Clathrin assembly regulation of glucose metabolism

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

Organs and tissues are made up of cells that must be able to respond to their environment in order to carry out many tasks, including regulating growth, fighting infection and controlling nutrition. A critical step in nutritional regulation is the ability to maintain blood sugar levels after eating. The body does this by moving a protein called GLUT4 in fat and muscle cells from within the cell to the cell's surface in response to insulin, which is released when blood sugar levels are high. GLUT4 acts to shuttle glucose (the form of sugar in the blood) into the cell, thereby reducing blood sugar levels. As such, in a fasting state, GLUT4 is retained inside the cell to prevent blood sugar levels becoming dangerously low, but is moved to the cell surface in response to the insulin released after you eat to prevent blood sugar levels becoming dangerously high. The GLUT4 is then moved back inside the cell when blood sugar levels have returned to normal. Disruption of this ability to control blood sugar levels can lead to diabetes.

Key to controlling blood sugar levels, therefore, is the ability to move GLUT4 from its specialised storage site to the cell surface in response to insulin, and then remove it from the cell surface after enough glucose has been removed from the blood. For this, a protein called clathrin is critical. Named for its clathrate (lattice-like) structure, clathrin acts to ensure that a huge range of proteins are in the right place in the cell at any time. Multiple molecules of clathrin self-assemble into basket-like coats that form and wrap around 'vesicles' that pinch off from membranes and are then transported to another part of the cell, in a mechanism known as 'membrane trafficking'. There are two forms of clathrin. The originally discovered form, referred to as CHC17, is best known for a stage in membrane traffic called 'endocytosis', the process by which proteins are moved from the cell surface to the interior of the cell. More recently, a second form of clathrin, CHC22, has been identified and been shown to be involved in a separate process. Instead of functioning at the cell surface, CHC22 operates entirely within the cell to move GLUT4 to its storage compartment, from where it is able to respond to insulin. Therefore, both forms of clathrin are critical to ensure that GLUT4 is trafficked properly in response to changing blood sugar levels. Recently, it has been discovered that humans have evolved two different forms of CHC22, and these two variants change how individuals are able to respond to blood glucose levels.

A key step in understanding clathrin function is to understand how clathrin self-assembles into coats. Here, two laboratories that have studied clathrin biology for many years are joined by two other experts in analysing the structures and interactions of proteins to answer this question. Thanks to exciting recent advances in the structural knowledge of clathrin led by one of the investigators collaborating here, detailed structures of CHC17 assemblies, including many key interacting points, are now known for the first time. This work aims to build on this knowledge to compare the contacts that are critical to assemblies of CHC17 and CHC22, and test how these contact points affect assembly rates of both clathrins. The structure of CHC22 assemblies is less well known than that of CHC17. This work aims to rectify that, and will determine the structures of the two major CHC22 variants. We will use this information to investigate how the regulation of clathrin assembly affects GLUT4 trafficking in response to insulin in cells. Therefore, this work will help to understand the mechanisms by which clathrin functions, which could in turn aid in the development of therapeutic strategies to help alter blood glucose clearance, for instance in diabetic patients.

Technical Summary

Clathrins are self-assembling vesicle coat proteins that mediate many membrane traffic pathways. There are two forms of clathrin, CHC17 and CHC22. CHC22 acts to sequester the GLUT4 glucose transporter in an insulin-responsive compartment. Recent work by the Brodsky lab has shown there are two major genetic variants of CHC22 that differ in assembly dynamics and alter GLUT4 sequestration, and thus insulin response. The recent 4.7 Å resolution cryo-EM model of CHC17 baskets from the Smith lab now reveals molecular contact points in assembled clathrin that can be tested for function for both clathrins and structurally validated for CHC22. This project has three specific aims, building on research from the two collaborating labs, to elucidate functional contact points in CHC17 and CHC22 baskets, and investigate how stability of CHC22 assembly alters GLUT4 traffic.

Aim 1 Identify functional contact points in CHC17 and CHC22: The roles of identified contact points in clathrin assembly will be tested in light scattering assays and cross-linking mass spectrometry will be used to compare the self-assembly contacts for CHC17 and CHC22. X-ray crystallography will be used refine the CHC17 cryo-EM model.
Aim 2 Map the molecular structure of CHC22: Protocols for expression and purification of recombinantly produced CHC22 proteins and fragments will be optimised. CHC22 baskets isolated from HeLa cells and recombinant proteins will be used to solve high resolution structures of CHC22 by combination of cryo-EM and X-ray crystallography.
Aim 3 Determine effects of CHC22 assembly on GLUT4 traffic: Mutagenised CHC22 will be expressed in relevant cell models to determine how CHC22 assembly modulates the translocation of GLUT4 in response to insulin, using microscopy and FACS.

This project significantly advances our understanding of molecular mechanisms controlling clathrin assembly and how they regulate cargo transport, and will produce the first structural analysis of CHC22.

Planned Impact

There are two forms of clathrin proteins in human cells, CHC17 and CHC22. Both are essential to the function of the membrane traffic pathway by which the GLUT4 glucose transporter removes glucose from blood after a meal in response to insulin. The proposed research will deliver advanced knowledge of CHC17 structure to understand its assembly mechanism and establish the structure and assembly mechanism of CHC22. Manipulation of the assembly mechanisms of CHC22 will be tested for its effects on the GLUT4 transport pathway and its response to insulin. These studies will reveal fundamental features of how muscle and fat tissue acquire glucose during a healthy diet. As this study addresses normal human nutrition, as well as basic mechanisms of cell biology, there are several groups who stand to benefit from the outcomes of this research:
1. Industry and private enterprise: One approach to dealing with the increase in glucose metabolism disorders is to understand the fundamental mechanisms of glucose metabolism. This could lead to dietary supplements that enhance glucose uptake from the modern diet, potentially curbing the alarming trend towards insulin resistance and resulting diabetic conditions. Understanding the molecular mechanisms of assembly for both CHC17 and CHC22 clathrin is essential for the development of drugs to specifically improve uptake of glucose into cells. As such, pharmaceutical companies interested in this preventative approach could directly benefit from this work.
2. Medical professionals: This project aims to understand how the clathrin genetics alters human GLUT4 trafficking, and consequently glucose tolerance. Therefore a variety of medical professionals, including healthcare providers, nutritionists and epidemiologists are likely to benefit from this improved understanding of the molecular regulation of GLUT4 transport.
3. Early career researchers: Those associated with the project will benefit from collaborations across the disciplines of cell biology, biochemistry, structural biology and biophysics and from the excellent training that they will gain in sought after skills such as cryo-electron microscopy, protein chemistry and data analysis. These skills will transfer into their future careers in whatever sector they work and the UK as a whole will benefit from such well-trained cross-disciplinary scientists. In addition, the early career members of the team will receive valuable training through opportunities at the University of Warwick and UCL for developing outreach skills.
4. Societal benefits: This research is likely to provide additional longer term benefits, including reducing cost to the NHS, increasing commercial success for companies designing drugs based on this knowledge and improving the health and well-being of the general public. For example, the Brodsky lab has established a connection with diabetic patients who have become actively engaged with their group, providing input into their work on disease mechanisms.
5. Wider public audience: The rise in incidence of insulin resistance in the UK has increased interest in food and metabolism amongst the general population. For example, the recent publication from the Brodsky lab investigating the genetic diversity of CHC22 and its role in glucose metabolism received coverage in several non-scientific national publications, including The Guardian, The Daily Mail and the Economist. The academics and early stage researchers will continue their programme of science communication with the general public, engaging with local schools, local and national media, science fairs, departmental public science events, etc. The investigators and co-PI's have significant experience and training in media engagement, serving as role models for team members to take part in such activities early in their careers and establishing lasting habits and expertise for benefitting the general public. This impact will be both immediate and far-reaching.

Publications

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