Team:UCopenhagen/Design
Remember to write about the background first (ie that there even is a conc in sweat that correlates to the amount in blood) wait this should probably be under the project description? I think?
IL-1
As mentioned above, the mechanism of action of IL-1 binding and signaling relies on the association of two or more receptors and the interleukin itself. The receptors in question are the IL-1R and the accessory receptor IL1RAcP. Formation of the heterotrimer and binding of the interleukin results in activation of the pathway in the native setting.
Our first step when working with IL-1 was to look into the IL-1R family. Through our search, we found that there are some receptors that function as inhibitors of the IL-1 system, and that could be more useful in our context compared to the signaling IL-1RI receptor. Particularly, the IL-1RII decoy receptor was of interest to us. The IL-1RII receptor differs from the type 1 receptor in that it lacks an intracellular toll-like receptor essential for normal signal relay, but it has some nice features of interest to us. For example, it has lower affinity for the IL-1 antagonist compared to the type 1 receptor (SOURCE), which is great for us, as it increases the selectivity for the signaling IL-1 molecules. It’s because of this increased sensitivity, and because the intracellular signaling domains are of no importance in our context, we decided to use the IL-1RII extracellularly.
IL-6
IL-10
The receptors for IL-10 form heterotrimers like the receptors for IL-1. Here, the IL-10R type 1 associated with IL-10, and subsequently recruits IL-10R type II. The reason we decided to look into IL-10 is to have a non acute-phase cytokine in our system. This receptor is also much smaller than the previous two receptors, increasing the chance of correct folding in S. cerevisiae, and is located closer to the membrane compared to the IL-6 receptor for instance.
Very good article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4489423/ Source????? Is this it???
Intro middle text:
The extracellular portions of the human interleukin receptors were then fused to a transmembrane domain to secure localization to the membrane. For this, we researched a lot of different endogenous yeast transmembrane domains (hereafter called TMDs) to find the one with the highest predicted localization to the membrane, based on sequence analyses. We ran a sequence analysis on 13 different endogenous single-pass type I transmembrane proteins as part of this endeavor, and using our knowledge of the characteristics of the phospholipid bilayer and transmembrane proteins in general, we came up with our own candidate for a transmembrane protein. This TMD would have hydrophobic amino acid residues pointing “inwards”, with the polar amino acid residues on either side, and tryptophan in-between those areas. Our candidate had the following sequence of amino acids:
IAGIVIGVVLGVIFILIAILFAFW
And proved to have a higher predicted localization to the membrane than the TMDs we compared it to (SOURCE? PIC? DRY LAB?).
As this added a level of unpredictability though, we decided to use the TMD Wsc1 for our project design henceforth, as some of our designs build on papers that use the Wsc1 domain.
The next step was to imitate an intracellular transduction pathway, following the extracellular association of our receptor complexes. As all our interleukin receptors’ normal mechanisms of action build on two non-identical receptors associating, we thought to integrate a similar protein/protein interaction as the key part of our own transduction system. This gave rise to our first design: the split-ubiquitin design.
Ubiquitin
This was the first design we developed, after being introduced to tools for identifying protein-protein interactions by our supervisor. Here we found the split-ubiquitin based Membrane Yeast Two-Hybrid method (MYTH) (SOURCE: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2818708/ ), where two different proteins of interest are fused to one half of a modified ubiquitin molecule each. The modifications made to the two halves of ubiquitin renders them unable to spontaneously bind to each other and reconstitute, without first being brought into close proximity of each other by another protein. This means that if the proteins of interest, that the ubiquitin halves are fused to, have a natural affinity for each other and consequently associate, the ubiquitin halves will also associate on the intracellular side of the membrane and reconstitute, as they’re being brought closer together by the association on the extracellular side. This suits our purposes perfectly, as we already know that the interleukin receptor parts that we’re using have a natural affinity for each other in the native setting (and of course in the presence of the fitting interleukin). Thus, by fusing ubiquitin to each of our receptors, we know that an extracellular association will result in an intracellular reconstitution of ubiquitin.
BOX: Modifying the ubiquitin halves happens through switching amino acid number 13 out from isoleucin to glycine. The C-terminal half (Cub) will then contain 42 aa, while the N-terminal half (Nub) will have a length of 51 aa, following the MYTH system proposed by Snider, J. et al. 2010.
At the same time as we fuse the two halves of ubiquitin to our two receptor proteins, we fuse a transcription factor to the C-terminal part of ubiquitin. As ubiquitin is recognized by deubiquitinating enzymes upon reconstitution, this means that the deubiquitinating enzymes will cleave off the transcription factor bound to ubiquitin, resulting in the release of the free transcription factor into the cytosol and ultimately the cell nucleus, where it can exert its effect.
For our purposes, we’ve using a synthetic transcription factor developed by Dossani, Z. Y. et al. 2018. This consists of the bacterial LexA DNA binding protein, fused with the viral activator domain VP16. A corresponding hybrid promoter with operator regions replaced with sequences that are recognized by LexA is also used, to avoid transcription of yeast-native genes (SOURCE: https://pubmed.ncbi.nlm.nih.gov/29084380/ ).
INSERT: picture of ubiquitin design
However, as mentioned in the project description page, the concentrations of interleukins found in sweat are rather low, and as of now, this design has no real signal amplification step. On the other hand, the close resemblance to the
Under normal circumstances we would’ve tested our design in the wet lab to gauge the importance of amplification at this point, or waited for the dry lab results to see if they confirmed our suspicions, but due to the restrictions in the lab we instead decided to re-think our design from the get-go. This meant that the next step for us would be to make another design, now with preferably more amplification.
INSERT: engineering cycle figure
Transcription
Our intermediate design utilizes the same receptor-system as the ubiquitin-based design, but with few modifications. Thanks to our supervisor’s guidance, we were introduced to another kind of split-protein: the split TEV-protease. This was another method developed by Wehr, M. C. et al. In 2006 to monitor protein-protein interactions. Here, we again have two engineered inactive halves of the TEV-protease, that only regain activity when coexpressed as fusion constructs with interacting proteins (SOURCE: https://www.nature.com/articles/nmeth967 ). Therefore, we again utilize the receptor/TMD domains from the previous designs, but now each of our receptors will be fused to one half of the TEV-protease instead.
BLAHBLAHBLAH write about the linker between the protease and the other shit with how you make it blah blah. From benchling:
The TEV N1a protease design is to have N-TEV with the aa residues 1-118 from the TEV protein, and a C-TEV from 119-242, as laid out by the original designers of the protease (Wehr, M. C. 2006), which will then be fused to the C-terminal ends of the transmembrane proteins. The N-TEV can also be fused to something else on the N-terminal without loss of function. According to a later article by the same author though (Wintgen, J. P. et al. 2017) there's also a substitution at residue 219, where a Serine is changed out for a Proline (Ser --> Pro, also covered by Tropea, J. E. 2009). Additionally, an optimization can be made where the protease is truncated after amino acid 221 to remove the inhibitory C-terminal tail, which blocks the active site of the TEV protease, and TEVs up to 231 aa are sold on genscript.
In parallel, we also express the Wsc1 TMD, which will be sorted and localized to the membrane. To this TMD, we’ll fuse the same transcription factor from the previous design (LexA-VP16), and use the recognition sequence for the TEV-protease as the linker between the two. This means that the TEV-protease, upon reconstitution, will be able to cleave the transcription factor and free it into the cytosol. In theory, the TEV-protease will be able to cut many transcription factors loose, meaning that one interleukin (by extension of the association of our two receptors) will result in the cleavage of multiple transcription factors and thus an amplification of the signal.
Again, this extra amplification step made us hopeful, but in order to achieve the highest level of amplification possible we moved on to other venues.
G alpha
Our last and most ambitious design hinges on hijacking the pheromone pathway in yeast. We had no doubt that this would give us the most amplification, which is especially important in our case given the low concentrations of interleukins in sweat (SOURCE????). The pheromone pathway is XXXXX EXPLAIN HERE OR NO?
In order to hijack the pheromone pathway, we initially set out to understand how the Ste5 scaffold protein was recruited to the membrane by the beta/gamma complex. Soon though, we saw that the interaction between the beta/gamma complex and the scaffold was more complicated than previously thought, and introducing changes to this step seemed risky (SOURCE about how we don’t know what beta/gamma does exactly here). Since the exact mechanism by which the beta/gamma complex recruits Ste5 was unknown to us, we decided to take a more conservative approach instead of changing beta/gamma for another recruiting maneuvre. Here, we finally thought to introduce an inhibitory sequence on beta, that could be removed at will, so as to keep the beta/gamma complex as the recruiting element. This inhibitory sequence would then, building onto the previous design, contain the TEV protease cleavage site, so an extracellular signal could trigger the TEV protease to cut off the inhibitory sequence and start the signaling. The next step from here would be to design such an inhibitory sequence, and through talking to our supervisor, we agreed that we should stay in the same conservative vein and look at natural inhibitors of the signaling. Here, the alpha subunit of the G protein was an obvious choice, as the alpha subunit binds to beta/gamma in the absence of an extracellular signal, and hinders them from recruiting Ste5 and completing the pheromone pathway signaling. As a natural inhibitor, G alpha was a great choice for us.
The next step from here was to find places to insert the TEV protease cleavage sites in G alpha. Our goals when doing this was to have a G alpha that
1) could keep its GTPase activity, so it could be used in other contexts in the future
2) could bind to and exert an inhibitory function on the beta/gamma complex’s ability to recruit Ste5
3) could, once cut by the TEV protease in the presence of a signal, dissociate from beta/gamma again, enabling regular signaling and recruitment of Ste5
For this, we had many approaches. Our first thought was to look at the yeast G alpha’s sequence, and change very few amino acids in places that already resembled the TEV recognition site. During this first iteration, we used the following recognition site:
EXLYΦQ\φ where X is any residue, Φ is any large or medium hydrophobic and φ is any small hydrophobic or polar residue (S/G; https://pubmed.ncbi.nlm.nih.gov/15477088/).
(INSERT PRELIMINARY RESULTS?)
However, we found that introducing different amino acids (albeit with the same properties) in the different cleavage sites would prove to make subsequent wet lab troubleshooting harder, as well as change the dry lab output in unreasonable ways, as the TEV protease would then have different efficiencies depending on the used recognition site (INSERT SOURCE DAVID’S ARTICLE FROM BENCHLING). In order to limit the amount of unknowns, and to have the same affinity of our TEV protease to our recognition site, we opted to use the same recognition site everywhere; ENLYFQG.
Using mutagenesis results from uniprot and other literature (SOURCE???? May be weird, I’m sleepy) we found the residues that have been found to give constitutive activity in the pheromone pathway as a result of alpha not being able to bind to beta/gamma (SOURCE insert Gladue, D. P. 2008) etcetera, and through extensive computer modeling described under our dry lab section, we eventually landed on some G alpha mutants where the TEV recognition site is inserted into tactical, most promising areas that should not interfere with normal G alpha function.
So, to summarize, our last and final design entails:
1) A signaling interleukin reaches our two receptors
2) The receptors associate extracellularly, giving rise to the intracellular complementation of a split TEV protease
3) The now active TEV protease can cut a mutant G alpha protein (LINK TO ENGINEERING SUCCESS PAGE!!!) into smaller peptide fragments, that’ll dissociate from the beta/gamma complex
4) The beta/gamma complex can exert its recruiting function on the Ste5 scaffold protein and recruit it to the membrane, thus triggering the pheromone cascade
5) In the end, our modified transcription factor, LexA-VP16, will bind to X promoter and result in the transcription and translation of our reporter protein
This last and final design has a lot of strengths compared to the two prior designs, but also some drawbacks....
HOW DO WE SAY THAT WE USED THE ENGINEERING CYCLE PERFECTLY, AND WHERE DO I PUT MY FIGURE???????
----- (notes)
Remember to say that our idea was to put an inhibitory sequence on the beta because we don’t know what beta/gamma does to recruit the Ste5 protein thing (source!!!), and then thanks to Karel we know something now.
Remember to say that in order to limit the amount of unknowns (different affinity sites and cleavage) we used the same recognition site.
So... what are you doing, exactly?
How nice of you to ask! Our aim is to ease the lives of people with chronic inflammatory diseases by providing them with an easy way to monitor their inflammation level. For this, we’ve developed a yeast-based biosensor located within a patch that’ll sit on the patient’s skin, where it’ll continuously gather sweat to analyze. If the yeast detects the presence of a certain interleukin in the sweat, it’ll start a signaling cascade ultimately resulting in the production of a pigment, which will reflect the amount of interleukins found in the sweat.
Venturing deeper into this page will show you how we hope to achieve this by developing our very own receptor-systems!
Interleukin Receptors
Most interleukin receptors fall under the category of Receptor Tyrosine Kinases (RTKs), and thus rely on the dimerization of at least one primary receptor and an accessory receptor . As similar RTKs aren’t normally found in S. Cerevisiae , we hypothesize that the intracellular signal transduction native to the RTKs will be hard to replicate in our yeast cells. Instead, we’ll attempt to design our own interleukin receptors, while taking advantage of the natural affinity the two extracellular portions of the human interleukin receptors have for each other.
This year, we’re simultaneously looking into three different interleukin receptors, each with their own unique properties.
IL-1
IL-1
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IL-6
IL-6
Lots of good things here
IL-10
IL-10
Oh nono da da
Three Tiers of Biosensor Designs
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Ease Pease
Lorem Ipsum is simply dummy text of the printing and typesetting industry. Lorem Ipsum has been the industry's standard dummy text ever since the 1500s, when an unknown printer took a galley of type and scrambled it to make a type specimen book. It has survived not only five centuries, but also the leap into electronic typesetting, remaining essentially unchanged. It was popularised in the 1960s with the release of Letraset sheets containing Lorem Ipsum passages, and more recently with desktop publishing software like Aldus PageMaker including versions of Lorem Ipsum.
Contrary to popular belief, Lorem Ipsum is not simply random text. It has roots in a piece of classical Latin literature from 45 BC, making it over 2000 years old. Richard McClintock, a Latin professor at Hampden-Sydney College in Virginia, looked up one of the more obscure Latin words, consectetur, from a Lorem Ipsum passage, and going through the cites of the word in classical literature, discovered the undoubtable source. Lorem Ipsum comes from sections 1.10.32 and 1.10.33 of "de Finibus Bonorum et Malorum" (The Extremes of Good and Evil) by Cicero, written in 45 BC. This book is a treatise on the theory of ethics, very popular during the Renaissance. The first line of Lorem Ipsum, "Lorem ipsum dolor sit amet..", comes from a line in section 1.10.32.
Bruh
Lots of good things here
Heyyyy
Oh nono da da
- Zola H. Analysis of receptors for cytokines and growth factors in human disease. Dis Markers. 1996;12(4):225-240. doi:10.1155/1996/807021 ↩
- Gunde T, Barberis A. Yeast growth selection system for detecting activity and inhibition of dimerization-dependent receptor tyrosine kinase. Biotechniques. 2005;39(4):541-549. doi:10.2144/000112011↩