It was a tough ride, but I’d take it again in a heartbeat. The only thing I’d change? Embracing every turn as an opportunity. Pursuing ideas is a privilege (yes, I know that's controversial), but if we never dare to "offend," life would be far too boring. #PhD

1️⃣7️⃣ Engineering a Chimeric LPS-Binding Protein
We fused TIRAP’s phosphoinositide-binding domain to caspase-11’s CARD. The aligned TIRAP-CARD bound LPS and aggregated. This supports electrostatic interactions as key for CARD-lipid binding. #Caspase11

1️⃣6️⃣ Caspase-11 Binds Phosphorylated Lipids
Caspase-11’s N-terminal positive charges suggest aggregation depends on negatively charged headgroups. PIP-strip assays confirmed binding to phosphorylated lipids, including PI(3,4,5)P3 expanding its ligand repertoire. #Caspase11 #Lipid

1️⃣5️⃣ Two Mutations Convert AcCARD into a Dual Receptor
Introducing D20K/E29K into AcCARD converted it from a PGPC receptor to a dual PGPC/LPS receptor, mimicking caspase-11. These mutations enabled LPS-induced aggregation and trypsin
resistance #Caspase11 #ProteinEngineering

1️⃣4️⃣ Engineering an LPS Receptor from AcCARD
By fusing caspase-11’s N-terminal region to AcCARD’s PGPC-binding domain, we created Casp11-AcCARD, which gained LPS-induced aggregation and trypsin resistance. #Immunology #Caspase11 #ProteinEngineering

1️⃣3️⃣ AcCARD is a Selective PGPC Receptor
We identified AcCARD, a CARD-containing protein from Amphilophus citrinellus, which binds PGPC but not LPS. This supports the idea that CARD functions can be selectively engineered to recognize different ligands. #Caspase11

1️⃣2️⃣Binding vs Aggregation
Caspase-11’s CARD uses distinct regions for lipid binding and oligomerization. Phe76 is crucial for binding both LPS and PGPC, but only LPS triggers aggregation. Deletions in residues 10–25 reveal key differences in how these lipids interact. #Caspase11

1️⃣1️⃣ Phe76 is Critical for LPS-Induced Signaling
We reconstituted caspase-11-/- iBMDMs with WT, C254A, and F76E variants. While WT triggered IL-1β and LDH release, F76E showed impaired responses at low LPS doses, confirming Phe76’s role in LPS binding and signaling. #Caspase11

🔟 Acyl Chain Binding is Conserved Across Mammals
PGPC protected 16 of 22 caspase-4 CARDs. Phe45 and Phe76 were enriched in PGPC-protected CARDs, but some protected variants lacked both, suggesting additional binding determinants. #Immunology #Caspase4 #LipidBinding

9️⃣ Acyl Chain Length Determines Binding
To examine the role of acyl chain length, we tested LysoPCs (6–18 carbons). Only LysoPCs with ≥14 carbons protected caspase-11. This protective effect also occurred with the CARD, similar to LPS and PGPC interactions. #Immunology #Caspase11 #LipidBinding

8️⃣ Caspase-11 Interacts with Diverse Lipid Headgroups
We tested 16-carbon monoacylated lipids with various negatively charged headgroups. Most protected caspase-11 from trypsin digestion, suggesting caspase-11 interacts broadly with monoacylated lipids.

7️⃣ Mapping Ligand Protection Sites
Mass spectrometry of caspase-11 after trypsin digestion showed that the 20 kDa fragment lacked peptides before K62, while the 35 kDa band retained the full N-terminal sequence. K62, located in the CARD, is protected upon lipid binding. #Caspase11

6️⃣ Trypsin Resistance Reveals Ligand Binding
Unbound caspase-11 degrades to a 20 kDa fragment within 15 min. However, LPS-EB, LPS-RS, and PGPC induce a trypsin-resistant state, yielding a 35 kDa band, suggesting ligand binding alters caspase-11 conformation. #ProteinStructure #LipidBinding

5️⃣ Ligand Binding Affects Protease Sensitivity
CD analysis suggested caspase-11-lipid interactions alter protein conformation, influencing protease accessibility. Limited trypsin digestion revealed ligand-dependent cleavage patterns, detected via immunoblotting. #Immunology #Caspase11

4️⃣ Ligand Binding Alters Caspase-11 Structure
CD analysis revealed a shift toward a more α-helical conformation upon binding non-aggregating ligands LPS-RS and PGPC. This suggests distinct ligand-induced structural changes. #Immunology #Caspase11 #ProteinStructure

3️⃣ Aggregation Isn’t the Whole Story
Lipid size and properties affect caspase-11 aggregation. PGPC, a small monoacylated lipid from oxPAPC, doesn’t aggregate caspase-11 but blocks LPS-induced aggregation, suggesting direct interaction. #Immunology #LPS

2️⃣ Not All LPS Induce Aggregation
Caspase-11 aggregation is often used as a proxy for LPS binding, but not all LPS that bind caspase-11 induce aggregation. For instance, LPS from Rhodobacter sphaeroides doesn’t trigger aggregation but inhibits E. coli LPS-induced aggregation.