Investigating the mechanisms of phosphatidylcholine sensing

Biological membranes are essential for all aspects of life; they separate the interior of cells from their environment; they organize the intracellular space into several compartments, or organelles, each with its own specific role; and they also function as platforms onto which complex chemical reactions take place. The basic building blocks of membranes are lipids. Therefore, it is critical that cells produce lipids at the right time and place. The amount and type of membrane lipids must be carefully controlled to match the specific requirements of each cell: for example, rapidly proliferating cells or cells expanding their intracellular organelles require more lipid synthesis to support the growth of the corresponding membranes. In this project we will investigate how cells monitor and adjust the levels of phosphatidylcholine (PC), the most abundant lipid in eukaryotic membranes.

The enzyme that controls the rate of PC production is called CCTa. Because PC is a cylindrical lipid, its depletion disrupts proper lipid packing within the membrane; this is sensed by CCTa, which is then activated to correct the defect. We recently found that this process takes place inside the nucleus of many different eukaryotic cells: when inactive, CCTa is intranuclear, while when cells need to make more PC, the enzyme associates with the inner side of the nuclear membrane, which separates the nucleus from the cytoplasm. This finding is surprising given that lipid synthesis is mostly a cytoplasmic process. In the proposed work, we will determine how CCTa associates with the inner nuclear membrane, why this process is restricted within the nucleus and how it is coordinated with the final step of PC synthesis which takes place in the cytoplasm.
Our recent work shows that the sensing of PC is highly conserved from simple unicellular organisms to humans. Therefore, we will carry out the initial discovery phase of our research in budding yeast as a model organism that provides rapid insights; in a second phase we will determine how these outcomes apply in human cells. We believe that this work will contribute to the understanding of a fundamental step in lipid metabolism and could guide future therapeutic approaches against lipid metabolic and visual disorders, which are caused by PC deficiency.

Phosphatidylcholine (PC) is the most abundant phospholipid of eukaryotic cell membranes, comprising 30%-60% of total phospholipid mass. Accordingly, PC synthesis and turnover are critical for cell growth and organelle function and their dysfunction leads to a number of human pathologies. Despite our relatively detailed knowledge of how PC is generated, how cells sense and adjust their membrane PC content in vivo remains unclear. Decrease of the PC content in membranes is known to cause lipid packing defects. We have recently found that the choline phosphate cytidylyltransferase (CCTa), the rate-limiting enzyme of PC synthesis, is intranuclear and re-locates to the inner nuclear membrane in response to surface lipid packing defects. CCTa binds the nuclear membrane of cell types with strong requirements for membrane synthesis to induce PC synthesis. This response is conserved in yeast, fly, and mammalian cells, suggesting an ancient mechanism by which CCTa senses PC levels inside the nucleus.
Here we propose to investigate the mechanisms by which nuclear CCTa detects PC. Our preliminary data suggest that the final step in PC synthesis takes place in the cytoplasm. We will test the hypothesis that by keeping the sites of sensing and synthesis of PC separate, cells can induce more efficiently bulk PC production for cell growth. We will then apply genetic and biochemical approaches to identify and characterize the key factors that regulate the activity of CCTa at the inner nuclear membrane. Because nuclear PC sensing is highly conserved in evolution, we will use genetically tractable models in budding yeast to guide our experiments in mammalian cells.

Proper regulation of phosphatidylcholine (PC) synthesis is essential for many aspects of cell physiology, with clinical and biotechnological applications. Therefore the work described in this proposal has significant potential to impact both the bioscience and industrial sectors with particular relevance to the strategic priorities of BBSRC in food, nutrition and health, bioenergy and industrial biotechnology.

(a) Our study of PC sensing and the regulators of CCTa in vivo can benefit the design of new compounds to modify CCTa subcellular localization and activity. It should be emphasized that the crystal structure and in vitro enzymology of CCTa have been studied, making it a good candidate for the future development of small molecule modifiers, once its in vivo regulation and activation is better established. This synergy would be particularly relevant for the following prevalent diseases:
(i) Inherited retinal degeneration (IRDs) disorders, as PC synthesis and CCTa activity appear critical for cone-rod function and mutations in the gene encoding CCTa have been associated with two types of IRD; retinal degeneration affects millions of people worldwide and can have a significant impact on their daily lives.
(ii) metabolic disorders such as obesity, fatty liver and type 2 diabetes, since CCTa activity has been shown to control lipid droplet dynamics, the organelles that store fat in cells; in addition loss of CCTa activity disrupts normal liver function in patients carrying mutations in the gene encoding CCTa; the increasing prevalence of these metabolic disorders in the UK represents a major health threat and results in rapidly growing costs on the national health system.
(b) The project has also the potential to impact the development of drugs combating eukaryotic pathogens, including fungal species that are a major cause of infections worldwide: specific inhibition of membrane targeting and activation of fungal orthologues of CCTa, based on knoweledge gained from the budding yeast enzyme, has the potential to decrease membrane biogenesis and consequently their proliferative potential and infectivity. A number of antifungals target lipid metabolism.
(b) Metabolic engineering of microorganisms and plants to increase TAG production is attracting growing attention as a means to generate new sources of (1) feedstock for biodiesel production, a carbon-neutral and environmentally beneficial alternative to fossil fuels and (2) fatty acids for nutritional use by the biotech and pharmaceutical industry. The generation of sustainable new energy supplies is a priority of the UK government. CCTa is highly conserved in eukaryotic microorganisms, algae (an emerging biodiesel feedstock for oil production) and plants and therefore the mechanisms that govern its activity are prime targets for metabolic engineering strategies that could lead to an increase in lipid droplet biogenesis.

(c) Benefit to the public is generated through a combination of the above factors. Specifically, the public will benefit because of the advancement of basic knowledge and the positive impact on improving health in the UK.