A study of the role of anionic lipids in cell signalling: a mechanism for the modulation of ABC multidrug transporters.

All cells are surrounded by a membrane so thin that it cannot be seen using a conventional microscope. This membrane is made of molecules called lipids which give the membrane one of its key properties; it forms a barrier that prevents the movement of many important biological molecules into and out of the cell. Embedded in the membrane are proteins and these proteins provide the machinery that allows material and information to cross the membrane. Therefore proteins provide the cell with the ability to take in nutrients and remove waste and toxic molecules such as drugs.

It was once assumed that the lipids simply provided a barrier in which membrane proteins were located. However, membranes are composed of a range of different lipid molecules and it is now becoming apparent that certain groups of lipid, called anionic lipids, play a role in controlling the biological function of certain membrane proteins.

We are interested in a group of proteins called the multidrug transporters that remove drugs from cells. Normally these proteins protect cells from toxins in the environment, including drugs that would poison the cell, but multidrug transporters also prevent anticancer drugs from killing cancer cells, particularly since the amount of these proteins is increased in cancer cells when they encounter such drugs. Related proteins also remove antibiotics from bacteria resulting in antibiotic resistant bacteria and remove herbicides from the plant cells of weeds leading to herbicide resistant weeds.

The aim of this research is to study the way in which the membrane lipids can affect the function of multidrug transporters. To do this we need to be able to examine the way membrane proteins, like the multidrug transporters, come together with the membrane lipids that surround them and determine how this relationship affects their ability to transport drugs. This is achieved by labeling the protein and lipid with agents that give a particular signal which occurs only when the lipid and the protein are in contact. We measure this signal, which is in the form of light, using a machine called a fluorometer, which measures the light given out by the protein and lipid in combination. By labeling different lipids we can determine which lipids have a closer relationship with the multidrug transporter and in similar experiments we can evaluate what this association does to the drug transporting ability of the protein. Because we know that the anionic lipids in the membrane change in amount and distribution when cells respond to changes in their environment we will be able to deduce what affects these lipid changes will have on drug transport.

These studies will tell us how signals are transmitted to membrane proteins by changes in membrane lipid composition and distribution. In addition a greater understanding of how multidrug transporters are controlled by lipids, may suggest ways in which these proteins can be controlled by the use of novel drugs that would also interact with these multidrug transporters. This could help to tackle treatment failures caused by the serious problems of antibiotic resistance in bacteria and resistance to anticancer drugs seen with repeated rounds of chemotherapy. A similar approach could be taken to provide strategies for reducing the resistance seen with a whole range of important molecules, including pesticides, herbicides, anti-malarials, etc.

There is evidence that the activities of many membrane proteins are modulated by anionic phospholipids in the bilayer. The levels and distribution of anionic lipids are modified in response to cellular events and this effect on membrane proteins provides a largely unexplored mechanism of cell signalling. We will characterise the binding of anionic lipids to ABC multidrug transporters SAV1866, from S. aureus, and mouse P-glycoprotein. Potential anionic lipid binding sites will be revealed by inspection of their crystal structures and coarse-grain molecular dynamic simulations of anionic lipids with these proteins. Anionic lipid binding sites will be established by replacing the three Trp residues in SAV1866 with single Trp residues at positions adjacent to potential anionic lipid binding sites. These single Trp mutants will be reconstituted into defined lipid bilayers containing brominated lipids that will quench Trp fluorescence by contact quenching. An analysis of the fluorescence quenching profiles in the presence of zwitterionic and anionic brominated lipids will identify the location of specific anionic lipid binding sites. Anionic lipid binding will also be correlated with effects on SAV1866 multidrug transport by measuring drug transport and ATPase activity in defined lipid bilayers and we will use mutagenesis of positively charged residues associated with the anionic lipid binding sites to further characterise these sites. We will also extend our studies to examine potential anionic lipid binding sites on P-glycoprotein.

These studies will demonstrate how alterations in lipid composition and distribution (as occurs during the transition from log to stationary phase in bacteria or in lymphocyte signalling) can act to alter membrane protein function. In addition the characterisation of anionic lipid binding sites will identify targets that could be exploited in reducing drug resistance encountered in the treatment of cancer, microbial infections, etc.

Beneficiaries of this research

Commercial sector: This proposal deals with cell signalling at a fundamental level by examining a largely unexplored mechanism by which membrane protein function may be modulated through a direct interaction with anionic lipids. However, we have chosen to examine this phenomenon in the context of two multidrug ABC transporters, one from the prokaryotic kingdom, SAV1866 and the other a mammalian homologue, P-glycoprotein. Although it is difficult to extrapolate, any insight into how multidrug transporters are modulated may be of value in areas of agriculture and medicine where such transporters reduce the effectiveness of selectively toxic compounds including, antibiotics, anthelmintics (for human and agricultural use), anti-malarials, pesticides, herbicides and anticancer drugs. By studying SAV1866 and P-glycoprotein it may be possible to identify drug targets for the development of agents that could act synergistically alongside antibiotics, anthelmintics (for human and agricultural use), anti-malarials, pesticides, anticancer drugs and herbicides to reduce resistance.

General public: Antibiotic resistance and drug resistance exhibited after multiple rounds of chemotherapy are major problems in the National Health Service. As discussed above these studies may provide target sites for the design of drugs that could act synergistically with antibiotics and anticancer drugs to ameliorate the problem of drug resistance.

Nature of the benefits stemming from this research

Given that this is basic research and at an early stage of development it is difficult to estimate to what degree and on what timescale benefits might emerge, beyond what has been discussed above. However this research will promote the study of membrane protein/lipid interactions at the molecular level in a manner that will help identify novel signalling pathways and novel drug targets on a range of membrane proteins in addition to the ABC multidrug transporters. The postdoc working on this project will pick up a range of specialist and transferable skills that would be of value in future employment, but we are particularly keen to ensure that they play a central role in communicating this work to a wider audience.