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- Giorgi, Laura, et al.
(författare)
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Mechanistic Insights into the Activation of Lecithin-Cholesterol Acyltransferase in Therapeutic Nanodiscs Composed of Apolipoprotein A-I Mimetic Peptides and Phospholipids
- 2022
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Ingår i: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8392 .- 1543-8384. ; 19:11, s. 4135-4148
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Tidskriftsartikel (refereegranskat)abstract
- The mechanistic details behind the activation of lecithin-cholesterol acyltransferase (LCAT) by apolipoprotein A-I (apoA-I) and its mimetic peptides are still enigmatic. Resolving the fundamental principles behind LCAT activation will facilitate the design of advanced HDL-mimetic therapeutic nanodiscs for LCAT deficiencies and coronary heart disease and for several targeted drug delivery applications. Here, we have combined coarse-grained molecular dynamics simulations with complementary experiments to gain mechanistic insight into how apoA-Imimetic peptide 22A and its variants tune LCAT activity in peptide-lipid nanodiscs. Our results highlight that peptide 22A forms transient antiparallel dimers in the rim of nanodiscs. The dimerization tendency considerably decreases with the removal of C-terminal lysine K22, which has also been shown to reduce the cholesterol esterification activity of LCAT. In addition, our simulations revealed that LCAT prefers to localize to the rim of nanodiscs in a manner that shields the membrane-binding domain (MBD), αA-αA′, and the lid amino acids from the water phase, following previous experimental evidence. Meanwhile, the location and conformation of LCAT in the rim of nanodiscs are spatially more restricted when the active site covering the lid of LCAT is in the open form. The average location and spatial dimensions of LCAT in its open form were highly compatible with the electron microscopy images. All peptide 22A variants studied here had a specific interaction site in the open LCAT structure flanked by the lid and MBD domain. The bound peptides showed different tendencies to form antiparallel dimers and, interestingly, the temporal binding site occupancies of the peptide variants affected their in vitro ability to promote LCAT-mediated cholesterol esterification.
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- Niemela, Tuomas Aarni Akseli, et al.
(författare)
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Mechanistic Insights into the Activation of Lecithin-Cholesterol Acyltransferase in Therapeutic Nanodiscs Composed of Apolipoprotein A-I Mimetic Peptides and Phospholipids
- 2023
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Ingår i: European Biophysics Journal. - 1432-1017 .- 0175-7571. ; 52:SUPPL 1, s. S98-S98
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Lecithin-cholesterol acyltransferase (LCAT) maturates high density lipoprotein (HDL) particles enabling reverse cholesterol transport (RCT). Its primary cofactor on HDL is apolipoprotein A1 (apoA1). In order to boost RCT efficiency to combat atherosclerotic cardiovascular diseases, apoA1 mimetic peptides have been developed, such as 22 amino acid long 22A. Despite being ~10 % the size of apoA1, when 22A is formulated to a synthetic HDL particle, called a nanodisc, it is able to activate LCAT as well as apoA1 based HDL. However, the mechanism of how 22A, or apoA1, activates LCAT is not known. We set out to explain this mechanism with a combination of coarse-grained molecular dynamics simulations, LCAT activity assays and electron microscopy (EM). The computational data matched the experimental data in two ways. First, EM imaging showed that LCAT binds to the perimeter of 22A based nanodiscs, which was replicated by the simulations. The overall binding pose was also consistent. Second, by simulating and assaying a set of 22A mutants, we were able to see a correlation between the in silico binding kinetics of a peptide to aspecific site in LCAT and how well the peptide activates LCAT in vitro. Taken together, we’ve shown that these simulated systems can be exploited for designing peptides with increased efficacy. A more detailed description is found in our similarly titled article in Mol. Pharmaceutics 19: 4135 -4148, 2022.
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- Olsén, Erik, 1994, et al.
(författare)
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Dual-Angle Interferometric Scattering Microscopy for Optical Multiparametric Particle Characterization
- 2024
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Ingår i: NANO LETTERS. - 1530-6984 .- 1530-6992. ; 24:6, s. 1874-1881
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Tidskriftsartikel (refereegranskat)abstract
- Traditional single-nanoparticle sizing using optical microscopy techniques assesses size via the diffusion constant, which requires suspended particles to be in a medium of known viscosity. However, these assumptions are typically not fulfilled in complex natural sample environments. Here, we introduce dual-angle interferometric scattering microscopy (DAISY), enabling optical quantification of both size and polarizability of individual nanoparticles (radius <170 nm) without requiring a priori information regarding the surrounding media or super-resolution imaging. DAISY achieves this by combining the information contained in concurrently measured forward and backward scattering images through twilight off-axis holography and interferometric scattering (iSCAT). Going beyond particle size and polarizability, single-particle morphology can be deduced from the fact that the hydrodynamic radius relates to the outer particle radius, while the scattering-based size estimate depends on the internal mass distribution of the particles. We demonstrate this by differentiating biomolecular fractal aggregates from spherical particles in fetal bovine serum at the single-particle level.
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- Parkkila, Petteri, 1990, et al.
(författare)
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Protein A/G-based surface plasmon resonance biosensor for regenerable antibody-mediated capture and analysis of nanoparticles
- 2022
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Ingår i: Colloids and Surfaces A: Physicochemical and Engineering Aspects. - : Elsevier BV. - 1873-4359 .- 0927-7757. ; 654
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Tidskriftsartikel (refereegranskat)abstract
- Characterization of nanoparticles (NPs) and their subpopulations in heterogeneous samples is of utmost importance, for example, during the initial design of targeted NP therapies and the different phases of their production cycle. Biological NPs such as extracellular vesicles (EVs) have shown promise in improving the drug delivery capabilities compared to traditional NP-based therapies, for example, in treating cancer and neurodegenerative diseases. This work presents a general antibody-mediated surface capture and analysis protocol for NPs using a Protein A/G-functionalized surface plasmon resonance biosensor. The use of anti-streptavidin antibodies allows regenerable capture of biotin-containing NPs such as large unilamellar vesicles commonly used as drug delivery vehicles. Furthermore, the use of antibodies directed against glycophorin A and B (CD235a and b) enabled diffusion-limited specific surface capture of red blood cell-derived extracellular vesicles (RBC EVs). RBC EVs showed the efficacy of the biosensor in the determination of size and bulk concentration of NP subpopulations isolated from a complex biological matrix. The mean size of the surface-captured RBC EVs was comparable to the corresponding sizes derived for the entire EV population measured with well-established NP sizing techniques, namely, nanoparticle tracking analysis and dynamic light scattering. Taken together, the Protein A/G-functionalized biosensor provides a generic alternative to the existing NP-capturing sensors based on, for example, covalent antibody attachment, hydrophobic surfaces or biotin-capped self-assembled monolayers.
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- Pirc, Katja, et al.
(författare)
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An oomycete NLP cytolysin forms transient small pores in lipid membranes
- 2022
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Ingår i: Science advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 8:10, s. eabj9406-
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Tidskriftsartikel (refereegranskat)abstract
- Microbial plant pathogens secrete a range of effector proteins that damage host plants and consequently constrain global food production. Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) are produced by numerous phytopathogenic microbes that cause important crop diseases. Many NLPs are cytolytic, causing cell death and tissue necrosis by disrupting the plant plasma membrane. Here, we reveal the unique molecular mechanism underlying the membrane damage induced by the cytotoxic model NLP. This membrane disruption is a multistep process that includes electrostatic-driven, plant-specific lipid recognition, shallow membrane binding, protein aggregation, and transient pore formation. The NLP-induced damage is not caused by membrane reorganization or large-scale defects but by small membrane ruptures. This distinct mechanism of lipid membrane disruption is highly adapted to effectively damage plant cells.
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