Difference between revisions of "Part:BBa K3617000"

Latest revision as of 02:23, 28 October 2020

sIL-6R-Nub

This biobrick is a part of a 2-protein system that is designed for detection of human interleukin-6 and transduction of the signal by means of a reconstituted ubiquitin. It is mainly comprised of the extracellular part of the human soluble interleukin-6 receptor and of the N-terminal part of split 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 [1]. 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 split-ubiquitin parts are fused. Human interleukin-6 receptor was expressed in Saccharomyces cerevisiae for the first time in 1996, and further improvements have paved the way for our own chimeric transmembrane proteins [2].

Sequence and Features

This biobrick consists of multiple parts; An endoplasmic reticulum import signal peptide from the S. cerevisiae cell wall integrity and stress response component 1 (Wsc1) . The second and third domain of human soluble interleukin-6 receptor subunit alpha (sIL-6R). The transmembrane domain of Wsc1, as well as the N-terminal part of the split version of ubiquitin, constituting the first 34 amino acids of ubiquitin. The ubiquitin domain possesses an Ile13Gly mutation that inhibits spontaneous association of the two split protein halves, by reducing their affinity to each other. A flexible 2XXGGGGS linker was used to link the sIL-6R domains to the transmembrane domain [3], whereas a 2XGGGGS linker was used to link the transmembrane domain to the N-terminal split-ubiquitin.

Sequence optimization

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

Structure and function

This part is designed to function as a human IL-6 receptor together with BBaK3617001. Compared to the human IL-6 receptor, only two out of three extracellular domains are included, and the intracellular domains are replaced with the N-terminal part of split-ubiquitin. The signal peptide and transmembrane domain constitute the backbone of the modular framework of the UCopenhagen 2020 team (CIDosis). These are used for localizing receptor proteins for interleukin-1, interleukin-6 and interleukin-10 at the plasma membrane of S. cerevisiae as type I single pass transmembrane proteins. As a type I transmembrane protein, the soluble interleukin receptor domains localizes extracellularly while the N-terminal part of the split protein is intracellular. Ivanusic et al. (citation) introduced the use of the signal peptide and transmembrane domain in a split-ubiquitin system for screening for protein-protein interactions at the plasma membrane in S. cerevisiae.

Figure 1: General system design of using complementation for intracellular signaling. The interchangeable, extracellular receptor may be any receptor of interest that either forms a homo- or heteromer, thereby leading to complementation of the interchangeable intercellular domains and downstream activation.

BBa_K3617000 consists of two out of three extracellular domains from the human IL-6 receptor, the transmembrane domain of Wsc1, and the N-terminal part of split-ubiquitin. Wsc1 signal peptide is used for proper localization to the plasma membrane (Figure 1). BBa_K3617000 constitutes a type I single pass transmembrane protein. As a type I transmembrane protein, the soluble interleukin receptor domains localizes extracellularly while the N-terminal part of the split protein is intracellular. [4] introduced the use of the signal peptide and transmembrane domain in a split-ubiquitin system for screening for protein-protein interactions at the plasma membrane in S. cerevisiae.

Figure 2: Mechanism for signal transduction by IL-6 receptor proteins. BBa_K361700 (marked in yellow) is designed to locate to the plasma membrane. Upon IL-6 binding it associates with BBa_K3617001 (marked in red), 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 release of the LexA-VP16 synthetic transcription factor.

The two fibronectin type III soluble interleukin-6 receptor subunit alpha domains mediates the binding of the receptor to interleukin-6, as demonstrated on figure 2. The outer Ig-like domain of the receptor mediates other functions of the receptor.[2]

BBa_K3617001 is designed to work together with BBa_K3617000 and constitute a functional human IL-6 receptor. BBa_K3617001 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 E. 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 deubiquitinizing enzyme, which facilitates releases of the transcription factor. The transcription factor then relocates to the nucleus and activates expression of a reporter gene (Figure 2).

Confocal flourescence microscopy

In order to investigate the cellular localization of our protein, superfolding 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.

Figure 2a: Confocal fluorescence microscopy of sIL-6R-Nub-sfGFP. 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.

Figure 2b: Confocal fluorescence microscopy of sIL-6R-Nub-sfGFP. Pictures were taken with a 150 μm pinhole. Here, the inclusion bodies are also evident in the brightfield image.

Figure 2c: Confocal fluorescence microscopy of sIL-6R-Nub-sfGFP. Pictures were taken with a 150 μm pinhole. Fluorescence can be observed in inclusion bodies and very faintly at the plasma membrane and around the nucleus.

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) that 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 4a & 4b).

Figure 4a: IL-6 Luciferase assay. 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.

Figure 4b: IL-10 split-ubiquitin biosensor luciferase assay.

This suggests that the two extracellular domains have an affinity to each other even without the presence of IL-6. This further implies that the proteins produced from BBa_K3617000 and BBa_K3617001 are localized at the same subcellular compartment(s). The high amount of luminescence may also be caused by partial degradation of BBa_K3617001 may also be partially degraded, after which the synthetic transcription factor, lexA-VP16, is released and re-localizes to the nucleus. This could be verified by integrating only the BBa_K3617001 and reporter gene into S. cerevisiae and performing an additional luciferase assay. Alternatively, one may perform a western blot with primary antibody against GFP on the strain used for the localization assays.

References

[1] Johnsson, N., & Varshavsky, A. (1994). Split ubiquitin as a sensor of protein interactions in vivo. Proceedings of the National Academy of Sciences of the United States of America, 91(22), 10340–10344. https://doi.org/10.1073/pnas.91.22.10340 https://www.pnas.org/content/pnas/91/22/10340.full.pdf

[2] Vollmer, P., Oppmann, B., Voltz, N., Fischer, M., & Rose-John, S. (n.d.). 438±446 (1999) q FEBS 1999. In Eur. J. Biochem (Vol. 263). https://www.sciencedirect.com/science/article/abs/pii/S0022175996001639

[3] Chen, X., Zaro, J. L., & Shen, W. C. (2013). Fusion protein linkers: Property, design and functionality. In Advanced Drug Delivery Reviews (Vol. 65, Issue 10, pp. 1357–1369). https://doi.org/10.1016/j.addr.2012.09.039

[4 ]Ivanusic, D., Heinisch, J. J., Eschricht, M., Laube, U., & Denner, J. (2015). Improved split-ubiquitin screening technique to identify surface membrane protein-protein interactions. BioTechniques, 59(2), 63–73. https://doi.org/10.2144/000114315