| 1. |
- Petersen, Daniel, et al.
(författare)
-
Binding and intracellular transport of 25-hydroxycholesterol by Niemann-Pick C2 protein
- 2020
-
Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 1879-2642. ; 1862:2
-
Tidskriftsartikel (refereegranskat)abstract
- Side-chain oxidized cholesterol derivatives, like 25-hydroxycholesterol (25-OH-Chol) are important regulators of cellular cholesterol homeostasis. How transport of oxysterols through the endo-lysosomal pathway contributes to their biological function is not clear. The Niemann-Pick C2 protein (NPC2) is a small lysosomal sterol transfer protein required for export of cholesterol from late endosomes and lysosomes (LE/LYSs). Here, we show that 25-hydroxy-cholestatrienol, (25-OH-CTL), an intrinsically fluorescent analogue of 25-OH-Chol, becomes trapped in LE/LYSs of NPC2-deficient fibroblasts, but can efflux from the cells even in the absence of NPC2 upon removal of the sterol source. Fluorescence recovery after photobleaching (FRAP) of 25-OH-CTL in endo-lysosomes was rapid and extensive and only partially dependent on NPC2 function. Using quenching of NPC2's intrinsic fluorescence, we show that 25-OH-Chol and 25-OH-CTL can bind to NPC2 though with lower affinity compared to cholesterol and its fluorescent analogues, cholestatrienol (CTL) and dehydroergosterol (DHE). This is confirmed by calculations of binding energies which additionally show that 25-OH-CTL can bind in two orientations to NPC2, in stark contrast to cholesterol and its analogues. We conclude that NPC2's affinity for all sterols is energetically favored over their self-aggregation in the lysosomal lumen. Lysosomal export of 25-OH-Chol is not strictly dependent on the NPC2 protein.
|
|
| 2. |
- Eriksson, Ida, 1985-
(författare)
-
Dealing with damaged lysosomes : Impact of lysosomal membrane stability in health and disease
- 2022
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The lysosome is the main unit for degradation and plays important roles in various cellular processes, such as nutrient sensing, cholesterol regulation and cell death. Consequently, altered lysosomal function contributes to, or even causes, several diseases. Lysosomal membrane permeabilization (LMP) and release of lysosomal content to the cytosol can induce cell death, and is implicated in inflammation and neuronal decline in several neurodegenerative diseases. It has also emerged as a potential target in cancer therapy. Due to the detrimental effects of LMP, cells harbor several mechanisms to protect and prevent lysosomal membrane damage. The aim of this thesis was to elucidate how lysosomal membrane stability and repair mechanisms affect cell death and survival. We find that lysosomal cholesterol is upregulated in response to an increased load of reactive oxygen species in a Parkinson’s disease cell model, and that augmented cholesterol protects from LMP. However, cholesterol also induces accumulation of α-synuclein and inhibits lysosome-mediated degradation, which can destabilize the lysosomal membrane and accelerate the course of disease. Further, we demonstrate that lysosomal membrane damage is counteracted by a calcium-dependent repair mechanism to prevent LMP. Lysosomes damaged beyond repair are instead sequestered in an autophagosome and degraded by intact lysosomes in a process called lysophagy. As a result, small vesicles containing lysosomal membrane proteins are generated, which we believe are used to restore lysosomal function. We show that malignant cells are more sensitive to LMP, and that they differ in their activation of damage-response mechanisms compared to normal cells. Moreover, in malignant cells, the intracellular position of the lysosomes determines the susceptibility to lysosomal damage. Peripherally located lysosomes are less sensitive, and by relocating lysosomes to the perinuclear area in the cell, we can sensitize lysosomes to LMP induction. In summary, this thesis demonstrates the importance of damage-response mechanisms to protect from lysosomal membrane damage and maintain cellular function. It also indicates that targeting of lysosomal stability and repair is a potential therapeutic strategy in both neurodegenerative diseases and in cancer.
|
|
| 3. |
- Nabo, Lina J., et al.
(författare)
-
Embedding beyond electrostatics-The role of wave function confinement
- 2016
-
Ingår i: Journal of Chemical Physics. - : American Institute of Physics (AIP). - 0021-9606 .- 1089-7690. ; 145:10
-
Tidskriftsartikel (refereegranskat)abstract
- We study excited states of cholesterol in solution and show that, in this specific case, solute wave-function confinement is the main effect of the solvent. This is rationalized on the basis of the polarizable density embedding scheme, which in addition to polarizable embedding includes non-electrostatic repulsion that effectively confines the solute wave function to its cavity. We illustrate how the inclusion of non-electrostatic repulsion results in a successful identification of the intense pi ->pi* transition, which was not possible using an embedding method that only includes electrostatics. This underlines the importance of non-electrostatic repulsion in quantum-mechanical embedding-based methods.
|
|
| 4. |
- Poongavanam, Vasanthanathan, et al.
(författare)
-
Computational Analysis of Sterol Ligand Specificity of the Niemann Pick C2 Protein
- 2016
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 55:36, s. 5165-5179
-
Tidskriftsartikel (refereegranskat)abstract
- Transport of cholesterol derived from hydrolysis of lipoprotein associated cholesteryl esters out of late endosomes depends critically on the function of the Niemann Pick Cl (NPC1) and C2 (NPC2) proteins. Both proteins bind cholesterol but also various other sterols and both-With strongly varying affinity: The molecular mechanisms under lying this multiligand specificity are not known. On the basis:Of the crystal structure of NPC2, we have here investigated structural details of NPC2 sterol interactions using molecular mechanics Poisson-Boltzmann surface area (MM-PBSA): calculations. We found that an aliphatic side chain in the sterol ligand results in strong binding to NPC2, while side chain oxidized sterols gave weaker binding. Estradiol and the hydrophobic amine U18666A had the lowest affinity of all lested ligands and at the same time showed the highest flexibility within the NPC2 binding pocket. The binding affinity of all ligands correlated highly with their calculated partitioning coefficient (logP) between octanol/water phases and with the potential of sterols to stabilize the protein backbone. prom molecular dynamics simulations, we suggest a general mechanism for NPC2 mediated sterol transfer, in which Phe66, Val96, and Tyr100 act as reversible gate keepers. These residues stabilize the sterol in the binding pose via pi-pi stacking but move transiently. apart during sterol release. A computational mutation analysis revealed that the binding of various ligands depends critically on the same specific amino acid residues within the binding pocket providing shape complementary to sterols, but also on residues in distal regions of the protein.
|
|
| 5. |
- Poongavanam, Vasanthanathan, et al.
(författare)
-
Computational Modeling Explains the Multi Sterol Ligand Specificity of the N-Terminal Domain of Niemann-Pick C1-Like 1 Protein
- 2019
-
Ingår i: ACS Omega. - : American Chemical Society (ACS). - 2470-1343. ; 4:25, s. 20894-20904
-
Tidskriftsartikel (refereegranskat)abstract
- Niemann-Pick C1 like 1 (NPC1L1) is a sterol transporter expressed in the apical membrane of enterocytes and hepatocytes. NPC1L1 resembles the lysosomal NPC1 protein including an N-terminal domain (NTD), which binds a variety of sterols. The molecular mechanisms underlying this multiligand specificity of the NTD of NPC1L1 (NPC1L1-NTD) are not known. On the basis of the crystal structure of NPC1L1-NTD, we have investigated the structural details of protein-sterol interactions using molecular mechanics Poisson Boltzmann surface area calculations here. We found a good agreement between experimental and calculated binding affinities with similar ranking of various sterol ligands. We defined hydrogen bonding of sterol ligands via the 3'-beta-hydroxy group inside the binding pose as instrumental in stabilizing the interaction. A leucine residue (LEU213) at the mouth of the binding pocket transiently opens to allow for the access of sterol into the binding pose. Our calculations also predict that NPC1L1-NTD binds polyene sterols, such as dehydroergosterol or cholestatrienol with high affinity, which validates their use in future experiments as close intrinsically fluorescent cholesterol analogs. A free energy decomposition and computational mutation analysis revealed that the binding of various sterols to NPC1L1-NTD depends critically on specific amino acid residues within the binding pocket. Some of these residues were previously detected as being relevant for intestinal cholesterol absorption. We show that clinically known mutations in the NPC1L1-NTD associated with lowered risk of coronary heart disease result in strongly reduced binding energies, providing a molecular explanation for the clinical phenotype.
|
|
