2️⃣0️⃣ Possibility 1 - Caspase-11 as a 3D Threat Sensor
Membrane disruptions can expose acyl chains, providing a context-dependent trigger for caspase-11 activation. Caspase-11 may also bind phosphoinositides to guard against bacterial escape. #Immunology #Caspase11 #DAMPs
1️⃣9️⃣ Self-Ligand Detection: Evolutionary Error or Advantage?
Caspase-11’s ability to detect both LPS and self-lipids aligns with Janeway’s PAMP concept. But is self-ligand detection an evolutionary error or an advantage? #Immunology #Caspase11 #DAMPs
1️⃣8️⃣ Caspase-11 Detects Lipids via Two Cues
Caspase-11 recognizes lipids with ≥14C acyl chains and a negatively charged phosphate headgroup. Chimeric proteins converting AcCARD and TIRAP-CARD support this bipartite model. #Immunology #Caspase11 #LipidBinding
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
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
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
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
Finally, our study on caspase-11 and its CARD-recognized molecular pattern is out in Science Advances! A small step, but we hope it opens new directions for studying caspase-11 and its ligands. 🔬🚀 #Immunology #Caspase11 #ScienceAdvances
🔗 science.org/doi/10.1126/sciadv.adt9027
