Control of dynamic palmitoylation: Identification of de-palmitoylating enzymes and their substrates in plants

Regulating protein function in plants - identification of new regulatory enzymes and their substrates

Proteins are the key functional molecules in the cell, forming structural building blocks and performing the many specialized functions a cell requires to survive. Making sure that individual proteins perform the right task at the right time is achieved by modifying them with various small molecules at specific places within the protein structure. This modification can alter where in the cell proteins are found, how they interact with other proteins, target them for recycling or alter how active they are. The modification of proteins that is key to this work is known as palmitoylation and involves the addition or removal of fatty acids from proteins to activate or suppress different responses. These cycles of addition and removal can promote responses through a wide range of different physical mechanisms depending on the individual protein. As these modifications are vital for regulating how a cell operates it is crucial for us to understand the mechanisms and processes that control and regulate these modifications. I have already identified and characterized the mechanisms by which palmitoyl groups are added to proteins in plants. This project aims to identify and characterize the mechanisms by which palmitoyl groups are removed from proteins in plants. This is essential for understanding the regulatory framework surrounding palmitoylation.

Once we have this knowledge we will then be in a position to identify all of the proteins that are regulated by palmitoylation. This will open the door to greater understanding of how cellular function is regulated and helps the organism operate as a whole. Palmitoylation affects nearly 15% of all proteins within the cell but how it regulates their function is essentially unknown. Using the tools we have developed we will identify proteins that undergo de-palmitoylation. This will allow us to specifically study proteins regulated by changes in palmitoylation state and begin to understand how changes in palmitoylation state alter protein function.

Palmitoylated proteins are involved in virtually every process in the cell that involves signaling or transport of molecules from outside of the cell to the inside. These proteins are therefore extremely important for interactions with the outside world and understanding how they are regulated to correctly perform their function will be extremely important for understanding how plants interact with their environment, fight off pathogens, build their cell walls and take up nutrients. This is in turn important for improving the food yield of crops, improving water, land and nutrient use efficiency and helping increase biofuel production - all essential parts of future food and energy security.

Palmitoylation (also known as S-acylation) is a post-translational modification of membrane and membrane-associated proteins that involves the addition of fatty acids to cysteine residues. Our recent work in Arabidopsis indicates that ~40% of the membrane proteome is palmitoylated with almost all membrane associate processes containing palmitoylated proteins. Palmitoylation is unique because it is the only reversible lipid modification of proteins. Consequently, a protein's palmitoylation state can go up or down in response to stimuli. This reversibility of palmitoylation has revealed its regulatory role and has been dubbed "fatty phosphorylation".

This proposal aims to identify and characterize the de-palmitoylating enzymes from plants. We have found that plants do not contain homologues of mammalian, fungal or trypanosomal depalmitoylating enzymes. The serine hydrolase targeting general de-palmitoylation inhibitor HDFP is however effective at preventing de-palmitoylation in Arabidopsis indicating that de-palmitoylation in plants is enzymatic. We will use HDFP in a competitive activity based protein profiling approach to allow the identification of HDFP sensitive de-palmitoylating activities by mass spectrometry. We will then validate and characterize the in vivo and in vitro activity of the identified enzymes against Type-I ROP small GTPases, the best studied reversibly palmitoylated proteins in plants. Finally we will use HDFP inhibition and mutants in identified de-palmitoylating enzymes to identify the dynamic palmitoyl proteome of plants. This will illustrate the range of dynamically palmitoylated proteins and define new areas of plant biology regulated by dynamic palmitoylation

To meet projected global food requirements in 2050 the Food and Agriculture Organisation (FAO) of the United Nations estimates that food production needs to increase by 70% overall (and by 100 % in developing countries). Pests/disease and lack of water are both major causes of crop loss (up to 65% in developing countries) and provide a substantial barrier to global food security. Improved and regulated plasticity in plant architecture, cell wall composition and cellular biochemistry are recognized means to ensure that yields are maximised under changeable growing conditions such as drought, flooding or pathogen attack. Carbon dioxide driven climate change also threatens food security and requires changes to our energy use policies; (waste) plant biomass as fuel feed stock is a key component of achieving carbon neutral energy. The majority of these processes directly involve membrane proteins; understanding how membrane proteins and associated pathways and processes are regulated is therefore required for targeted breeding or manipulation of crops to deliver desired improvements. The proposed research will be exploited as detailed in the Pathways to Impact and is expected to benefit the following areas.

1. Breeders, biotechnology, synthetic biology and industry - This work seeks to characterize an entirely new mechanism key to all aspects of plant life. This work will provide greater understanding of mechanisms at work during plant perception and response to stimuli and allow for greater subtlety and accuracy in manipulating desired traits such as yield, architecture, pathogen resistance or water use efficiency and reducing unwanted effects. This work has the potential to maximize land use, reduce losses pre- and post-harvest and reduce uncertainty in food production. This can be achieved through informed breeding, genetic modification or synthetic biology routes.
2. Consumers - Improved food production efficiency thereby reducing food costs and reducing the use of potentially harmful chemical control measures.
3. Global and UK economic competitiveness - Crops showing increased adaptability to environmental change, increased digestible biomass, resistance to pathogens or improved yield will prove profitable to breeders and biotech companies. Jobs will be created to implement any novel advantageous mechanisms found. Reduced expenditure on disease control and irrigation or increased yield per unit area of land will lead to greater profit margins for growers while ensuring costs are kept low for consumers.
4. Environment - By reducing chemical control use ecological diversity can be maintained or improved and will prevent contamination of watercourses, reduce buildup of chemicals in the soil and reduce greenhouse gas emissions from chemical production, transport and application. Improved water use will reduce soil salination thereby increasing sustainability and will provide more fresh water for human consumption thereby improving health. Carbon neutral biofuels will help stop increases in global carbon dioxide.
5. Policy makers - This work will help keep policy makers informed of progress towards safeguarding food supplies against current and emerging pathogens and environmental change.
6. Research Staff - Staff on the project will be trained in public speaking, presentation preparation, presenting data and information to expert and lay audiences, analytical processes, accurate record keeping and collaborative work. These are widely transferable skills applicable to all employment sectors.

This work is aligned to the "Sustainably enhancing agricultural production" and "Synthetic biology" strategic priorities.

Timescales
Due to the fundamental nature of this work the initial impact will primarily be within academia. In the medium to long term (5-25 years) non-academic parties will likely benefit from translation of these important fundamental data.