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Quantitative analysis of protein S-palmitoylation in host resistance to intracellular pathogens.

Periodic Reporting for period 2 - QuantPalm_immunity (Quantitative analysis of protein S-palmitoylation in host resistance to intracellular pathogens.)

Reporting period: 2017-07-01 to 2018-06-30

The failure in the treatment of infectious diseases by antibiotics, also known as antimicrobial resistance (AMR) is estimated to be responsible for 25,000 deaths per year in the EU and 700,000 deaths per year worldwide. The European commission recognizes the pressing need for new antimicrobial drugs and recently adopted a new action plan against AMR (June 2017). The new action plan aims to boost research and to address knowledge gaps with the aim to develop new antibiotics exploiting novel modes of action. “QuantPalm_Immunity” addresses one of the knowledge gap.
The immune system is a network of cells, tissues and organs responsible of defending the body against attacks by foreign invaders that can cause infections. The foreign invaders are known as pathogens and they include bacteria, parasites and viruses. It is not fully understood how cells can recognize and destroy pathogens or alternatively how pathogens can escape host defense and lead to infection. Understanding the mechanisms by which host cells resists to pathogens and alternatively how pathogens achieve increased virulence against their host, provides our greatest weapon in the fight against microbial infection and may reveal new classes of antimicrobial targets.
Proteins are synthesized based on their DNA sequence and are often modified by small molecules such as lipids and sugars. These small modifications are essential for proteins to function properly in cells. The first objective of the action “QuantPalm_Immunity” was to find which proteins involved in the fight to pathogens infection (immune proteins) are modified with a lipid, palmitic acid, and to study if levels of this lipid modification change upon infection. The second objective of the research was to understand why the lipid is important for the protein function and correct localization in cells. The third objective was to understand how this lipid modification is important to destroy the pathogen via a process called autophagy.
Research addressing the first and second objectives has been carried out for two years at the partner organization, The Rockefeller University (New York, USA), in a lab specialized in lipid modifications of proteins. The third objective was implemented at the Crick (London, UK) in a lab specialized in autophagy.
The action “QuantPalm_Immunity”, was implemented at the partner organization, The Rockefeller University (New York, USA) for 2 years, in a lab specialized in lipid modifications of proteins and for one year at the host organization, the Crick (London, UK) in a lab specialized in autophagy. The first objective of the action was to find which proteins involved in the fight to pathogen infection (immune proteins) are modified with palmitic acid, and to study if levels of this lipid modification changes upon infection. Palmitic acid is typically added to Cysteine residues of proteins. The first objective could be accomplished with the development of a method allowing to 1) detect which proteins are modified by the lipid, 2) detect the protein and Cysteine modified and 3) quantify the amount of protein modified by the lipid. This could be done by “mass-spectrometry-based quantitative proteomic”. This analysis method allows to identify which peptides are found in the sample by detecting their mass, which protein they belong to and in what relative quantity they were found (relative quantity compared to other proteins or other samples).
The development of a mass-spectrometry (MS) based quantitative proteomic method proved to be highly challenging due to low solubility of the proteins modified with palmitic acid. We thus developed a proteomic method for the identification of the lipidated proteins and sites. Extensive optimizations were performed to increase the peptide solubility and recovery for MS analysis. Our increased knowledge of peptide solubility will be used in the future to develop a quantitative method to quantify sites modified with palmitic acid. The new method was successfully applied to detect known and novel sites modified with palmitic acid. Detection of modified sites is essential to 1) confirm that the protein is modified by palmitic acid and 2) identify which Cysteines is/are modified, as it could be time consuming to identify the modified Cysteine by more traditional methods.
To study if levels of this lipid modification change upon infection, we used MS based proteomics again and compared the amount of palmitic acid modified proteins in naïve and inflammatory stimulated immune cells (RAW264.7). These changes might reflect changes in protein expression or abundance that often occur following immune stimulation and might not reflect an increase of palmitic acid modification on one specific protein.
Our screen allowed us to map proteins modified with palmitic acid in two different cell lines, RAW264.7 and HeLa cells. We also identified a protein involved in autophagy-mediated clearance of pathogen which was found to be modified with palmitic acid. While its enrichment did not change between stimulated and non-stimulated cells, we hypothesized that this protein might play a key role in host-defense to pathogen infection. These results have recently been published (open access).
The second objective of the work was to study the role of the lipid modification on the protein localization and function. We first confirmed that the protein was indeed modified by palmitic acid and we confirmed the modification site. Levels of palmitic acid on the protein were found unchanged following different stimulus (immune stimulation with IFN-alpha or autophagy activation in HeLa cells). Localization and function of the protein did not change when palmitic acid was removed from the protein. The role of palmitic acid is sometime challenging to study and to understand. However, unpublished data suggests that palmitic acid plays an important role in protein trafficking. The role of palmitic acid modification was further studied at the host institution in the context of Mycobacterium tuberculosis (MTb). We hypothesize that palmitic acid is key for the recruitment of the protein to vesicles containing MTb and this could be an important mechanism for the autophagy-mediated clearance of pathogens.
Work carried out has allowed to identify proteins involved in autophagy-mediated clearance of pathogen that are modified with palmitic acid. Methods to detect the lipid modification on proteins were improved and these new methods are available to the scientific community and the public thanks to open access publication. The work carried out at the Rockefeller university or at the Crick has also provided many tools to study these important proteins. All these tools were used at the Crick to understand the role of these proteins in host defense to pathogen infection and will be shared with the scientific community following publication.
The project addresses the knowledge gap in pathogen infection. Understanding how cells can recognize and destroy pathogens via autophagy or alternatively how they can escape host defense and lead to infection, is key to develop new antimicrobials exploiting novel modes of action.
The research complies with one of Horizon 2020 societal challenges, “Health, Demographic Change and Wellbeing”, and to the new European commission action plan against antimicrobial resistance.