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Protein-Protein Interactions in Human Aquaporin Regulation

Roche, Jennifer Virginia LU (2018)
Abstract
Water is an essential compoment of every living orgamism and forms a major part of the human body. Regulated water transport is crucial for proper cell functioning and body homeostasis. Cell, the smallest structural and functional unit of life, uses specialized water conducting pores made up of proteins to transport water across the plasma membrane by facilitated diffusion.The discovery of one of these proteins in red blood cells was the first evidence of cellular water conducting channels and the protein was later named aquaporin 1 (AQP1). Since then, thirteen human aquaporin isoforms have been identified and classified as orthodox AQPs, those that transport water only, and aquaglyceroporins, those that also transport glycerol, ammonia,... (More)
Water is an essential compoment of every living orgamism and forms a major part of the human body. Regulated water transport is crucial for proper cell functioning and body homeostasis. Cell, the smallest structural and functional unit of life, uses specialized water conducting pores made up of proteins to transport water across the plasma membrane by facilitated diffusion.The discovery of one of these proteins in red blood cells was the first evidence of cellular water conducting channels and the protein was later named aquaporin 1 (AQP1). Since then, thirteen human aquaporin isoforms have been identified and classified as orthodox AQPs, those that transport water only, and aquaglyceroporins, those that also transport glycerol, ammonia, urea or other small solutes.
In this thesis, we focus on human AQP0 and AQP2, with an aim to deepen our understanding of how they are regulated by protein-protein interactions and the effect of phosphorylation, a post translational modification. Human AQP2 is found in the kidney where it plays an important role in regulating urine volume and is regulated by trafficking. The phosphorylation of the C-terminal residues in AQP2 and its interaction with lysosomal trafficking regulator interacting protein 5 (LIP5) plays a major role in AQP2 trafficking and targeting for lysosomal degradation. AQP0 has dual role in the eye lens, functioning as a water transporter as well as an adhesion protein in cell junctions in lens fibre cells. Previous studies have demonstrated that the binding of calmodulin (CaM) to the C-terminal of AQP0 in the presence of calcium inhibits water transport in a calcium dependent manner.
In our studies, the interaction between interacting proteins (LIP5 and CaM) and full length (FL) AQPs and AQP peptides was measured using Micro Scale Thermophoresis (MST) and fluorescence anisotropy. While we were able to show the interaction and determine the binding affinities for both FL AQPs and peptides, we also could demonstrate that using FL proteins was advantageous compared to peptides. Interaction studies between FL AQP2 and LIP5 showed that AQP2 binds the LIP5 N-terminal domain (ND-LIP5) and that one ND-LIP5 molecule bound per AQP2 tetramer. We also showed that phosphorylation of the distal C-terminus of AQP2 allosterically regulates its interaction with LIP5. The stability of AQP2 and AQP2 phoshpho mimics as well as soluble proteins in the presence of detergents was demonstrated by Circular Dichroism (CD). Small angle X-ray scattering (SAXS) measurements provided information on AQP2 in nanodiscs. However, more experimental data would be required to characterize the structural aspects of AQP2:LIP5 interaction. Our data shows that recombinantly expressed AQP0 functions as a water channel when reconstituted into proteoliposomes. We demonstrated for the first time that direct binding of CaM reduces its water permeability in vitro and that phosphorylation of S229 and S235 in AQP0 abolishes the interaction with CaM while S231 shared similar binding affinities as FL AQP0. We further showed that CaM binds AQP0 in a cooperative manner. (Less)
Abstract (Swedish)
Every living organism is made up of cells, the smallest structural and functional unit of life. These organisms can further be classified on the basis of cell number as single-cell or multi-cell organism. Over time these primitive organisms have evolved leading to the formation of more complex machinery in higher organism. While it is tough and tedious to calculate the exact number of cells in the human body, researchers have been able to estimate it to 37.2 trillion cells consisting of specialised organelles to perform everyday functions.
Why are cells so important? How does it affect the everyday activity? Imagine a factory or industry with few employees, where each employee or a group of employees are specialized and responsible to... (More)
Every living organism is made up of cells, the smallest structural and functional unit of life. These organisms can further be classified on the basis of cell number as single-cell or multi-cell organism. Over time these primitive organisms have evolved leading to the formation of more complex machinery in higher organism. While it is tough and tedious to calculate the exact number of cells in the human body, researchers have been able to estimate it to 37.2 trillion cells consisting of specialised organelles to perform everyday functions.
Why are cells so important? How does it affect the everyday activity? Imagine a factory or industry with few employees, where each employee or a group of employees are specialized and responsible to perform a specific task. Often a combination of expertise may be required necessitating coordination among co-workers. Safe working environment with measures to avoid unauthorised people from performing tasks or trespassing is essential. In a simplistic way, a cell may be compared to this small factory or industry and the organelles as employees performing specific tasks. Coordination between organelles is essential to perform certain functions. The presence of plasma membrane, also known as cell membrane acts as a barrier to the external environment and maintains internal cellular integrity. In situations where the external involvement is required, specialised organelles facilitate this need.
The cell membrane is made up of a phospholipid bilayer with proteins acting as key players for interaction between the internal and external environment. Some proteins are partially embedded into the bilayer, known as peripheral proteins and some may span across the bilayer, known as integral membrane proteins. Here we work with integral membrane proteins from humans called aquaporins (AQP). As the name suggests it is a water transporting channel with a pore to facilitate its action. The structure (architecture) of AQPs comprises four monomeric units forming a tetramer. Each monomer has a central pore to allow the passage of water or other small solute molecules. The aquaporins are classified as orthodox aquaporins, those that facilitate water transport only and aquaglyceroporins, those that also transport glycerol, urea, ammonia and other small solutes.
A large portion of the human body consists of water and it is crucial to maintain water balance for proper functioning. In this thesis, we focus on human AQP2 and AQP0. Human AQP2 plays a crucial role in maintaining body water homeostasis. It is mainly found in the kidney collecting duct and helps to concentrate urine. AQP0 mainly found
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in the eye lens helps maintain lens water homeostasis. Truncated AQP0 can stack together and function as a cell adhesion molecule in cell junctions.
Proteins are made up of amino acid residues and mutations in these residues can affect the structure. Since an intact protein structure is important for proper functioning, such mutations can lead to disease states. Mutations or dysfunction in AQP2 leads to nephrogenic diabetes insipidus (NDI), a state where the individual lacks the ability to concentrate urine leading to dehydration. Mutations in AQP0 can cause cataract, a condition where the eye lens become opaque leading to blurry vision.
As protein may not always be present at the site of action, proper trafficking becomes crucial. This is true for AQP2, which is stored in storage compartment under water saturating conditions and transported to the cell membrane during periods of dehydration. Signalling receptors and interacting proteins trigger the action, initiating a signalling cascade and eventually translocating the protein to the desired site. When the protein is no longer required it is shuttled back. The interaction of AQP2 with Lysosomal Trafficking Regulator Interacting Protein 5 (LIP5) helps in sorting AQP2 for lysosomal degradation. AQP0 is the only AQP in lens fibre cells and the mutation can affect lens structure. AQP0 interacting with calmodulin (CaM) in the presence of calcium inhibits water permeability by causing pore closure.
In this study we focus on the interaction between AQP2:LIP5 and AQP0:CaM with an aim to elucidate the underlying mechanism for their interaction. Predominant focus on the previously proposed C-terminal (the carboxylic part of the protein) of AQP2 and AQP0 and the role of phosphorylation (a post-translational protein modification) on protein-protein interaction is emphasized. From our data, we have demonstrated the affinities of interaction using complimentary techniques like micro scale thermophoresis (MST), fluorescence anisotropy and Far Western blot and shown that phosphorylation can affect the interaction by lowering the binding affinity. We demonstrated the stability of aquaporins, phospho mimicking mutants of aquaporins and soluble proteins in detergents using circular dichroism spectroscopy. Structural insights into the interaction mechanism of AQP2:LIP5 were attempted using small angle X-ray scattering. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Fenton, Robert A., Department of Biomedicine, Aarhus University, Denmark
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Membrane proteins, Human Aquaporins, water channels, Protein-protein interaction, Regulation, AQP2, AQP0, LIP5, CaM
pages
180 pages
publisher
Lund University, Faculty of Science, Department of Chemistry
defense location
Hall B, Kemicentrum, Naturvetarvägen 14, Lund
defense date
2018-11-09 09:15:00
ISBN
978-91-7422-604-1
language
English
LU publication?
yes
id
29d70a61-9bc2-4832-8982-6d6cf1b08297
date added to LUP
2018-10-15 10:13:14
date last changed
2018-11-21 21:42:26
@phdthesis{29d70a61-9bc2-4832-8982-6d6cf1b08297,
  abstract     = {Water is an essential compoment of every living orgamism and forms a major part of the human body. Regulated water transport is crucial for proper cell functioning and body homeostasis. Cell, the smallest structural and functional unit of life, uses specialized water conducting pores made up of proteins to transport water across the plasma membrane by facilitated diffusion.The discovery of one of these proteins in red blood cells was the first evidence of cellular water conducting channels and the protein was later named aquaporin 1 (AQP1). Since then, thirteen human aquaporin isoforms have been identified and classified as orthodox AQPs, those that transport water only, and aquaglyceroporins, those that also transport glycerol, ammonia, urea or other small solutes.<br/>In this thesis, we focus on human AQP0 and AQP2, with an aim to deepen our understanding of how they are regulated by protein-protein interactions and the effect of phosphorylation, a post translational modification. Human AQP2 is found in the kidney where it plays an important role in regulating urine volume and is regulated by trafficking. The phosphorylation of the C-terminal residues in AQP2 and its interaction with lysosomal trafficking regulator interacting protein 5 (LIP5) plays a major role in AQP2 trafficking and targeting for lysosomal degradation. AQP0 has dual role in the eye lens, functioning as a water transporter as well as an adhesion protein in cell junctions in lens fibre cells. Previous studies have demonstrated that the binding of calmodulin (CaM) to the C-terminal of AQP0 in the presence of calcium inhibits water transport in a calcium dependent manner.<br/>In our studies, the interaction between interacting proteins (LIP5 and CaM) and full length (FL) AQPs and AQP peptides was measured using Micro Scale Thermophoresis (MST) and fluorescence anisotropy. While we were able to show the interaction and determine the binding affinities for both FL AQPs and peptides, we also could demonstrate that using FL proteins was advantageous compared to peptides. Interaction studies between FL AQP2 and LIP5 showed that AQP2 binds the LIP5 N-terminal domain (ND-LIP5) and that one ND-LIP5 molecule bound per AQP2 tetramer. We also showed that phosphorylation of the distal C-terminus of AQP2 allosterically regulates its interaction with LIP5. The stability of AQP2 and AQP2 phoshpho mimics as well as soluble proteins in the presence of detergents was demonstrated by Circular Dichroism (CD). Small angle X-ray scattering (SAXS) measurements provided information on AQP2 in nanodiscs. However, more experimental data would be required to characterize the structural aspects of AQP2:LIP5 interaction. Our data shows that recombinantly expressed AQP0 functions as a water channel when reconstituted into proteoliposomes. We demonstrated for the first time that direct binding of CaM reduces its water permeability in vitro and that phosphorylation of S229 and S235 in AQP0 abolishes the interaction with CaM while S231 shared similar binding affinities as FL AQP0. We further showed that CaM binds AQP0 in a cooperative manner.},
  author       = {Roche, Jennifer Virginia},
  isbn         = {978-91-7422-604-1},
  language     = {eng},
  publisher    = {Lund University, Faculty of Science, Department of Chemistry},
  school       = {Lund University},
  title        = {Protein-Protein Interactions in Human Aquaporin Regulation},
  url          = {https://lup.lub.lu.se/search/ws/files/52998736/Thesis_Jennifer_Roche.pdf},
  year         = {2018},
}