Update results.md

docs/project/results.md

@@ -4,7 +4,7 @@
 
 After evaluating several well-known human E3 ligases that could be engineered, we decided to work on evolving SIAH1 and SIAH2 (collectively referred to as SIAH1/2 in this text), which are primarily associated with cellular stress response, such as hypoxia [^E3_1][^E3_2]. Both belong to the RING family of E3 ligases and share 86% sequence identity with nearly identical substrate-binding domains[^E3_3]. Their small sizes—282 amino acids for SIAH1 and 324 for SIAH2—are optimal for phage-assisted continuous evolution (PACE), as this allows for efficient expression in E. coli and packaging into M13 phages. Moreover, this small size compared to other E3 ligases also reduces the theoretical library size which is usually advantageous in directed evolution experiments. In general, neither SIAH1 nor SIAH2 are individually characterised to an extent we would like them to be but together they generate a full picture. SIAH2 has already been used successfully in E. coli ubiquitination assays without any partner proteins except E1 and E2, suggesting that additional regulatory proteins aren’t required for its ubiquitination activity [^E3_4]. This streamlines the evolution processes by reducing potential points of failure in the selection system and keeps the plasmid sizes within an acceptable range. Given how similar SIAH1 is to SIAH2 in structure and function, we anticipate it will behave similarly in E. coli as well. While these studies are missing for SIAH1, the binding of SIAH1 to specific peptide sequences has been well-studied via X-ray crystallography [^E3_5][^E3_6], which is missing for SIAH2. Together, the data available for SIAH1/2 offer a solid foundation for targeting new sequences and evolving either protein to recognize non-canonical substrates.
 
-The SIAH family recognizes its targets through a PXAXVXP degron motif [^E3_7], where conserved residues Pro, Ala, Val, and Pro face the SIAH binding pocket (Figure X). Specificity is mainly determined by the Ala and Val residues in positions 3 and 5, as these pockets are too small to accommodate larger side chains [^E3_8]. Additionally, the Pro residue at position 7 interacts with Trp178 in SIAH, contributing further specificity. Among canonical substrates, the VXP sequence is highly conserved [^E3_6][^E3_9], while other residues show more variability, making them prime candidates to alter SIAH1/2 specificity for these positions.
+The SIAH family recognizes its targets through a PXAXVXP degron motif [^E3_7], where conserved residues Pro, Ala, Val, and Pro face the SIAH binding pocket (Figure 1). Specificity is mainly determined by the Ala and Val residues in positions 3 and 5, as these pockets are too small to accommodate larger side chains [^E3_8]. Additionally, the Pro residue at position 7 interacts with Trp178 in SIAH, contributing further specificity. Among canonical substrates, the VXP sequence is highly conserved [^E3_6][^E3_9], while other residues show more variability, making them prime candidates to alter SIAH1/2 specificity for these positions.
 
 <figure markdown>
 ![Figure_E3](https://idec-teams.github.io/2024_Evolution_Suisse/img/result figures/SIAH1_peptidebinding.png)
@@ -30,11 +30,11 @@ Based on these five criteria, we searched published ubiquitination databases (10
 | EGLN1  | 426 | 69-VGP-72, 376-VQP-379* | K256 | SIAH1/2 |
 | ɑ-Synuclein  | 140  | 118-VDP-120 | K(6,10,12,21,23,32,34) | SIAH1/2 |
 
-* For EGLN1 accurate degron was not identified, though two VXP motifs exist of which at least one should act as SIAH1/2 degron.
+*For EGLN1 accurate degron was not identified, though two VXP motifs exist of which at least one should act as SIAH1/2 degron.
 
 ɑ-Synuclein is well known for causing Parkinson's disease (PD) and multiple systems atrophy (MSA). Therefore, optimization of SIAH1/2 binding to ɑ-Synuclein could be an attractive alternative evolution strategy in case reprogramming SIAH1/2 specificity  does not work as planned.
 
-NLRP3 was selected as an evolutionary target, for its mentioned clinical relevance in hepatic  and neurodegenerative diseases. Furthermore, NLRP3 is an attractive target as it contains two VXP motifs (at positions 200 and 707), located in disordered regions on the surface of NLRP3 (Figure). This motif seems to be highly conserved among canonical SIAH1/2 substrates. Therefore, we assume our target choice is limited to proteins naturally displaying this motif on its surface, while other residues involved in binding could be changed. This would allow an evolution of SIAH1/2 to adapt for recognizing different VXP surrounding residues, while retaining the conserved VXP binding motif. Furthermore, lysins are found within the VXP surrounding residues, which are needed for ubiquitination. Remarkably, one of these lysins (K689) is natively polyubiquitinated and leads to the canonical degradation of NLRP3 (Liang, Damianou, Di Daniel, & Kessler, 2021), hence suggesting that polyubiquitination of K689 is a viable strategy of inducing NLRP3 degradation. With 1036 amino acids and its potential for oligomerization, NLRP3 could constrain our evolutionary system. Thus two peptide fragments of NLRP3 containing the VXP motif as well as surrounding residues were incorporated in the selection system.
+NLRP3 was selected as an evolutionary target, for its mentioned clinical relevance in hepatic  and neurodegenerative diseases. Furthermore, NLRP3 is an attractive target as it contains two VXP motifs (at positions 200 and 707), located in disordered regions on the surface of NLRP3 (Figure 2). This motif seems to be highly conserved among canonical SIAH1/2 substrates. Therefore, we assume our target choice is limited to proteins naturally displaying this motif on its surface, while other residues involved in binding could be changed. This would allow an evolution of SIAH1/2 to adapt for recognizing different VXP surrounding residues, while retaining the conserved VXP binding motif. Furthermore, lysins are found within the VXP surrounding residues, which are needed for ubiquitination. Remarkably, one of these lysins (K689) is natively polyubiquitinated and leads to the canonical degradation of NLRP3 [^substr_1], hence suggesting that polyubiquitination of K689 is a viable strategy of inducing NLRP3 degradation. With 1036 amino acids and its potential for oligomerization, NLRP3 could constrain our evolutionary system. Thus two peptide fragments of NLRP3 containing the VXP motif as well as surrounding residues were incorporated in the selection system.
 
 <figure markdown>
 ![Figure_nlrp3](https://idec-teams.github.io/2024_Evolution_Suisse/img/result figures/NLRP3_figure.png)
@@ -44,9 +44,10 @@ NLRP3 was selected as an evolutionary target, for its mentioned clinical relevan
 
 ## Development of an E3 ligase PACE evolutionary system
 ### Ubiquitination-dependent selection logic
+
 To evolve the SIAH1/2 E3 ubiquitin ligases using the PACE system, we needed to link the ligase’s activity directly to phage propagation. To achieve this, we utilised a modified T7 bacteriophage RNA polymerase (RNAP) that had been split into two halves. Normally, this split RNAP is inactive unless both halves are brought close together within the cell, forming a complete, functional complex. We designed a system where the RNAP halves would only assemble if the E3 ligase successfully ubiquitinated its target.
 
-In this setup, one half of the RNAP is fused to ubiquitin, while the other half is fused to a substrate that can be ubiquitinated. If SIAH1/2 (E3) ubiquitinates the substrate, the two RNAP halves are brought close enough to combine, forming a complete RNAP capable of transcribing genes. We took advantage of this by placing the gene _gIII_ (coding for pIII, a protein crucial for phage propagation) under the control of a T7 promoter, which only the assembled RNAP can activate. As a result, phages can only propagate if SIAH1/2 effectively ubiquitinates the target, tying the phage’s propagation to the ligase’s activity. Additionally, we also designed a system that could select against specific off-target effects (Figure X). To this end, we would add an additional genetic circuit which ‘punished’ E3 ligases that recognised unwanted targets. Instead of the substrate towards which we want to evolve, we fuse the unwanted protein (dubbed the mock substrate) to the C-terminal  RNAP subunit. This could be the original canonical substrate of the E3 or a protein recognized as an off-target in later stages of the SIAH1/2 evolution. If this mock substrate is recognized by a SIAH1/2 variant it would also trigger the formation of a functioning RNAP through the ubiquitination of the mock substrate. Crucially, the C-terminal RNAP fused to the mock substrate differs from the one present in the positive selection as it does not bind this T7 promoter sequence, but a slightly altered one. This ensures that one can perform the positive and negative selection at the same time. The altered T7 promoter sequence (*PT7; Figure X) is then recognized by the negative selection RNAP which leads to the transcription of a mock _gIII_. Phages that incorporate the encoded protein are essentially unable to infect new cells, leading to the washout of phages carrying SIAH1/2 variants that recognize unwanted substrates.
+In this setup, one half of the RNAP is fused to ubiquitin, while the other half is fused to a substrate that can be ubiquitinated. If SIAH1/2 (E3) ubiquitinates the substrate, the two RNAP halves are brought close enough to combine, forming a complete RNAP capable of transcribing genes. We took advantage of this by placing the gene _gIII_ (coding for pIII, a protein crucial for phage propagation) under the control of a T7 promoter, which only the assembled RNAP can activate. As a result, phages can only propagate if SIAH1/2 effectively ubiquitinates the target, tying the phage’s propagation to the ligase’s activity (Figure 3a). Additionally, we also designed a system that could select against specific off-target effects (Figure 3b). To this end, we would add an additional genetic circuit which ‘punished’ E3 ligases that recognised unwanted targets. Instead of the substrate towards which we want to evolve, we fuse the unwanted protein (dubbed the mock substrate) to the C-terminal  RNAP subunit. This could be the original canonical substrate of the E3 or a protein recognized as an off-target in later stages of the SIAH1/2 evolution. If this mock substrate is recognized by a SIAH1/2 variant it would also trigger the formation of a functioning RNAP through the ubiquitination of the mock substrate. Crucially, the C-terminal RNAP fused to the mock substrate differs from the one present in the positive selection as it does not bind this T7 promoter sequence, but a slightly altered one. This ensures that one can perform the positive and negative selection at the same time. The altered T7 promoter sequence (*PT7; Figure X) is then recognized by the negative selection RNAP which leads to the transcription of a mock _gIII_. Phages that incorporate the encoded protein are essentially unable to infect new cells, leading to the washout of phages carrying SIAH1/2 variants that recognize unwanted substrates.
 
 <figure markdown>
 ![Figure_positive_negative_selection](https://idec-teams.github.io/2024_Evolution_Suisse/img/result figures/E3_selection_V1.png)
@@ -55,7 +56,7 @@ In this setup, one half of the RNAP is fused to ubiquitin, while the other half
 </figure>
 
 ### Assembling the PACE system
-To implement this system, we split the evolution process across three plasmids. The first plasmid is the selection phage (SP), which carries the SIAH1/2 gene and the phage genome but lacks the _gIII_, preventing the phage from propagating without the ligase’s activity. The second plasmid, accessory plasmid 1 (AP1), contains the genes for the E1 and E2 enzymes (which are required for ubiquitination but not normally present in bacteria), the N-terminal half of RNAP fused to ubiquitin, and the gIII controlled by the T7 promoter. The third plasmid, accessory plasmid 2 (AP2), contains the substrate fused to the C-terminal half of RNAP.
+To implement this system, we split the evolution process across three plasmids (Figure 4). The first plasmid is the selection phage (SP), which carries the SIAH1/2 gene and the phage genome but lacks the _gIII_, preventing the phage from propagating without the ligase’s activity. The second plasmid, accessory plasmid 1 (AP1), contains the genes for the E1 and E2 enzymes (which are required for ubiquitination but not normally present in bacteria), the N-terminal half of RNAP fused to ubiquitin, and the gIII controlled by the T7 promoter. The third plasmid, accessory plasmid 2 (AP2), contains the substrate fused to the C-terminal half of RNAP.
 
 This modular system allows us to easily swap out the substrate on AP2, enabling it to be applied to different E3 ligases or substrates. It also supports performing “substrate walks,” a process where we incrementally alter the amino-acid sequence of the substrate recognition motif to shift from a canonical target to a novel target of therapeutic interest. By doing this stepwise, we can control the selection pressure and gradually evolve SIAH1/2 to recognize new substrates.
 
@@ -143,3 +144,4 @@ To this end, we propagated the SIAH1 SP phages in bacterial cells that contain a
 [^E3_8]:Briant DJ, Ceccarelli DF, Sicheri F. I Siah Substrate! Structure. 2006;14: 627–628. doi:10.1016/j.str.2006.03.003
 
 [^E3_9]:House CM, Frew IJ, Huang H-L, Wiche G, Traficante N, Nice E, et al. A binding motif for Siah ubiquitin ligase. Proc Natl Acad Sci USA. 2003;100: 3101–3106. doi:10.1073/pnas.0534783100
+[^substr_1]:Liang Z, Damianou A, Di Daniel E, Kessler BM. Inflammasome activation controlled by the interplay between post-translational modifications: emerging drug target opportunities. Cell Commun Signal. 2021;19: 23. doi:10.1186/s12964-020-00688-6