Human Glut3 Production for Structural Studies
(2023) MOBN03 20222Degree Projects in Molecular Biology
- Abstract
- Glucose transporters, known as Gluts, play an important role in facilitating the transport of sugar across the cell membrane. Of these, 14 types are known, one of which is Glut3, which is highly expressed in the brain, testicles and nervous system.
Its importance lies in the fact that it is responsible for the uptake of glucose in cells with high sugar requirements, such as neurons. Glucose uptake by Glut3 is significantly increased in cancer cells, a phenomenon discovered by Otto Warburg in the 1950s, as they depend on glucose metabolism for their growth and proliferation.
Targeting glucose metabolism in cancer cells has become an important research area, as blocking glucose uptake is a potential therapeutic strategy. A class of drugs... (More) - Glucose transporters, known as Gluts, play an important role in facilitating the transport of sugar across the cell membrane. Of these, 14 types are known, one of which is Glut3, which is highly expressed in the brain, testicles and nervous system.
Its importance lies in the fact that it is responsible for the uptake of glucose in cells with high sugar requirements, such as neurons. Glucose uptake by Glut3 is significantly increased in cancer cells, a phenomenon discovered by Otto Warburg in the 1950s, as they depend on glucose metabolism for their growth and proliferation.
Targeting glucose metabolism in cancer cells has become an important research area, as blocking glucose uptake is a potential therapeutic strategy. A class of drugs has been found that work to inhibit Glut proteins and thus prevent glucose uptake into cells. Salicylketoxime derivatives show an inhibitory effect on Gluts.
The aim of this thesis is to express, purify, and crystallize human Glut3 in complex with salicylketoxime inhibitors to gain insight into the molecular details of their binding and interactions with Glut3, which would facilitate further compound development.
The protein was expressed in insect cells using the Baculovirus Expression Vector System, solubilized, and purified using metal affinity and size exclusion chromatography. The purified protein was crystallized by a hanging drop vapor diffusion method, and initial crystals were obtained. The crystals were tested at the BioMAX beamline at MAX IV and diffracted to around 13Å. This is a promising result, indicating that with further optimization higher resolution could be achieved and the Glut3-inhibitor complex structure determined.
Overall, the study of Glut3 and its relationship to glucose transport, cancer, and other diseases provides insights into the development of therapeutics and targeted therapies to prevent glucose uptake in cancer cells and improve understanding of glucose metabolism in various health conditions. (Less) - Popular Abstract
- Glucose transporters are proteins found in the cell membranes that transport glucose into cells, which is the primary source of energy. There are many types of Gluts, one of which is Glut3. Glut3 is expressed in specific tissues such as the brain, nerve cells, and testes, and plays an important role in transporting glucose to these tissues in order to provide them with energy. In cancer cells, the rate of glucose uptake is higher, because they depend heavily on glucose metabolism for rapid growth and reproduction. Researchers are studying the metabolism of cancer cells to find ways to target and treat cancer. A very promising method is to block glucose uptake or metabolism in cancer cells.
Scientists are developing drugs that target... (More) - Glucose transporters are proteins found in the cell membranes that transport glucose into cells, which is the primary source of energy. There are many types of Gluts, one of which is Glut3. Glut3 is expressed in specific tissues such as the brain, nerve cells, and testes, and plays an important role in transporting glucose to these tissues in order to provide them with energy. In cancer cells, the rate of glucose uptake is higher, because they depend heavily on glucose metabolism for rapid growth and reproduction. Researchers are studying the metabolism of cancer cells to find ways to target and treat cancer. A very promising method is to block glucose uptake or metabolism in cancer cells.
Scientists are developing drugs that target specific glucose transporters that are overexpressed in cancer cells, such as Glut1 and Glut3. These drugs, called Gluts inhibitors, block the activity of Glut proteins, thereby preventing glucose uptake into cancer cells. For example, a family of Glut inhibitors called salicylketoxime derivatives has been developed as a potential treatment for cancer.
Glut3 consists of a unique structure that are transmembrane helices that form a sugar-binding cavity. Glucose is transported in an alternate transport mechanism, which opens and closes to allow glucose to enter the cell. Glut3 has a high affinity for glucose and is suitable for glucose transport even at low concentrations. Glut3 is not only related to cancer but also to other diseases such as Alzheimer's and Huntington's disease. Therefore, understanding the role of Glut3 in these diseases could lead to detection of them early and targeted therapies.
This thesis provides an overview of Glut3 and its expression in tissues and its role in glucose transport and energy metabolism. It spotlights on the relationship between Glut3 and cancer. The thesis also discusses the molecular structure of Glut3 and its alternating transport mechanism. The importance of Glut3 and its role as a neuronal glucose transporter was emphasized, as Glut3 has a high affinity for glucose and is of great importance in conditions of low glucose concentration.
The Glut3 protein was produced using Sf9 cells derived from an insect. When the Sf9 cells are infected with the specific virus, modified to carry a Glut3 gene, they start making large amounts of the protein of interest, Glut3. The cells are then used to extract Glut3 from their membranes by various protein separation techniques. Once the pure Glut3 sample is obtained it is further used to grow protein crystals, which are needed to obtain X-ray diffraction data. By processing this data, the 3D molecular structure of the protein can be resolved which allows to examine how the protein interacts with potential drug compounds. This provides scientists with the necessary information to further develop the therapeutics.
The result was successful expression, purification, and production of Glut3 for further research on its structure and function. This research contributes to the development of targeted therapies and potential interventions for cancer treatment. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9136019
- author
- Makhzoum, Iman
- supervisor
- organization
- course
- MOBN03 20222
- year
- 2023
- type
- H2 - Master's Degree (Two Years)
- subject
- language
- English
- id
- 9136019
- date added to LUP
- 2023-09-01 14:55:47
- date last changed
- 2023-09-01 14:55:47
@misc{9136019,
abstract = {{Glucose transporters, known as Gluts, play an important role in facilitating the transport of sugar across the cell membrane. Of these, 14 types are known, one of which is Glut3, which is highly expressed in the brain, testicles and nervous system.
Its importance lies in the fact that it is responsible for the uptake of glucose in cells with high sugar requirements, such as neurons. Glucose uptake by Glut3 is significantly increased in cancer cells, a phenomenon discovered by Otto Warburg in the 1950s, as they depend on glucose metabolism for their growth and proliferation.
Targeting glucose metabolism in cancer cells has become an important research area, as blocking glucose uptake is a potential therapeutic strategy. A class of drugs has been found that work to inhibit Glut proteins and thus prevent glucose uptake into cells. Salicylketoxime derivatives show an inhibitory effect on Gluts.
The aim of this thesis is to express, purify, and crystallize human Glut3 in complex with salicylketoxime inhibitors to gain insight into the molecular details of their binding and interactions with Glut3, which would facilitate further compound development.
The protein was expressed in insect cells using the Baculovirus Expression Vector System, solubilized, and purified using metal affinity and size exclusion chromatography. The purified protein was crystallized by a hanging drop vapor diffusion method, and initial crystals were obtained. The crystals were tested at the BioMAX beamline at MAX IV and diffracted to around 13Å. This is a promising result, indicating that with further optimization higher resolution could be achieved and the Glut3-inhibitor complex structure determined.
Overall, the study of Glut3 and its relationship to glucose transport, cancer, and other diseases provides insights into the development of therapeutics and targeted therapies to prevent glucose uptake in cancer cells and improve understanding of glucose metabolism in various health conditions.}},
author = {{Makhzoum, Iman}},
language = {{eng}},
note = {{Student Paper}},
title = {{Human Glut3 Production for Structural Studies}},
year = {{2023}},
}