOTOC__		__NOTOC__
−	<partinfo>BBa_K3617001 short</partinfo>	+	==<partinfo>BBa_K3617001 short</partinfo>==
		+	This biobrick is a part of a two-protein system in Saccharomyces cerevisiae, which is designed for detection of human interleukin-6 and transduction of the signal by means of a reconstituted ubiquitin. Development of split-ubiquitin as a tool for study of protein-protein interactions in vivo was first published in 1994 and has been an essential feature in biologists’ toolbox ever since (source: https://www.pnas.org/content/pnas/91/22/10340.full.pdf). A specific mutation in the N-terminal part protects it from binding spontaneously to the C-terminal part. However, re-association can be facilitated by binding of a pair of proteins to which the ubiquitin parts are fused. Human signal transducer gp130 was expressed in yeast for the first time in 1997 and further improvements paved the way to our own chimeric transmembrane proteins (source: https://pubmed.ncbi.nlm.nih.gov/9271090/).
		+	==<span class='h3bb'>Sequence and Features</span>==
		+	<partinfo>BBa_K3617001 SequenceAndFeatures</partinfo>
−	This BioBrick is an ORF encoding a fusion protein consisting of:	+	From N- to C-terminal the protein includes; an endoplasmic reticulum import signal peptide from the S. cerevisiae cell wall integrity and stress response component 1 (Wsc1), The first three domains of the soluble isoform of human interleukin-6 co-receptor, soluble glycoprotein 130 (sgp130). The transmembrane domain from Wsc1, as well as the C-terminal part of the split version of ubiquitin and the synthetic transcription factor LexA-VP16. LexA is a DNA binding domain from Escherichia Coli and VP16 is a transcriptional activation domain from Herpes simplex virus Type 1. Together LexA-VP16 functions as an orthogonal transcription factor in S. cerevisiae. Between the extracellular sgp130 domains and the transmembrane domain, a (2x)GGGGS-linker was added. Between the transmembrane domain and the C-terminal split ubiquitin domain, two basic amino acids (KR), and the (2x)GGGGS-linker was added.
−	<ul>	+
−	<li>The first 21 amino acids (Signal peptide for import to endoplasmatic reticulum) of the endogenous Cell wall integrity and stress response component 1 (Wsc1 [Jon: the protein is usually referred to as Wsc1 while the gene is called SLG1 what do we call it?) receptor in S. Cerevisiae.</li>	+
−	<li>The first and third domain (D1-D3; aa 22-323) of human IL-6 co-receptor soluble glycoprotein 130 (sgp130).</li>	+
−	<li>The transmembrane domain of Wsc1 (aa 327-351)</li>	+
−	<li>C-terminal part of a split version of ubiquitin. (aa 363-405)</li>	+
−	<li>Between the three domains of sgp130 and the transmembrane domain we added a GGGGS-linker and between the transmembrane domain (Wsc1) and the C-terminal split ubiquitin domain we added two basic amino acids; KR, and the GGGGS-linker again. [Jon: do you think we should explain the rational of these gene engineering choices or is that redundant?]</li>	+
−	</ul>	+
−	The C-terminal part (Cub) is the amino acids 35-76 of the split ubiquitin molecule and has the reporter protein cassette LexA-VP16 fused to Cub. LexA is a DNA binding domain from E. Coli (EG10533 (EcoCyc); P0A7C2 (UniProt)) and Herpes simplex virus Type 1 VP16 gene is a transcriptional activation domain.	+	==Sequence optimization==
		+	The sequence was codon-optimized for S. cerevisiae. Recognition sequences for SpeI, XbaI, NotI, EcoRI, PstI were avoided to follow RFC10 standard.
−	The LexA-VP16 protein cassette is used in yeast two-hybrid method (Y2H) to assay protein-protein interaction (PPI), where the LexA-VP16 is dissociated from the Cub by deubiquitinase when PPI occurs. The LexA-VP16 transcription factor will then be transported into the nucleus where it will trigger the expression of reporter genes.	+	==Structure and function==
		+	[[Image:T--UCopenhagen--parts-il6rnubflour.jpg|460px|thumb|<p align="justify"> ''' Figure 2: Mechanism for signal transduction by IL-6 receptor proteins. '''</p>]]
		+	BBa_K361701 (marked in red) is designed to locate to the plasma membrane. Upon IL-6 binding it associates with BBa_K3617000 (marked in yellow), forming a trimeric complex. Following extracellular binding, the two intercellular parts of ubiquitin (C-ub and N-ub) come together forming a full-length ubiquitin. This is then cleaved by a deubiquitinase, triggering the release of the LexA-VP16 synthetic transcription factor.
		+
		+	BBa_K361701 is designed to work together with BBaK3617000 and constitute a functional human IL-6 receptor. BBaK3617001 possesses domains 1-3 out of the 6 extracellular domains of the IL-6 co-receptor soluble glycoprotein 130 (sgp130), the C-terminal part of split-ubiquitin, and the LexA-VP16 synthetic transcription. The synthetic transcription factor is a fusion of the DNA binding domain of the LexA transcription factor from Escherichia coli, and an activation domain from the herpes simplex virus transcriptional regulatory protein VP16. LexA-VP16 is often used in yeast 2 hybrid assays as it does not affect endogenous S. cerevisiae genes, and therefore provide orthogonality. In the presence of interleukin-6, the extracellular domains of BBa_K3617000 and BBa_K3617001 (IL-6R and sgp130) associate, forming a heterotrimer consisting of IL-6, IL-6R, and sgp130. The trimerization causes intracellular complementation of the two ubiquitin parts allowing for recognition by an endogenous deubiquitinating enzyme, which facilitates releases of the transcription factor. The transcription factor then relocates to the nucleus and activates expression of a reporter gene (Figure 1).
−	The sequence was codon optimized for S. cerevisiae using Benchling.	+	==Confocal flourescence microscopy==
		+	In order to investigate the cellular localization of our protein, superfolder green fluorescent protein was fused to the C-terminal end of the protein. Following expression of our new fusion construct, the cells were observed with confocal fluorescence microscopy for visualization.
−	<strong>Expected function of the protein:</strong>	+	[[Image:T--UCopenhagen--002_ch02.png|700px|thumb|center|<p align="justify"> '''Figure 2a: <b> Confocal fluorescence microscopy of sgp130(D1-D3)-Cub-sfGFP. </b> Pictures were taken with a 150 μm pinhole. The image shows both a faint localization in the endoplasmic reticulum, and at the membrane, but most of the protein ends up in inclusion bodies/vacuoles.'''</p>]]
		+	[[Image:T--UCopenhagen--004_ch002.png|700px|thumb|center|<p align="justify"> '''Figure 2b: <b> Confocal fluorescence microscopy of sgp130(D1-D3)-Cub-sfGFP. </b> Pictures were taken with a 150 μm pinhole. The image shows both a faint localization in the endoplasmic reticulum, and at the membrane, but most of the protein ends up in inclusion bodies/vacuoles.'''</p>]]
		+	The majority of investigated cells had one or more fluorescent aggregates. These aggregates were predominantly positioned between the nucleus and the plasma membrane, which could indicate the formation of inclusion bodies. For some cells, the fluorescence signal accumulated close to the nucleus. A possible explanation could be that the protein may be stuck in the Golgi apparatus. Previous studies by Vollmer et al. (1999) have shown that removing the N-terminal Ig-like domain of the IL-6 receptor leads to retention of the protein in the secretory pathway. To circumvent this localization issue, one could add back the N-terminal Ig-domain of the IL6-R.
−	The signal peptide and transmembrane domain constitute the backbone of our modular framework for localizing our receptors at the plasma membrane as type I single pass transmembrane proteins. As a type I transmembrane protein the soluble interleukin receptor domains would be localized extracellularly while the N-terminal part of the split protein would be the intracellular. Ivanusic et al. (citation) introduced the use of the signal peptide and transmembrane domain in a split-ubiquitin system for screening for PPIs at the plasma membrane in S. cerevisiae. The two fibronectin type III soluble interleukin-6 receptor subunit alpha domains are mediating the binding of the receptor to interleukin-6 as seen in crystal structures (see fig. 1). The outer Ig-like domain of the receptor mediates other functions of the receptor (Vollmer et al. PMID: 10406952) and it is OMITTED in this part - this might cause the unwanted localization as addressed later! This BioBrick is intended to work together with (BioBrick) which has the outer three domains of the IL-6 co-receptor soluble glycoprotein 130 (sgp130), extracellularly and the C-terminal part of split ubiquitin intracellularly with the synthetic transcription factor linked to the C-terminal of the split ubiquitin domain. We hypothesized that BBa_K3617001 (this BioBrick) and (andet BioBrick nummer) would both localize to the same membrane but that they would be dissociated in the absence of interleukin 6. In the presence of interleukin 6, we imagined that the extracellular domains of the two parts; IL-6R and sgp130, would associate into a heterotrimer consisting of IL-6, IL-6R and sgp130. Unfortunately, our assays indicated that the BioBricks do not work together as intended.	+	==Biosensor assays==
		+	To test the functionality of the part, it was stably transformed into chromosome x site 3 of S. cerevisiae and constitutively expressed by the pTDH3 promoter together with BBa_K3617001. The latter was under constitutive expression by the pPCCW12 promoter. In addition, NanoBit luciferase, which luminesces 100 times brighter than firefly and Renilla luciferase, was also expression under the control of the lexA-VP16 promoter. After growing the cell cultures to an OD600=0,5, the cells were incubated at 30°C with different concentrations of commercially supplied IL-6 for 1, 3, 14, and 22 hours. Proteins were extracted from the cell cultures using YeastBuster, an industrial protein extraction reagent, and a luminescence assay was performed in order to analyze luciferase expression (Figure 3a & 3b)
		+
		+	[[Image:T--UCopenhagen--results-IL6assay.jpg|700px|thumb|center|<p align="justify"> '''Figure 3a: <b> Il-6 luciferase assay. '''</p>]] </b> Cells expressing BBa_K3617000, BBa_K367001, and luciferase under control of the LexA-VP16 promoter, were induced for varying amount of time with different concentrations of IL-6. Proteins were subsequently extracted, and luminescence measured in order to evaluate luciferase expression.
−	<!-- Add more about the biology of this part here	+	[[Image:T--UCopenhagen--results-IL10assay.jpg|700px|thumb|center|<p align="justify"> '''Figure 3b: <b> Il-10 split ubiquitin biosensor luciferase assay. '''</p>]] </b> No correlation between IL-6 concentration and luminescence intensity was observed at any time point. This indicates that the biosensor does not work as intended for the concentrations and experimental conditions of the experiment. A similar assay was performed with the IL-10 biosensor strain also developed by the UCopenhagen 2020 team. Compared with the IL-10 biosensor, the amount of luminescence was between 3-10 times higher at all concentrations and incubation times. This suggests that the two extracellular domains have an affinity towards each other even without IL-6. As a result, this also implies that the two proteins produced from BBa_K3617000 and BBa_K3617001 localize to the same subcellular compartment(s). The high amount of luminescence may also be caused by partial degradation of BBa_K3617001, leading to release of lexA-VP16. This could be examined by expressing BBa_K3617001 and reporter gene together, without BBa_K361700. Alternatively, a western blot with primary antibody against GFP could be used on GFP-fusion constructs.
−	===Usage and Biology===	+
−		+
−	<!-- -->	+
−	<span class='h3bb'>Sequence and Features</span>	+
−	<partinfo>BBa_K3617001 SequenceAndFeatures</partinfo>	+
−	<!-- Uncomment this to enable Functional Parameter display	+	<!-- Uncomment this to enable Functional Parameter display
	===Functional Parameters===		===Functional Parameters===
−	<partinfo>BBa_K3617001 parameters</partinfo>	+	<partinfo>BBa_K3617000 parameters</partinfo>
	<!-- -->		<!-- -->
		+
		+	==References==
		+	<p>[1] Johnsson, Nils, and Alexander Varshavsky. 1994. “Split Ubiquitin as a Sensor of Protein Interactions in Vivo.” <i> Proceedings of the National Academy of Sciences of the United States of America. </i> https://doi.org/10.1073/pnas.91.22.10340.
		+	</p>
		+	<p>[2] Vollmer, Petra, Birgit Oppmann, Nicole Voltz, Martina Fischer, and Stefan Rose-John. 1999. “A Role for the Immunoglobulin-like Domain of the Human IL-6 Receptor: Intracellular Protein Transport and Shedding.” <i> European Journal of Biochemistry. </i> https://doi.org/10.1046/j.1432-1327.1999.00511.x. </p>
		+	<p>[3] Zhang, Jian Guo, Catherine M. Owczarek, Larry D. Ward, Geoffrey J. Howlett, Louis J. Fabri, Bronwyn A. Roberts, and Nicos A. Nicola. 1997. “Evidence for the Formation of a Heterotrimeric Complex of Leukaemia Inhibitory Factor with Its Receptor Subunits in Solution.” <i> Biochemical Journal. </i> https://doi.org/10.1042/bj3250693. </p>

---

Latest revision as of 01:52, 28 October 2020

sgp130(D1-D3)-Cub

This biobrick is a part of a two-protein system in Saccharomyces cerevisiae, which is designed for detection of human interleukin-6 and transduction of the signal by means of a reconstituted ubiquitin. Development of split-ubiquitin as a tool for study of protein-protein interactions in vivo was first published in 1994 and has been an essential feature in biologists’ toolbox ever since (source: https://www.pnas.org/content/pnas/91/22/10340.full.pdf). A specific mutation in the N-terminal part protects it from binding spontaneously to the C-terminal part. However, re-association can be facilitated by binding of a pair of proteins to which the ubiquitin parts are fused. Human signal transducer gp130 was expressed in yeast for the first time in 1997 and further improvements paved the way to our own chimeric transmembrane proteins (source: https://pubmed.ncbi.nlm.nih.gov/9271090/).

Sequence and Features

From N- to C-terminal the protein includes; an endoplasmic reticulum import signal peptide from the S. cerevisiae cell wall integrity and stress response component 1 (Wsc1), The first three domains of the soluble isoform of human interleukin-6 co-receptor, soluble glycoprotein 130 (sgp130). The transmembrane domain from Wsc1, as well as the C-terminal part of the split version of ubiquitin and the synthetic transcription factor LexA-VP16. LexA is a DNA binding domain from Escherichia Coli and VP16 is a transcriptional activation domain from Herpes simplex virus Type 1. Together LexA-VP16 functions as an orthogonal transcription factor in S. cerevisiae. Between the extracellular sgp130 domains and the transmembrane domain, a (2x)GGGGS-linker was added. Between the transmembrane domain and the C-terminal split ubiquitin domain, two basic amino acids (KR), and the (2x)GGGGS-linker was added.

Sequence optimization

The sequence was codon-optimized for S. cerevisiae. Recognition sequences for SpeI, XbaI, NotI, EcoRI, PstI were avoided to follow RFC10 standard.

Structure and function

BBa_K361701 (marked in red) is designed to locate to the plasma membrane. Upon IL-6 binding it associates with BBa_K3617000 (marked in yellow), forming a trimeric complex. Following extracellular binding, the two intercellular parts of ubiquitin (C-ub and N-ub) come together forming a full-length ubiquitin. This is then cleaved by a deubiquitinase, triggering the release of the LexA-VP16 synthetic transcription factor.

BBa_K361701 is designed to work together with BBaK3617000 and constitute a functional human IL-6 receptor. BBaK3617001 possesses domains 1-3 out of the 6 extracellular domains of the IL-6 co-receptor soluble glycoprotein 130 (sgp130), the C-terminal part of split-ubiquitin, and the LexA-VP16 synthetic transcription. The synthetic transcription factor is a fusion of the DNA binding domain of the LexA transcription factor from Escherichia coli, and an activation domain from the herpes simplex virus transcriptional regulatory protein VP16. LexA-VP16 is often used in yeast 2 hybrid assays as it does not affect endogenous S. cerevisiae genes, and therefore provide orthogonality. In the presence of interleukin-6, the extracellular domains of BBa_K3617000 and BBa_K3617001 (IL-6R and sgp130) associate, forming a heterotrimer consisting of IL-6, IL-6R, and sgp130. The trimerization causes intracellular complementation of the two ubiquitin parts allowing for recognition by an endogenous deubiquitinating enzyme, which facilitates releases of the transcription factor. The transcription factor then relocates to the nucleus and activates expression of a reporter gene (Figure 1).

Confocal flourescence microscopy

In order to investigate the cellular localization of our protein, superfolder green fluorescent protein was fused to the C-terminal end of the protein. Following expression of our new fusion construct, the cells were observed with confocal fluorescence microscopy for visualization.

The majority of investigated cells had one or more fluorescent aggregates. These aggregates were predominantly positioned between the nucleus and the plasma membrane, which could indicate the formation of inclusion bodies. For some cells, the fluorescence signal accumulated close to the nucleus. A possible explanation could be that the protein may be stuck in the Golgi apparatus. Previous studies by Vollmer et al. (1999) have shown that removing the N-terminal Ig-like domain of the IL-6 receptor leads to retention of the protein in the secretory pathway. To circumvent this localization issue, one could add back the N-terminal Ig-domain of the IL6-R.

Biosensor assays

To test the functionality of the part, it was stably transformed into chromosome x site 3 of S. cerevisiae and constitutively expressed by the pTDH3 promoter together with BBa_K3617001. The latter was under constitutive expression by the pPCCW12 promoter. In addition, NanoBit luciferase, which luminesces 100 times brighter than firefly and Renilla luciferase, was also expression under the control of the lexA-VP16 promoter. After growing the cell cultures to an OD600=0,5, the cells were incubated at 30°C with different concentrations of commercially supplied IL-6 for 1, 3, 14, and 22 hours. Proteins were extracted from the cell cultures using YeastBuster, an industrial protein extraction reagent, and a luminescence assay was performed in order to analyze luciferase expression (Figure 3a & 3b)

Cells expressing BBa_K3617000, BBa_K367001, and luciferase under control of the LexA-VP16 promoter, were induced for varying amount of time with different concentrations of IL-6. Proteins were subsequently extracted, and luminescence measured in order to evaluate luciferase expression. No correlation between IL-6 concentration and luminescence intensity was observed at any time point. This indicates that the biosensor does not work as intended for the concentrations and experimental conditions of the experiment. A similar assay was performed with the IL-10 biosensor strain also developed by the UCopenhagen 2020 team. Compared with the IL-10 biosensor, the amount of luminescence was between 3-10 times higher at all concentrations and incubation times. This suggests that the two extracellular domains have an affinity towards each other even without IL-6. As a result, this also implies that the two proteins produced from BBa_K3617000 and BBa_K3617001 localize to the same subcellular compartment(s). The high amount of luminescence may also be caused by partial degradation of BBa_K3617001, leading to release of lexA-VP16. This could be examined by expressing BBa_K3617001 and reporter gene together, without BBa_K361700. Alternatively, a western blot with primary antibody against GFP could be used on GFP-fusion constructs.

References

<p>[1] Johnsson, Nils, and Alexander Varshavsky. 1994. “Split Ubiquitin as a Sensor of Protein Interactions in Vivo.” Proceedings of the National Academy of Sciences of the United States of America. https://doi.org/10.1073/pnas.91.22.10340.

[2] Vollmer, Petra, Birgit Oppmann, Nicole Voltz, Martina Fischer, and Stefan Rose-John. 1999. “A Role for the Immunoglobulin-like Domain of the Human IL-6 Receptor: Intracellular Protein Transport and Shedding.” European Journal of Biochemistry. https://doi.org/10.1046/j.1432-1327.1999.00511.x.

[3] Zhang, Jian Guo, Catherine M. Owczarek, Larry D. Ward, Geoffrey J. Howlett, Louis J. Fabri, Bronwyn A. Roberts, and Nicos A. Nicola. 1997. “Evidence for the Formation of a Heterotrimeric Complex of Leukaemia Inhibitory Factor with Its Receptor Subunits in Solution.” Biochemical Journal. https://doi.org/10.1042/bj3250693.