Oxidative stress produced in response to infection or sterile injury activates the innate immune response. We found that extracellular vesicles (EVs) isolated from the plasma of patients with rheumatoid arthritis or secreted from cells subjected to oxidative stress contained oxidized phospholipids that stimulated cells expressing Toll-like receptor 4 (TLR4). EVs from healthy subjects or reconstituted synthetic EVs subjected to limited oxidation gained the ability to stimulate TLR4-expressing cells, whereas prolonged oxidation abrogated this property. Furthermore, we found that 15-lipoxygenase generated hydro(pero)xylated phospholipids stimulated TLR4-expressing cells. Molecular modeling suggested that the mechanism of activation of TLR4 by oxidized phospholipids in EVs was structurally similar to that of the TLR4 ligand lipopolysaccharide (LPS). This was supported by experiments showing that EV-mediated stimulation of cells required MD-2, that mutations that block LPS binding to TLR4 abrogated the stimulatory effect of EVs, and that EVs induced TLR4 dimerization. On the other hand, analysis of gene expression profiles showed that genes encoding factors that resolve inflammation were more abundantly expressed in responses to EVs than in response to LPS. Together, these data suggest that EVs act as an oxidative stress-induced endogenous danger signal that underlies the pervasive role of TLR4 in inflammatory diseases.
COBISS.SI-ID: 5706266
The mechanism of prion protein (PrP) conversion, the key event in prion diseases, is still not understood. We investigated how perturbations of interactions between the subdomains β1-α1-β2 and α2-α3 affect PrP conversion. In vitro fibrillization and biophysical methods were used to relate mouse PrP conversion kinetics to thermodynamic stability. We show that pathologic mutations H187R and E196K destabilize PrP and accelerate fibrillization. At acidic pH, the major contribution to the destabilization of PrP comes from the protonation of histidine 187 because its replacement by tyrosine led to more stable protein with slower fibrillization. Furthermore, we show that the introduction of a novel histidine residue into the subdomain interface (F198H) acts as a pH-inducible switch that promotes conversion upon histidine protonation, whereas this effect is not observed when His residue is introduced at the protein surface (Y155H). We observed a strong correlation between the stability of native structure and kinetics of fibrillization of PrP variants. Our results show that pathologic mutations promote subdomain separation and suggest that stabilization of the native structure might be a viable strategy for the development of novel therapeutics for prion diseases.
COBISS.SI-ID: 5610266
The high mutation rate of the human immunodeficiency virus type 1 (HIV-1) virus is a major problem since it evades the function of antibodies and chemical inhibitors. Here, we demonstrate a viral detection strategy based on synthetic biology principles to detect a specific viral function rather than a particular viral protein. The resistance caused by mutations can be circumvented since the mutations that cause the loss of function also incapacitate the virus. Many pathogens encode proteases that are essential for their replication and that have a defined substrate specificity. A genetically encoded sensor composed of a fused membrane anchor, viral protease target site, and an orthogonal transcriptional activator was engineered into a human cell line. The HIV-1 protease released the transcriptional activator from the membrane, thereby inducing transcription of the selected genes. The device was still strongly activated by clinically relevant protease mutants that are resistant to protease inhibitors. In the future, a similar principle could be applied to detect also other pathogens and functions.
COBISS.SI-ID: 5607450