Prof Geraint Thomas
Professor of Biochemistry
My research currently focuses on three main areas:
The role of Arf G-proteins in the control of phospholipid metabolism and signalling
Arfs (ADP-ribosylation factors) are a family of ras-like GTP-binding proteins with roles in cellular signal transduction, adhesion and cargo trafficking. Recently, we have completed a rigorous study of the selectivity of two classes of downstream Arf effectors phospholipase D1 (PLD1) and phosphatiylinositol 4 phosphate 5-kinases (PtdIns4P 5-kinases) between two different Arf isoforms, Arf1 and 6. Arfs 1 and 6 are the most divergent members of this family both structurally and functionally. Existing data on selectivity was not synoptic, principally because of problems associated with the production of recombinant enzymes or Arfs or significant differences in the assay conditions. We have concluded that Arf1/PLD1 is a strongly preferred coupling where as PtdIns4P 5-kinase are entirely promiscuous. We have also established that the likely mode of activation of PtdIns4P 5-kinases by Arfs is by membrane recruitment from the cell cytosol so increasing the apparent substrate on-rate, rather than through significant changes in the kinases specific activity. Simulation studies indicate profound consequences will arises from this Arf isoform selectivity in the coupled, cooperative behaviour of Arf/PLD1/ PtdIns4P 5-kinase triples.
|A molecular model of our ARF1-GDP obtained as part of a single crystal X-ray diffraction study|
In further work we have concentrated on the specific Arf1/PLD1 interaction and studied selectivity for coupled Arf1/PLD1 amongst upstream guanine nucleotide exchange factors (GEFS) of the ARNO/Cytohesin/Grp1 family. We have established that structural differences between The GEF proteins in this family are redundant in the regulation of PLD1. More recently we have reconstituted the regulation of the Cytohesin1/ARF1/PLD1 triple by polyphosphoinositides. Surprisingly the system responds best to PtdIns(3,4,5)P3, indicating either key roles for still unidentified PtdIns(3,4,5)P3-binding proteins or that this specificity is an emergent property of this system.
The hepatocyte cis-Golgi compartment
surrounds the nucleus (stained
The hepatocyte trans-Golgi compartment
(stained with anti-TGN38)
The control of polyphosphoinositide metabolism
Many cell signalling events induce the hydrolysis of phosphatidyinositol 4,5-bisphosphate [PtdIns(4,5)P2] by phospholipase C and the accumulation of the products inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and 1,2-diacylglycerol (DAG). Indeed, many times more PtdIns(4,5)P2 is broken down than is initially present. Consequently, the control of the resynthesis of PtdIns(4,5)P2 by PtdIns4P 5-kinases to meet this increased flux is essential. We have now established that DAG is a direct activator of all known active isoforms of PtdIns4P 5-kinases. This is the shortest and most direct positive feedback loop (a single arc in any model network) to the control of PtdIns(4,5)P2 resynthesis yet discovered and as such is likely to be both the most stable and fastest mechanism of control. Simulation studies incorporating this new regulatory arc model experimentally determined PtdIns(4,5)P2 concentration changes nearly identically.
Control of constitutive protein secretion from the liver
The biggest endocrine protein secretory event in the body of mature mammals is the continuous secretion of albumin in to the blood. For many years this process was not thought to be regulated but to proceed by a default pathway described as constitutive secretion. In the constitutive model the direction (exit face from the hepatocytes) but not rate of secretion is thought to be controlled. Recently we have shown that protein tyrosine kinases and phosphatases play an essential permissive role in this process and that their inhibition leads to blockade of albumin secretion at the level of Trans Golgi Network (TGN). This is the first demonstration of any role for these key signalling proteins in any constitutive secretory process.
More recently by reconstituting the complete albumin processing and secretory pathway in permeabilised hepatocytes we have resolved two chloride dependent processes in the trafficking and maturation of albumin within cells. This has led us to the discovery of a novel chloride-dependent trigger for the destruction of cargos during trafficking. Interestingly, this breakdown cannot easily be ascribed to lysosomal proteolysis and appears to act on properly folded proteins.
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