Abstract
Cells require a constant supply of sugar and carbon source to power all the metabolic processes that maintain life. Photosynthetic organism can synthesized sugar through carbon fixation. This sugar is hydrolyzed and distributed throughout the plant body, but can also be consumed and used by other organisms. Glucose is the preferential source of chemical energy. The GLUTs and the SWEET1 transporters are integral membrane proteins in humans that facilitate the transport of glucose and other hexoses across the cell membrane. The GLUTs are a wide group of proteins and among the 14 members, GLUT1-4 are of particular interest due to their relevance in several metabolic diseases. The GLUTs belong to the Sugar Porter (SP) family, which represents a large subfamily of the Major Facilitator Superfamily (MFS). The MFS also includes several plant transporter proteins. In plants, sucrose is synthesized in source tissues and after being transported to the apoplastic space is hydrolyzed to monosaccharides and distributed by the MFS, Sugar Transporter Proteins (STP). This study explores the expression profile, function, binding affinity and structural insights of the two systems that function as passive uniporters in humans (The GLUTs and SWEETs) and the plant STP proteins that function as symporters.
The hGLUT1 transporter is probably the most well study protein among the GLUTs and this work describes the crystal structure of hGLUT1 in the presence of glucose, at 2.4 Å resolution. This structure is identical to the previous solved structures with the addition of a secondary glucose-binding site in the cytosolic domain, which can give a new interpretation regarding the GLUTs mechanism of regulation. However, the possible physiological role of this internal binding site is out of the scope of this study. Instead, the binding-site residues were inspected by mutational studies. Furthermore, a single point mutation converted GLUT1 kinetics into a GLUT3-like transporter. Further studies are required in order to inspect the oligomerization state and expression profiles of wild-type proteins and mutants. Nevertheless, this work has pinpointed several residues that are essential for the modulation of the conformation state of the transporter, and hence its kinetics.
This work also includes the 2.4 Å crystal structure of the Arabidopsis thaliana high affinity sugar transport protein, STP10. The structure was captured in an outward-occluded state with a glucose bound in the central cavity. These proteins were predicted to have the MFS fold and other structural features from the Sugar Porter family. However, significant variations were also found between the amino acid sequence of STP10 and its closest homologue in bacteria. Indeed, the structure revealed a cluster of aromatic residues forming a helix-helix-loop-helix, doubt Lid domain, at the extracellular side. This domain sits on top of both transmembrane domains, locked by a disulfide bridge. This structural feature has never been seen before in a MFS member but is fully conserved among all Arabidopsis thaliana STP proteins. The structure allowed for the extrapolation of a model that links proton and sugar transport. Furthermore, functional data suggests a shielding role for the Lid domain to allow efficient coupling of the proton gradient to drive glucose transport.
Moreover, this work describes strategies for successful purification of two additional human sugar transporters in the yeast Saccharomyces cerevisiae system. The human GLUT9 has a unique substrate affinity since, in addition to transporting hexoses, it also is a high-capacity uric acid transporter. Elucidating the homeostasis of uric acid through the role of GLUT9 could lead to discoveries with significant clinical implications. In humans, the SWEET family contains one member. The SWEET1 physiological role remains speculative and structural information would be greatly beneficial. This study also includes the numerous used approaches to crystallize SWEET1.
In summary, the work described in this dissertation provides a better understanding of the basic mechanism of sugar transport in humans and plants.
The hGLUT1 transporter is probably the most well study protein among the GLUTs and this work describes the crystal structure of hGLUT1 in the presence of glucose, at 2.4 Å resolution. This structure is identical to the previous solved structures with the addition of a secondary glucose-binding site in the cytosolic domain, which can give a new interpretation regarding the GLUTs mechanism of regulation. However, the possible physiological role of this internal binding site is out of the scope of this study. Instead, the binding-site residues were inspected by mutational studies. Furthermore, a single point mutation converted GLUT1 kinetics into a GLUT3-like transporter. Further studies are required in order to inspect the oligomerization state and expression profiles of wild-type proteins and mutants. Nevertheless, this work has pinpointed several residues that are essential for the modulation of the conformation state of the transporter, and hence its kinetics.
This work also includes the 2.4 Å crystal structure of the Arabidopsis thaliana high affinity sugar transport protein, STP10. The structure was captured in an outward-occluded state with a glucose bound in the central cavity. These proteins were predicted to have the MFS fold and other structural features from the Sugar Porter family. However, significant variations were also found between the amino acid sequence of STP10 and its closest homologue in bacteria. Indeed, the structure revealed a cluster of aromatic residues forming a helix-helix-loop-helix, doubt Lid domain, at the extracellular side. This domain sits on top of both transmembrane domains, locked by a disulfide bridge. This structural feature has never been seen before in a MFS member but is fully conserved among all Arabidopsis thaliana STP proteins. The structure allowed for the extrapolation of a model that links proton and sugar transport. Furthermore, functional data suggests a shielding role for the Lid domain to allow efficient coupling of the proton gradient to drive glucose transport.
Moreover, this work describes strategies for successful purification of two additional human sugar transporters in the yeast Saccharomyces cerevisiae system. The human GLUT9 has a unique substrate affinity since, in addition to transporting hexoses, it also is a high-capacity uric acid transporter. Elucidating the homeostasis of uric acid through the role of GLUT9 could lead to discoveries with significant clinical implications. In humans, the SWEET family contains one member. The SWEET1 physiological role remains speculative and structural information would be greatly beneficial. This study also includes the numerous used approaches to crystallize SWEET1.
In summary, the work described in this dissertation provides a better understanding of the basic mechanism of sugar transport in humans and plants.
| Originalsprog | Engelsk |
|---|
| Forlag | Århus Universitet |
|---|---|
| Antal sider | 188 |
| Status | Udgivet - maj 2019 |