Martinon, D. | bioRxiv Science

bioRxiv2024

Hypothermia protects against ventilator-induced lung injury by limiting IL-1beta release and NETs formation

Although mechanical ventilation is a critical intervention for acute respiratory distress syndrome (ARDS), it can trigger an IL-1{beta}-associated complication known as ventilator-induced lung injury. In mice, we found that LPS and high-volume ventilation, LPS-HVV, leads to hypoxemia with neutrophil extracellular traps (NETs) formation in the alveoli. Furthermore, Il1r1-/- LPS-HVV mice did not develop hypoxemia and had reduced NETs, indicating that IL-1R1 signaling is important for NETs formation and hypoxemia. Therapeutic hypothermia (TH) is known to reduce the release of inflammatory mediators. In LPS-HVV mice, TH (32 {degrees}C body temperature) prevented hypoxemia development, reducing albumin leakage, IL-1{beta}, gasdermin D (GSDMD) and NETs formation. We also observed that LPS-primed macrophages, when stimulated at 32{degrees}C with ATP or nigericin, release less IL-1{beta} associated with reduced GSDMD cleavage. Thus, hypothermia is an important modulating factor in the NLRP3 inflammasome activation, IL-1{beta} release and NETs formation, preventing LPS-HVV-induced acute respiratory failure.

immunology↗

bioRxiv2021

NMR Hydrogen Exchange and Relaxation Reveal Positions Stabilized by p53 Rescue Mutants N239Y and N235K

Inactivation of p53 is found in over 50% of all cancers; p53 disfunction is often caused by a single missense mutation localized in the DNA binding domain (DBD). Rescue mutants N235K and N239Y stabilize and restore function to multiple p53 cancer mutants. Here, we use NMR to compare protein dynamics between WT and rescue mutants to understand the mechanism of stabilization. We measured and compared folding dynamics by calculating protection factors (PFs) from NMR hydrogen exchange rates of backbone amides. We find that both rescue mutants impose a global stabilizing effect that dampens their motions compared to WT DBD, predominantly in the {beta}-sandwich. However, a few regions become more flexible in rescue mutants. Notably, positions that have increased PFs map to cancer mutants rescued by each mutant. We also compared relaxation results to obtain flexibility information in the ps to ns timescale regime. Protein sequence analysis was used to determine the occurrence of these rescue mutants in nature and showed that 235K is found in mice and rats, but there is no evidence of 239Y occurring naturally in any species. Understanding the mechanism by which stabilizing mutants rescue p53 may reveal novel avenues for the development of cancer therapeutics. Our findings suggest that cancer therapeutics aimed at restoring p53 function could consider protein dynamics as a metric of drug efficacy. Statement of SignificanceTwo mutations (N235K and N239Y) within the DNA binding domain of p53 are known to reverse the effects of multiple cancer mutants. However, the mechanism of rescue is not clear since the crystal structures of these mutants are virtually identical to that of the WT. Here we use NMR methods to show that the protein dynamics of the rescue mutants are significantly different compared to WT, allowing us to describe the stability on a per-residue basis. Although these mutations are only four residues apart, they stabilize the structure distinctly in different regions, consistent with the specific cancer mutations they to which they restore function.

biophysics↗