From b796a17b36562e55098136c9bad88daf70225002 Mon Sep 17 00:00:00 2001 From: KianBV <91744358+KianBV@users.noreply.github.com> Date: Tue, 8 Oct 2024 18:19:05 +0200 Subject: [PATCH] Update results.md --- docs/project/results.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/docs/project/results.md b/docs/project/results.md index e3d26c0..6b50372 100644 --- a/docs/project/results.md +++ b/docs/project/results.md @@ -2,7 +2,7 @@ ## Selection of of the E3 ligase -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. +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 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.