Browsing by Subject "HDL"
Now showing items 1-4 of 4
-
(2022)Coronary heart disease is a number one killer in westernized countries and the costs from it will continue to grow in the future. It is caused by atherosclerosis, build-up of plaque and chronic inflammation in the arteries of heart, and endogenous lipoproteins have a special role in its development. Among other atheroprotective properties, High density lipoproteins (HDL) have a role in intrinsic mechanism of the reverse cholesterol transport (RCT), of gathering and removing excess cholesterol from peripheral tissues. There have been several HDL raising strategies in the past for the treatment of atherosclerosis, but their success has been modest. Synthetic HDL (sHDL), comprising of various types of phospholipids and proteins or peptides, have been developed to mimic the properties of endogenous HDL. Despite some success in animal studies, failures in clinical studies have turned the focus on the HDL’s interaction with a specific enzyme lecithin:cholesterol acyl transferase (LCAT), responsible for cholesterol esterification, a key step in RCT. ApoA-I, the most abundant protein component of HDL, acts as LCAT cofactor in cholesterol esterification, and many LCAT activating peptides have been developed to mimic the features of apoA-I. The molecular level understanding behind LCAT activation is however still foggy. During enzymatic activation, LCAT goes through conformational changes specific regions, which are generated by interactions with apoA-I or synthetic peptides. These mechanisms have been studied widely with molecular dynamic simulations, in vitro experiments, and imaging. In this study, we investigated 22A (PVLDLFRELLNELLEALKQKLK), apoA-I mimetic peptide known for its as good LCAT activation potency as apoA-I, and four variations of it (21A, 22A-P, 22A-K22Q, and 22A-R7Q), and combined them with phospholipid DPPC to create sHDL nanodiscs by thermal cycling method. We examined the effect of small changes in peptide sequence on LCAT-sHDL binding strength with quartz crystal microbalance with dissipation (QCM-D). The interest was to further test the suitability of thermal cycling method on nanodisc assembly, test the binding strengths against the hypothesis of the role of salt-bridge forming amino acids R7 and K22 in peptide dimerization and its effect on LCAT binding and activation, and to see if QCM could act as a suitable method for the research of sHDL-LCAT interactions. All peptides formed similar sized sHDL particles with diameter of ~10 nm with thermal cycling method. As expected, the LCAT binding tendency of 22A-sHDL was highest, about double compared to four other peptide nanodiscs with almost identical results. The QCM results suggest that binding tendency between LCAT and sHDL is affected by small, one amino acid change in peptide sequence, but it does not necessarily have a big impact on LCAT’s esterification activity, but based on this experiment alone, we cannot make any further conclusions. Electron microscopy revealed exceptional breakdown of 21A-sHDL incubated with LCAT compared to 22A-sHDL. This phenomenon could indicate high lipolytic rate of LCAT but needs further investigation. There were some challenges with the measurement parameters in the beginning, and the variability between parallel measurements with QCM-D was high, which cause a little doubt about the method’s suitability for these kinds of precise measurements. More research for revealing the molecular mechanism behind LCAT activation is needed for the development of more effective treatments.
-
(2022)Coronary heart disease is a number one killer in westernized countries and the costs from it will continue to grow in the future. It is caused by atherosclerosis, build-up of plaque and chronic inflammation in the arteries of heart, and endogenous lipoproteins have a special role in its development. Among other atheroprotective properties, High density lipoproteins (HDL) have a role in intrinsic mechanism of the reverse cholesterol transport (RCT), of gathering and removing excess cholesterol from peripheral tissues. There have been several HDL raising strategies in the past for the treatment of atherosclerosis, but their success has been modest. Synthetic HDL (sHDL), comprising of various types of phospholipids and proteins or peptides, have been developed to mimic the properties of endogenous HDL. Despite some success in animal studies, failures in clinical studies have turned the focus on the HDL’s interaction with a specific enzyme lecithin:cholesterol acyl transferase (LCAT), responsible for cholesterol esterification, a key step in RCT. ApoA-I, the most abundant protein component of HDL, acts as LCAT cofactor in cholesterol esterification, and many LCAT activating peptides have been developed to mimic the features of apoA-I. The molecular level understanding behind LCAT activation is however still foggy. During enzymatic activation, LCAT goes through conformational changes specific regions, which are generated by interactions with apoA-I or synthetic peptides. These mechanisms have been studied widely with molecular dynamic simulations, in vitro experiments, and imaging. In this study, we investigated 22A (PVLDLFRELLNELLEALKQKLK), apoA-I mimetic peptide known for its as good LCAT activation potency as apoA-I, and four variations of it (21A, 22A-P, 22A-K22Q, and 22A-R7Q), and combined them with phospholipid DPPC to create sHDL nanodiscs by thermal cycling method. We examined the effect of small changes in peptide sequence on LCAT-sHDL binding strength with quartz crystal microbalance with dissipation (QCM-D). The interest was to further test the suitability of thermal cycling method on nanodisc assembly, test the binding strengths against the hypothesis of the role of salt-bridge forming amino acids R7 and K22 in peptide dimerization and its effect on LCAT binding and activation, and to see if QCM could act as a suitable method for the research of sHDL-LCAT interactions. All peptides formed similar sized sHDL particles with diameter of ~10 nm with thermal cycling method. As expected, the LCAT binding tendency of 22A-sHDL was highest, about double compared to four other peptide nanodiscs with almost identical results. The QCM results suggest that binding tendency between LCAT and sHDL is affected by small, one amino acid change in peptide sequence, but it does not necessarily have a big impact on LCAT’s esterification activity, but based on this experiment alone, we cannot make any further conclusions. Electron microscopy revealed exceptional breakdown of 21A-sHDL incubated with LCAT compared to 22A-sHDL. This phenomenon could indicate high lipolytic rate of LCAT but needs further investigation. There were some challenges with the measurement parameters in the beginning, and the variability between parallel measurements with QCM-D was high, which cause a little doubt about the method’s suitability for these kinds of precise measurements. More research for revealing the molecular mechanism behind LCAT activation is needed for the development of more effective treatments.
-
(2022)Streptococcus pneumoniae is an opportunistic pathogen found in the normal flora of humans that can cause pneumonia and systemic infections. Pneumonia can trigger inflammation, which may lead to plaque rupture, or acute coronary syndrome in patients with atherosclerosis. Pneumolysin (PLY) S. pneumoniae virulence factor that causes cell lysis. High-density lipoprotein (HDL) reduces foam cell formation, and it possesses anti-inflammatory properties, protecting against atherosclerosis. My group has shown that PLY can modify HDL, causing chemical and structural modification to HDL. Modified HDL has an impaired cholesterol efflux potential and increased ability to activate complement and therefore can trigger inflammation. The study aimed to determine whether anti-PLY antibodies could protect against inflammatory effects and hemolysis caused by PLY. The hemolysis experiment showed that anti-PLY antibodies isolated from serum inhibit the hemolytic activity of PLY. The anti-PLY antibodies bound more to PLY-modified HDL compared to unmodified HDL; however, neither increased complement activation. These results demonstrate the protective role of anti-PLY antibodies against PLY. In conclusion, anti- PLY antibodies have the potential to be beneficial in preventing the impact of S. pneumoniae infection and its complications. In the future, these results could be used to investigate the effects of anti-PLY antibodies in atherosclerosis.
-
(2022)Streptococcus pneumoniae is an opportunistic pathogen found in the normal flora of humans that can cause pneumonia and systemic infections. Pneumonia can trigger inflammation, which may lead to plaque rupture, or acute coronary syndrome in patients with atherosclerosis. Pneumolysin (PLY) S. pneumoniae virulence factor that causes cell lysis. High-density lipoprotein (HDL) reduces foam cell formation, and it possesses anti-inflammatory properties, protecting against atherosclerosis. My group has shown that PLY can modify HDL, causing chemical and structural modification to HDL. Modified HDL has an impaired cholesterol efflux potential and increased ability to activate complement and therefore can trigger inflammation. The study aimed to determine whether anti-PLY antibodies could protect against inflammatory effects and hemolysis caused by PLY. The hemolysis experiment showed that anti-PLY antibodies isolated from serum inhibit the hemolytic activity of PLY. The anti-PLY antibodies bound more to PLY-modified HDL compared to unmodified HDL; however, neither increased complement activation. These results demonstrate the protective role of anti-PLY antibodies against PLY. In conclusion, anti- PLY antibodies have the potential to be beneficial in preventing the impact of S. pneumoniae infection and its complications. In the future, these results could be used to investigate the effects of anti-PLY antibodies in atherosclerosis.
Now showing items 1-4 of 4