An examination of the possible inhibition of the interaction between AQP4 and CaM using TFP, and the crystallization of AQP4
(2018) KEMK03 20181Department of Chemistry
- Abstract
- AQP4 is a protein in the brain which regulate the diffusion of water in and out of cells. Through studies using AQP4 null mice, a correlation between the regulation of AQP4 and the survival rate of cerebral edemas has been noted. Due to the fact that there are three different kinds of cerebral edemas, and that they require different kinds of regulation of AQP4 in order to lessen the problem, the regulation of AQP4 is of upmost relevance.
Through previous studies, it has been made clear that Calmodulin (CaM) affects the water permeability of the cell by regulating the amount of AQP4 available in the membrane. In Susanna Törnroth Horsefield’s group, it has also been proven that CaM interacts directly with AQP4 through the C-terminus of... (More) - AQP4 is a protein in the brain which regulate the diffusion of water in and out of cells. Through studies using AQP4 null mice, a correlation between the regulation of AQP4 and the survival rate of cerebral edemas has been noted. Due to the fact that there are three different kinds of cerebral edemas, and that they require different kinds of regulation of AQP4 in order to lessen the problem, the regulation of AQP4 is of upmost relevance.
Through previous studies, it has been made clear that Calmodulin (CaM) affects the water permeability of the cell by regulating the amount of AQP4 available in the membrane. In Susanna Törnroth Horsefield’s group, it has also been proven that CaM interacts directly with AQP4 through the C-terminus of AQP4.
In this study, the possible inhibition of the interaction between AQP4 and CaM through the FDA-approved drug Trifluoperazine (TFP) was examined using Microscale Thermophoresis (MST), as well as the interaction between just the C-terminus of AQP4 and CaM. In addition, the crystallization of the full length AQP4 was attempted, since the previous crystallization of AQP4 has excluded the C- and N-termini because of their flexibility, which makes the crystallization harder to achieve.
In the first studies where varying concentrations of TFP was added to constant concentrations of AQP4 and CaM, no inhibition could be seen. Several attempts were made in order to investigate the reason for this. Finally, when the solvent of the TFP was altered from Dimetylsulfoxid (DMSO) to water and TFP concentrations was kept constant at 10 mM while varying the concentration of AQP4, an inhibition could be detected. In future studies, a TFP dilution series will be tested, with an addition of a constant volume of AQP4 and CaM, where the concentration of AQP4 would be closer to the Kd value (Kd=[AQP4][CaM]/[AQP4*CaM] ) than in previous examinations.
Through the studies between the C-terminus of AQP4 and CaM, no clear binding curve could be seen. The result behind this could to be that the C-terminus is too small for MST to detect the complex formation. In future studies, a fluorescence-marked peptide could be used instead of CaM, in order to see if the size for the marked component would increase when the complex is formed, which would be clearer to see in the MST.
The crystallization of the full length AQP4 was achieved using a precipitant containing 0.1 M Tris pH 8.0, 44% v/v PEG and 0.2 M CaCl2 but have not yet been optimized. The obtained crystals had a resolution of 14 Å, which is too low to get any clear information in regards to the structure of the protein. In further studies, more experimental setups with conditions similar to the one which gave crystals would be tested, as well as further examination of the previously setup of additive screening. (Less) - Popular Abstract (Swedish)
- I dagens I-länder är neurologiska sjukdomar en av de främsta orsakerna bakom dödsfall. Enligt statistik från ”Disabled World” påverkas upp till 1 miljard människor världen över av någon form av neurologisk sjukdom, vilket inkluderar allt från epilepsi och migrän till Alzheimer och stroke.
Till följd av detta är medicin för att såväl förhindra som att motverka skador från neurologiska sjukdomar en prioritering inom forskarvärlden. För att kunna bota en sjukdom måste man först förstå vad det är som blir fel i kroppen, samt vad som kan göras för att motverka det.
Ett specifikt fall på en orsak på en neurologisk sjukdom är hjärnödem, också känt som ansamlingar av vatten vid hjärnan vilket kan leda till stroke, trauma, inflammation och... (More) - I dagens I-länder är neurologiska sjukdomar en av de främsta orsakerna bakom dödsfall. Enligt statistik från ”Disabled World” påverkas upp till 1 miljard människor världen över av någon form av neurologisk sjukdom, vilket inkluderar allt från epilepsi och migrän till Alzheimer och stroke.
Till följd av detta är medicin för att såväl förhindra som att motverka skador från neurologiska sjukdomar en prioritering inom forskarvärlden. För att kunna bota en sjukdom måste man först förstå vad det är som blir fel i kroppen, samt vad som kan göras för att motverka det.
Ett specifikt fall på en orsak på en neurologisk sjukdom är hjärnödem, också känt som ansamlingar av vatten vid hjärnan vilket kan leda till stroke, trauma, inflammation och tumörer. Via forskning kring hjärnödem har det framkommit att reglering av ett protein i hjärnan, vid namn aquaporin 4, kan ha en positiv påverkan på hjärnödem genom att påverka mängden vatten som tas in i hjärnans celler.
I denna rapport undersöks möjligheten av reglering av aquaporin 4 med hjälp av en redan existerande medicin vid namn Trifluoperazine (TFP), samtidigt som strukturen hos aquaporin 4 försöker kartläggas med hjälp av röntgen kristallografi, eftersom kunskap kring ett proteins struktur och uppbyggnad är viktigt för att känna till dess egenskaper. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/8949322
- author
- Torell, Tea LU
- supervisor
- organization
- course
- KEMK03 20181
- year
- 2018
- type
- M2 - Bachelor Degree
- subject
- keywords
- biochemistry, biokemi
- language
- English
- id
- 8949322
- date added to LUP
- 2018-06-21 11:54:01
- date last changed
- 2018-06-21 11:54:01
@misc{8949322,
abstract = {{AQP4 is a protein in the brain which regulate the diffusion of water in and out of cells. Through studies using AQP4 null mice, a correlation between the regulation of AQP4 and the survival rate of cerebral edemas has been noted. Due to the fact that there are three different kinds of cerebral edemas, and that they require different kinds of regulation of AQP4 in order to lessen the problem, the regulation of AQP4 is of upmost relevance.
Through previous studies, it has been made clear that Calmodulin (CaM) affects the water permeability of the cell by regulating the amount of AQP4 available in the membrane. In Susanna Törnroth Horsefield’s group, it has also been proven that CaM interacts directly with AQP4 through the C-terminus of AQP4.
In this study, the possible inhibition of the interaction between AQP4 and CaM through the FDA-approved drug Trifluoperazine (TFP) was examined using Microscale Thermophoresis (MST), as well as the interaction between just the C-terminus of AQP4 and CaM. In addition, the crystallization of the full length AQP4 was attempted, since the previous crystallization of AQP4 has excluded the C- and N-termini because of their flexibility, which makes the crystallization harder to achieve.
In the first studies where varying concentrations of TFP was added to constant concentrations of AQP4 and CaM, no inhibition could be seen. Several attempts were made in order to investigate the reason for this. Finally, when the solvent of the TFP was altered from Dimetylsulfoxid (DMSO) to water and TFP concentrations was kept constant at 10 mM while varying the concentration of AQP4, an inhibition could be detected. In future studies, a TFP dilution series will be tested, with an addition of a constant volume of AQP4 and CaM, where the concentration of AQP4 would be closer to the Kd value (Kd=[AQP4][CaM]/[AQP4*CaM] ) than in previous examinations.
Through the studies between the C-terminus of AQP4 and CaM, no clear binding curve could be seen. The result behind this could to be that the C-terminus is too small for MST to detect the complex formation. In future studies, a fluorescence-marked peptide could be used instead of CaM, in order to see if the size for the marked component would increase when the complex is formed, which would be clearer to see in the MST.
The crystallization of the full length AQP4 was achieved using a precipitant containing 0.1 M Tris pH 8.0, 44% v/v PEG and 0.2 M CaCl2 but have not yet been optimized. The obtained crystals had a resolution of 14 Å, which is too low to get any clear information in regards to the structure of the protein. In further studies, more experimental setups with conditions similar to the one which gave crystals would be tested, as well as further examination of the previously setup of additive screening.}},
author = {{Torell, Tea}},
language = {{eng}},
note = {{Student Paper}},
title = {{An examination of the possible inhibition of the interaction between AQP4 and CaM using TFP, and the crystallization of AQP4}},
year = {{2018}},
}