1,720,972 research outputs found
Phospholipases A2: structure and function
No full textPhospholipases A2 (PLA2) are an example of peripheral membrane proteins that must first bind to the phospholipid interface to allow phospholipid hydrolysis to occur. The interfacial (membrane) binding step plays a crucial role in the biological function of the enzyme and membrane affinity may be determined both by the phospholipid composition of the membrane and the properties of the interfacial binding surface of the protein. There are now three major categories of these enzymes, secreted PLA2 (sPLA2), cytosolic PLA2 (cPLA2) and Ca2+-independent PLA2 (iPLA2). The structure and function of each category is discussed highlighting how both membrane binding and phospholipid substrate specificity may contribute to the overall functions of these enzymes
Age, obesity and hyperglycaemia: Activation of innate immunity initiates a series of molecular interactions involving anionic surfaces leading to COVID-19 morbidity and mortality
No full textObesity and type 2 diabetes are major factors in COVID-19 causing a progression to excessive morbidity and mortality. An important characteristic of these conditions is poor glycaemic control leading to inappropriate chemical reactions and the production of glycated proteins in which positively charged lysine and arginine residues are neutralised. We propose that this protein glycation primes the inflammatory system as the presence of aspartate and glutamate residues in any glycated zwitterionic protein will thus increase its anionic characteristics. As a result, these macromolecules will be recognised by the innate immune system and identified as originating from infection or cell damage (sterile inflammation). Many proteins in the body exist to non-specifically target these anionic macromolecules and rely heavily on positively charged (cationic) binding-sites to produce a relatively non-specific interaction as the first step in the body's response. Proteins involved in this innate immunity are collectively referred to as damage associated molecular pattern molecules or pathogen associated molecular pattern molecules. A crucial player in this process is RAGE (Receptor for Advanced Glycation End products). RAGE plays a central role in the inflammatory response and on ligand binding stimulates many aspects of inflammation including the production of the key inflammatory mediator NF-κB, and the subsequent production of inflammatory cytokines. This process has the potential to show a positive feedback loop resulting in a dramatic response within the tissue. We propose that protein glycation primes the inflammatory system by generating negatively charged surfaces so that when a SARS-Cov-2 infection occurs within the lung the further release of negatively-charged macromolecules due to cell damage results in a potentially catastrophic inflammatory response resulting in the cytokine storm associated with COVID-19 morbidity and mortality. That part of the population who do not suffer from inflammatory priming (Phase 1), such as the young and the non-obese, should not be subjected to the catastrophic inflammatory response seen in others (Phase 2). This hypothesis further highlights the need for improved dietary intake to minimise the inflammatory priming resulting from poor glycaemic control.</p
The antibacterial properties of secreted phospholipases A2
No full textThere is a considerable body of evidence to support the antibacterial properties of the group IIa phospholipase A2 as an important physiological function. This enzyme is able to act as an acute phase protein and may be part of the innate defence system of the body, acting in concert with other antibacterial proteins and peptides. The enzyme is most effective against Gram-positive bacteria whereas penetration of the lipopolysaccharide coat of Gram-negative bacteria requires bactericidal/permeability-increasing protein (BPI) as an additional permeabilising factor. The global cationic nature of this protein (pI>10.5) appears to facilitate penetration of the anionic bacterial cell wall. In addition, the considerable preference of the enzyme for anionic phospholipid interfaces provides specificity toward anionic bacterial membranes as opposed to zwitterionic eucaryotic cell membranes
The interaction of liver fatty-acid-binding protein (FABP) with anionic phospholipid vesicles: is there extended phospholipid anchorage under these conditions?
No full textLiver FABP (fatty-acid-binding protein) binds a variety of non-polar anionic ligands including fatty acids, fatty acyl CoAs, lysophospholipids and bile acids. Liver FABP is also able to bind to anionic phospholipid vesicles under conditions of low ionic strength, and membrane binding results in the release of bound ligand. However, the molecular interactions involved in binding to the phospholipid interface and the mechanism of ligand release are not known. Ligand release could be due to a significant conformational change in the protein at the interface or interaction of a phospholipid molecule with the ligand-binding cavity of the protein resulting in ligand displacement. Two portal mutant proteins of liver FABP, L28W and M74W, have now been used to investigate the binding of liver FABP to anionic phospholipid vesicles, monitoring changes in fluorescence and also fluorescence quenching in the presence of brominated lipids. There is a large increase in fluorescence intensity when the L28W mutant protein binds to vesicles prepared from DOPG (dioleoyl-sn-phosphatidylglycerol), but a large decrease in fluorescence intensity when the M74W mutant binds to these vesicles. The Br4-phospholipid prepared by bromination of DOPG dramatically quenches both L28W and M74W, consistent with the close proximity of a fatty acyl chain to the tryptophan residues. The binding of liver FABP to DOPG vesicles is accompanied by only a minimal change in the CD spectrum. Overall, the results are consistent with a molecule of anionic phospholipid interacting with the central cavity of the liver FABP, possibly involving the phospholipid molecule in an extended conformation.<br/
Bacterial cell membrane hydrolysis by secreted phospholipases A2: a major physiological role of human group IIa sPLA2 involving both bacterial cell wall penetration and interfacial catalysis
No full textThe ability of human group IIa secreted phospholipase A2 (human sPLA2) to hydrolyse the phospholipid membrane of whole cell suspensions of Gram-positive bacteria is demonstrated in real time using a continuous fluorescence displacement assay. Micrococcus luteus is used as a model system and demonstrates an almost absolute specificity for this human enzyme compared with porcine pancreatic and Naja naja venom sPLA2s. This specificity is due to selective penetration of the highly cationic human sPLA2 through the highly anionic bacterial cell wall. Disruption of the peptidoglycan cell wall by treatment with lysozyme allows all three enzymes to express similar hydrolytic activity against the anionic bacterial cell membrane. Extensive (>50%) phospholipid hydrolysis was observed and this was confirmed by electrospray mass spectrometry that allowed the identification of several molecular species of phosphatidylglycerol as the targets for hydrolysis. However, the bactericidal activity of the human enzyme under these assay conditions was low, highlighting the capacity of the organism to survive a major phospholipid insult. In addition to pure enzyme, the human sPLA2 activity in tears was demonstrated using M. luteus as substrate. In comparison to M. luteus, cell suspensions of Staphylococcus aureus were highly resistant to hydrolysis by human sPLA2 as well as to the pancreatic and venom enzymes. Treatment of this organism with the specific cell wall protease lysostaphin resulted in a dramatic enhancement in cell membrane phospholipid hydrolysis by all three sPLA2s. Overall, the results highlight the potential of the human sPLA2 as a selective antimicrobial agent against Gram-positive bacteria in vivo because this enzyme is essentially inactive against mammalian plasma membranes. However, the enzyme will be most effective in combination with other antimicrobial agents that enhance the permeability of the bacterial cell wall and where potentiation of the effectiveness of other antibiotics would be expected
A catalytically independent physiological function for human acute phase protein group IIA phospholipase A2: cellular uptake facilitates cell debris removal
No full textHuman group IIA phospholipase A2 (IIA PLA2) is an acute phase protein first identified at high concentrations in synovial fluid from patients with rheumatoid arthritis. Its physiological role has since been debated; the enzyme has a very high affinity for anionic phospholipid interfaces but expresses almost zero activity with zwitterionic phospholipid substrates, because of a lack of interfacial binding. We have prepared the cysteine-containing mutant (S74C) to allow the covalent attachment of fluorescent reporter groups. We show that fluorescently labeled IIA was taken up by phorbol 12-myristate 13-acetate-activated THP-1 cells in an energy-dependent process involving cell surface heparan sulfate proteoglycans. Uptake concurrently involved significant cell swelling, characteristic of macropinocytosis and the fluorescent enzyme localized to the nucleus. The endocytic process did not necessitate enzyme catalysis, ruling out membrane phospholipid hydrolysis as an essential requirement. The enzyme produced supramolecular aggregates with anionic phospholipid vesicles as a result of bridging between particles, a property that is unique to this globally cationic IIA PLA2. Uptake of such aggregates labeled with fluorescent anionic phospholipid was dramatically enhanced by the IIA protein, and uptake involved binding to heparan sulfate proteoglycans on activated THP-1 cells. A physiological role for this protein is proposed that involves the removal of anionic extracellular cell debris, including anionic microparticles generated as a result of trauma, infection, and the inflammatory response, and under such conditions serum levels of IIA PLA2 can increase approximately 1000-fold. A similar pathway may be significant in the uptake into cells of anionic vector DNA involving cationic lipid transfection protocols.<br/
Could anionic LDL be a ligand for RAGE and TREM2 in addition to LOX-1 and thus exacerbate lung disease and dementia?
No full textWe recently highlighted the potential of protein glycation to generate anionic (electronegative) surfaces. We hypothesised that these anionic proteins are perceived by the innate immune system as arising from infection or damaged cell components, producing an inflammatory response within the lung involving the receptor RAGE. We now review two other pathologies linked to the innate immune response, cardiovascular disease and dementia that involve receptors LOX-1 and TREM2 respectively. Remarkable similarities in properties between RAGE, LOX-1 and TREM2 suggest that electronegative LDL may act as a pathogenic anionic ligand for all three receptors and exacerbate lung inflammation and dementia
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