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Membrane lipids and proteins form dynamic domains crucial for physiological and pathophysiological processes, including viral infection. Many plasma membrane proteins, residing within membrane domains enriched with cholesterol (CHOL) and sphingomyelin (SM), serve as receptors for attachment and entry of viruses into the host cell. Among these, human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), use proteins associated with membrane domains for initial binding and internalization. We hypothesized that the interaction of lipid-binding proteins with CHOL in plasma membrane could sequestrate lipids and thus affect the efficiency of virus entry into host cells, preventing the initial steps of viral infection. We have prepared CHOL-binding proteins with high affinities for lipids in the plasma membrane of mammalian cells. Binding of the perfringolysin O domain four (D4) and its variant D4 E458L to membrane CHOL impaired the internalization of the receptor-binding domain of the SARS-CoV-2 spike protein and the pseudovirus complemented with the SARS-CoV-2 spike protein. SARS-CoV-2 replication in Vero E6 cells was also decreased. Overall, our results demonstrate that the integrity of CHOL-rich membrane domains and the accessibility of CHOL in the membrane play an essential role in SARS-CoV-2 cell entry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

NLRP3 inflammasome is a multiprotein platform for the activation of caspase-1. Despite the increasing number of reports linking NLRP3 inflammasome to a variety of diseases, the mechanism behind the NLRP3 activation remains elusive, especially in terms of the early stages which are critical to the NLRP3 inflammasome assembly. In the present study we aimed to determine the minimal oligomerization state required for the NLRP3 inflammasome activation. For this purpose, NLRP3 pyrin domain (NLRP3 PYD ) was fused to various dimerization and trimerization domains. The constructs were expressed under the inducible promoter in mouse macrophages lacking endogenous NLRP3. Dimerization of the NLRP3 PYD either in parallel or in antiparallel orientation was insufficient for the inflammasome activation. Trimerization of the NLRP3 PYD with the foldon domain, however, induced pyroptosis and robust IL-1β maturation, which was caspase-1 dependent. Interestingly, foldon-induced constitutive activation is resistant to inhibition with NLRP3-specific inhibitor MCC950 and does not lead to ASC speck formation. Although we cannot exclude that wild-type NLRP3 forms higher oligomer species similar to NLRP1 or NLRC4, our results clearly demonstrate that efficient IL-1β response can be achieved by the induced trimerization of the NLRP3 PYD domain., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Prion diseases are a group of fatal neurodegenerative diseases caused by scrapie form of prion protein, PrP Sc . Prion protein (PrP) is bound to the cell via glycophosphatidylinositol (GPI) anchor. The role of GPI anchor in PrP Sc replication and propagation remains unclear. It has been shown that anchorless and truncated PrP accelerate the formation and propagation of prions in vivo and further increases the risk for transmission of prion diseases among species. To explain the role of anchorless forms of PrP in the development of prion diseases, we have prepared five C-terminal PrP truncated variants, determined their thermodynamic properties and analyzed the kinetics of conversion into amyloid fibrils. According to our results thermodynamic and kinetic properties are affected both by pH and truncation. We have shown that the shortest variant was the most destabilized and converted faster than other variants in acidic pH. Other variants converted with longer lag time of fibrillization than WT despite comparable or even decreased stability in acidic pH. Our results indicate that even the change in length for 1 amino acid residue can have a profound effect on in vitro conversion., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

In prion diseases cellular prion protein (PrP(C)) undergoes conformational transition into the β-sheet-rich form (PrP(Sc)). PrP(C) consists of the disordered N-terminal part and a C-terminal globular domain containing three α-helices (H1, H2, H3) and an antiparallel beta sheet (B1, B2). B2-H2 loop, which has a focal role in the species barrier, contains the highest density of asparagine (N) and glutamine (Q) residues in the whole sequence. Q/N-rich domains are essential for the conversion of yeast prions. We investigated the role of Q/N residues in the B2-H2 loop in PrP conversion. We prepared mouse PrP mutants with increasing number of consecutive Q/N residues in the B2-H2 loop. Stability of the mutants decreased with the increasing number of inserted glutamines. In vitro conversion of mutants yielded fibrils of similar morphology as the wild-type PrP. Q/N mutants accelerated fibrillization in comparison to the wild-type PrP, with mutant containing the most glutamines having the shortest lag phase. The effect of Q/N residues was specific for the B2-H2 loop and was not due to simple increase in flexibility as the introduction of Gly-Ser or Ala residues slowed the conversion despite their decreased stability. Our results thus suggest that Q/N residues in the B2-H2 loop of PrP promote protein conversion and may represent a link to conversion of Q/N-rich prions., (Copyright © 2011 Elsevier Inc. All rights reserved.)