Mechanisms of LAM-mediated intracellular sterol traffic and its regulation by conserved kinases

Understanding how cells control cholesterol traffic

The purpose of this project is to improve understanding of traffic of cholesterol inside cells, a poorly understood process of vital importance to the health of all cells, from humans to fungi and plants. Cells are separated from the outside world by the plasma membrane, which consists of a bilayer (two apposed layers) of fat molecules (lipids) that prevent the escape of molecules inside the cell. The key lipid for the plasma membrane is cholesterol, which strengthens the membrane and prevents it from dividing up into different regions, which would be toxic and may contribute to a wide variety of societally important diseases from atherosclerosis to Alzheimer's.

When the physical properties of the plasma membrane have to change, for example when body temperature increases, levels of cholesterol in the plasma membrane must change so that its physical properties match the new conditions. Cholesterol flow into and out of the plasma membrane is carried out by specialised sterol transfer proteins, which fold into the overall shape of a box with a hinged lid. They accommodate a single cholesterol molecule inside, and they can repeatedly shuttle sterol from one site inside cells to another. We recently discovered a large family of sterol transfer proteins called LAMs in all cells in humans, fungi and plants. LAMs transfer excess cholesterol away from the plasma membrane to an internal organelle called the endoplasmic reticulum, where is converted into an oil form that is stored in fat bodies for later use.

Cells have several pathways that respond to new conditions, each pathway containing multiple proteins. Most of these pathways are regulated by the activity of proteins called kinases, which physically modify downstream pathway components to switch them on. Among the major kinases found in all cells in all complex life forms is TORC2. Like a thermostat counteracting temperature changes, TORC2 responds to changes in the plasma membrane by modifying components in several pathways that all counteract the original changes. TORC2 is known to affect many different lipids of the plasma membrane, but so far it has never been linked directly to cholesterol.

This three year project is based on our new evidence that cells use TORC2 to control the activity of LAMs. We will find out if the important sterol trafficking function of LAMs is controlled by TORC2 as our data suggests. We will also test the paradigm that master regulators like TORC2 have multiple outputs that combine to create synergy. For this we will study how cells combine changes in cholesterol with other changes in lipids of the plasma membrane that TORC2 enacts. We have also found out that cells sometimes rapidly destroy LAMs. Targeted protein destruction is a very common way in which pathways are controlled; indeed a major way TORC2 works is by altering the activity of the machinery that destroys specific proteins. Therefore, we will find out how LAMs are destroyed and whether destruction of LAM proteins is a second layer of control imposed by TORC2 on cholesterol traffic.

Our work will be carried out in budding yeast where the LAM and TORC2 pathways are better understood than in human cells. However, all the participating proteins and lipids involved in yeast have counterparts in humans, and we will examine if the effects we discover in yeast apply in human cells.

The practical applications following on from our basic research will be to help devise ways to manipulate many processes where cholesterol movement is important. Because LAM proteins and TORC2 are found in all complex life forms, our research will produce knowledge that is applicable to plants, fungi, and animals. Our results will add to the understanding not only of healthy ageing (atherosclerosis, Alzheimer's, type II diabetes), but also for food security since cholesterol traffic is vital both to crop plants and to fungi that destroy crops.

Sterol is the most abundant lipid in the plasma membrane (PM), where it fulfils many vital functions such as rigidification and emulsification. Levels of PM sterol are homeostatically maintained, but the mechanism is poorly understood. When PM sterol is in excess, for example when delivered from outside, it is transferred to the endoplasmic reticulum (ER) for storage in lipid droplets, but the transfer pathway has been obscure. We recently discovered a large family of sterol-specific lipid transfer proteins conserved in all eukaryotes called LAMs. In yeast, three LAMs mediate sterol traffic from PM to ER: Lam1p, Ysp2p, and Sip3p. Our new preliminary data focus on Ysp2p, which is phosphorylated by Ypk1p, the main kinase downstream of the conserved PM stress sensor TORC2, known to regulate other PM lipids but not sterols. Ysp2p that cannot be phosphorylated by Ypk1p is more active in indirect assays. We also found a possible second route by which TORC2 regulates Ysp2p: its stability depends on forming complexes with Sip3p, and a region of Ysp2p critical for stability has multiple ubiquitination motifs.

Our most important objective is to determine how TORC2 regulates sterol traffic through phosphorylating Ysp2p. We will show how sterol traffic integrates with other lipid effectors of TORC2 to enact an overall PM stress programme. For the second layer of regulation, we will study ubiquitination of Ysp2p, and see how forming complexes with another LAM prevents degradation. Yeast is the ideal model system for rapid progress, but both these regulatory pathways are highly conserved from yeast to man, so we will study TORC2-mediated regulation of LAMs and stability of LAMs in human cells

Bioscience for Health:

The project studying yeast and human LAMs and their conserved regulation by TORC2 has many strands that impact on the BBSRC priority of healthy ageing across the lifecourse. Sterol metabolism is vital for animal health (incl. humans), where "bad cholesterol" is a major treatable cause of atherosclerosis, a huge cause of chronic ill-health and premature death. Dysfunctional cholesterol metabolism also underlies many other diseases, from Alzheimer's Disease to cancer. Little is known about LAMs in humans, except links to cancer and chemo-resistance, because no directed studies of LAMs have yet been carried out. TORC2, not previously studied as a regulator of sterol traffic, is important in many aspects of healthy ageing, including neuroprotection, metabolic syndrome/type II diabetes, and neoplasia. The fundamental insights we hope to obtain in this study may well have benefits for these areas of health, although we cannot yet predict these in detail.

Human harms arising from fungi include increasingly common lethal invasive fungal infections (Aspergillus, Candida, Cryptococcus, Pneumocystis) in immunosuppressed people. Fungal infections also occur in previously healthy people, both in LEDCs (Mucormycosis, Fusarium etc.), and in developed countries (C gattii). Many severe fungal infections are treated with AmBisome, a liposomal formulation of Amphotericin B (AmB) that lowers toxicity, but costs >100x base price of AmB. Toxicity, high cost and poor treatment outcome (~40% mortality, 4000 deaths/yr in UK) all justify major investment to find drugs that synergise with AmB. This project will provide fundamental insight into regulation of LAMs, which is a vital precursor for the development of small molecular LAM inhibitors that will render fungi highly sensitive to AmB. This could pave the way for a dual therapy inhibiting LAMs in combination with AmB, which could be well tolerated and highly effective . Thus, pharma companies developing new antifungals will be big beneficiaries. We collaborate with F2G Ltd, an SME dedicated to developing antifungals.

Agriculture and food security (Fungi):

The project will have an impact on important fungal pathogens, relating to BBSRC priorities both of combatting antimicrobial resistance and of sustainably enhancing agricultural production. This includes not only fungi that infect humans, but also those that blight crops: 10% of UK crops are lost to fungi, and LEDCs face higher losses (>$200Bn loss / year world-wide). Some fungal infections threaten to globally devastate host species: Fusarium wilt TR4 - bananas, Chytridiomycosis - frogs, Chalara fraxinea - Ash trees ("dieback"). All of these are important to the UK and globally, and the solutions lie in better understanding of fungal cell biology to exploit their vulnerabilities. The major sterol of fungi is ergosterol, which differs subtly from both animal cholesterol and plant phytosterol, producing a therapeutic window. LAMs show enormous promise as pharmaceutical targets, in particular because inhibitors of this protein superfamily are already in development (HPA-12).
Crop losses to fungi are minimised by repeated antifungal treatments. In the UK alone fungicides mainly targeting sterol pathways prevent £200M/y wheat wastage by septoria leaf blotch. However, resistance is increasing, and to ensure food security we must develop new antifungal strategies. Therefore, beneficiaries will include more secure crop yields.

In addition to fungal targets, LAMs in plants regulate their responses to pathogens. Our work will advance understanding of how economically important plants resist pathogens, and benefit food security in a second way.

Societal Impact:

The project will impact positively on society via communication of our results to the general public using WWW resources. In addition, we will use the reputation of UCL in teaching and research to communicate our work to a wide audience.