Investigation of Interaction between Aquaporin 5 and Cytoplasmic Proteins CLIP2 and β-catenin

Abstract

In all living organisms, proteins which transport water and other small solutes across the membrane exist and are known as aquaporins. Aquaporins consist out of four monomers, forming a homo-tetramer with a substrate conducting channel in each monomer. Aquaporins can be regulated by either gating or trafficking, which allows the cell to control the permeability of the membrane. Aquaporin 5 is one of the aquaporins which is believed to be regulated by trafficking, partly due to sequence homology to aquaporin 2, which is a well-studied membrane protein and known to be regulated by trafficking. Interactions with other proteins play an important role in the regulatory process of aquaporins and further studies and knowledge regarding their regulatory processes are of great interest, since aquaporins are involved in several pathophysiological conditions such as cancer, obesity, nephrogenic diabetes insipidus (NDI) and Sjögren’s syndrome.

Two proteins suggested to be involved in the trafficking machinery of aquaporin 5, are CLIP2 and β-catenin. CLIP2 and β-catenin are both located in the cytoplasm of the cell and previous studies have indications of interactions between CLIP2 and β-catenin and aquaporin 5. The goal of this study is to investigate whether there is an interaction and at some extent investigate what part of the protein that are interacting. This is done with two techniques, far Western Blot and Microscale Thermophoresis (MST), which both are methods used for protein-protein interaction investigation. Furthermore, the structure of CLIP2 is investigated with crystallization, and co-crystallization between CLIP2 and aquaporin 5 C-termini peptide.

The results from the MST measurements indicates an interaction between aquaporin 5 and CLIP2, where the dissociation constant (Kd) was determined to 97.4 ± 13.8 nM, while far Western Blot indicated the interaction, but the experiment was not reproducible. Further analysis is done on CLIP2 with only one domain present at a time (out of two). This shows no interaction with aquaporin 5, suggesting that it has no or very low affinity when only one domain is present. MST analysis between aquaporin 5 C-termini peptide and CLIP2, an indication of binding can be seen, but with much lower affinity. Regarding β-catenin and aquaporin 5, no interaction is seen in either analysis method. Protein crystals is seen for both crystallization attempts, of CLIP2 and CLIP2 with aquaporin 5 C-termini peptide, making way for further structural analysis.

Popular Abstract

An Investigation of Binding between a Water Channel Protein and Cytoplasmic Proteins

Water is vital for all living organisms. In human cells there are proteins which are specialized in transporting water in and out of the cells over the cell membrane, driven by an osmotic gradient. The proteins responsible for water transportation in cells are known as aquaporins. Earlier studies have shown that aquaporins play a major role in several pathophysiological diseases, where regulation and alteration can be of great interest for further medical development or fundamental knowledge in further research regarding these proteins. Aquaporins are known to be regulated by two different pathways: gating or trafficking. Gating is mainly regulated by conformational changes, while trafficking regulates aquaporins by triggers from the environment, including other proteins in the trafficking machinery. In this study, the focus is on one of the thirteen human aquaporins: aquaporin 5. This protein has a very high sequence similarity to aquaporin 2, which is a well-studied protein, known to be regulated by trafficking. Studies have suggested that aquaporin 5 may also be regulated by trafficking, where other proteins in the cytoplasm are involved in the translocation of aquaporin 5 to the plasma membrane. CLIP2 and β-catenin are two of the proteins which are believed to be involved in the translocation of aquaporin 5, suggested to interact with aquaporin 5 in its trafficking mechanism.

The aim of my thesis is to investigate the suggested interaction between aquaporin 5 and the cytoplasmic proteins CLIP2 and β-catenin. This is done by several purification steps, leading up to two analytical methods: far Western Blot and Microscale Thermophoresis (MST). Furthermore, the structure of CLIP2 is investigated by protein crystallization and co-crystallization with aquaporin 5 peptide. The findings indicate that there is an interaction between aquaporin 5 and CLIP2, which is seen with Microscale Thermophoresis and indicated with far Western Blot. Further analysis is done on CLIP2 with only one of its two domains present which does not show any indication of binding, suggesting that both domains are required for the binding with aquaporin 5 to occur. Analysis with only the C-termini peptide of aquaporin 5 and CLIP2 is also done, showing interaction but with much lower affinity. No protein-protein interaction is seen between aquaporin 5 and β-catenin with either MST or far Western Blot. Considering crystallization, protein crystals is seen for both attempts which is a useful starting point for further analysis of the protein structures.

• author: Nash, Angelica
• supervisor: Susanna Horsefield
• organization: Department of Chemistry
• course: KEMR30 20241
• year: 2024
• type: H1 - Master's Degree (One Year)
• subject: Chemistry
• keywords: Aquaporin 5, β-catenin, CLIP2, protein-protein interactions, trafficking, biochemistry
• language: English
• id: 9163474
• date added to LUP: 2024-06-20 14:34:25
• date last changed: 2024-06-20 14:34:25