Optimizing cross-linking to study the interactions between AQP0 and CaM and AQP2 and LIP5
(2018) KEMR33 20172Department of Chemistry
- Abstract
- Mammals express thirteen different isoforms of the membrane-bound water channel aquaporin in a tissue-dependent manner. From a medical point of view, two of the most studied isoforms are aquaporin (AQP0), also known as major intrinsic protein and aquaporin 2 (AQP2). AQP0 is exclusively expressed in the mammalian eye lens and responsible for the transport of water across lens fiber cell membranes to maintain lens clarity. The water transport via AQP0 is modulated by CaM, a small protein composed of N and C- terminal calcium (Ca2+) binding domains that connected by an internal flexible linker. AQP2 is expressed in the kidney collecting duct, where it plays a critical role in water reabsorption from urine. AQP2 apical membrane abundance is... (More)
- Mammals express thirteen different isoforms of the membrane-bound water channel aquaporin in a tissue-dependent manner. From a medical point of view, two of the most studied isoforms are aquaporin (AQP0), also known as major intrinsic protein and aquaporin 2 (AQP2). AQP0 is exclusively expressed in the mammalian eye lens and responsible for the transport of water across lens fiber cell membranes to maintain lens clarity. The water transport via AQP0 is modulated by CaM, a small protein composed of N and C- terminal calcium (Ca2+) binding domains that connected by an internal flexible linker. AQP2 is expressed in the kidney collecting duct, where it plays a critical role in water reabsorption from urine. AQP2 apical membrane abundance is regulated by trafficking in response to vasopressin, in a phosphorylation-dependent manner. Part of this process involves interacting with lysosomal trafficking regulator interacting protein-5 (LIP5), a small cytosolic protein with a molecular weight of 42 kDa. This interaction, which has been shown to be dependent on AQP2 phosphorylation, facilitates AQP2 lysosomal degradation and therefore balance the abundance of AQP2 in the body. In this project, the main purpose was to develop methods for chemical cross-linking of the AQP0-CaM and AQP2-LIP5 complexes in order to study the structural details of how human aquaporin’s interact with regulatory proteins. For this purpose, human AQP0, AQP2, LIP5 and CaM were overproduced and purified, and cross-linking experiments was set up using two different cross-linkers. For both complexes, successful cross-linking could be verified using Western blot. In addition, co-crystallization attempts of the AQP0 C- terminal peptide and CaM was explored, resulting in small crystals that will be further investigated. (Less)
- Popular Abstract
- The faulty interaction between integral membrane protein and regulatory proteins can lead to several diseases. Therefore, studies of these interactions help us understand and answer essential questions on the development and outcome of these disease states. In this project, we investigated two water channels, aquaporin 0 (AQP0) and aquaporin 2 (AQP2) and how they are regulated by calmodulin (CaM) and lysosomal trafficking regulator interacting protein-5 (LIP5), respectively. The aim was to structurally characterize the complexes of these proteins. To reach this aim, AQP0, AQP2, LIP5 and CaM were produced and their ability for interaction was tested using chemical cross-linking and crystallization. The results suggest cross-linking can be... (More)
- The faulty interaction between integral membrane protein and regulatory proteins can lead to several diseases. Therefore, studies of these interactions help us understand and answer essential questions on the development and outcome of these disease states. In this project, we investigated two water channels, aquaporin 0 (AQP0) and aquaporin 2 (AQP2) and how they are regulated by calmodulin (CaM) and lysosomal trafficking regulator interacting protein-5 (LIP5), respectively. The aim was to structurally characterize the complexes of these proteins. To reach this aim, AQP0, AQP2, LIP5 and CaM were produced and their ability for interaction was tested using chemical cross-linking and crystallization. The results suggest cross-linking can be used to stabilize the complexes of AQP0 and CaM and AQP2 and LIP5. Furthermore, small crystals were obtained of the complex between the AQP0 C-terminus and CaM. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/8940819
- author
- Hussein, Rawya LU
- supervisor
- organization
- course
- KEMR33 20172
- year
- 2018
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- biochemistry, biokemi
- language
- English
- id
- 8940819
- date added to LUP
- 2018-06-20 16:04:36
- date last changed
- 2018-06-20 16:04:36
@misc{8940819,
abstract = {{Mammals express thirteen different isoforms of the membrane-bound water channel aquaporin in a tissue-dependent manner. From a medical point of view, two of the most studied isoforms are aquaporin (AQP0), also known as major intrinsic protein and aquaporin 2 (AQP2). AQP0 is exclusively expressed in the mammalian eye lens and responsible for the transport of water across lens fiber cell membranes to maintain lens clarity. The water transport via AQP0 is modulated by CaM, a small protein composed of N and C- terminal calcium (Ca2+) binding domains that connected by an internal flexible linker. AQP2 is expressed in the kidney collecting duct, where it plays a critical role in water reabsorption from urine. AQP2 apical membrane abundance is regulated by trafficking in response to vasopressin, in a phosphorylation-dependent manner. Part of this process involves interacting with lysosomal trafficking regulator interacting protein-5 (LIP5), a small cytosolic protein with a molecular weight of 42 kDa. This interaction, which has been shown to be dependent on AQP2 phosphorylation, facilitates AQP2 lysosomal degradation and therefore balance the abundance of AQP2 in the body. In this project, the main purpose was to develop methods for chemical cross-linking of the AQP0-CaM and AQP2-LIP5 complexes in order to study the structural details of how human aquaporin’s interact with regulatory proteins. For this purpose, human AQP0, AQP2, LIP5 and CaM were overproduced and purified, and cross-linking experiments was set up using two different cross-linkers. For both complexes, successful cross-linking could be verified using Western blot. In addition, co-crystallization attempts of the AQP0 C- terminal peptide and CaM was explored, resulting in small crystals that will be further investigated.}},
author = {{Hussein, Rawya}},
language = {{eng}},
note = {{Student Paper}},
title = {{Optimizing cross-linking to study the interactions between AQP0 and CaM and AQP2 and LIP5}},
year = {{2018}},
}