Periodic Reporting for period 4 - C18Signaling (Regulation of Cellular Growth and Metabolism by C18:0)
Okres sprawozdawczy: 2021-09-01 do 2023-08-31
My lab studies how cells regulate their growth and metabolism during normal development and in disease, with a focus on how signaling pathways sense nutrients. Work in the lab prior to this grant had found that proteins can be modified with different lipids, in particular C16:0 and C18:0, and that this can affect their function.
To study C18:0 signaling and fatty acid modifications of proteins the aims of the project were to:
1) identify as complete a set as possible of proteins that are stearoylated in human and Drosophila cells, thereby characterizing the cellular 'stearylome',
2) study how stearoylation affects the molecular function of these target proteins, and thereby cellular growth and metabolism, and
3) study how stearoylation is added, and possibly removed, from target proteins.
Overall, the project was successful. We discovered that dozens of proteins can be post-translationally modified with lipids, and that in almost all cases single cysteine residues on proteins can be modified with a variety of lipids including C16:0 and C18:0. By studying in depth the functional consequences on some proteins, we discovered that the identity of the lipid that modifies a protein can determine the consequence of the modification on the protein's function. For instance, for GNAI proteins, we found that acylation with C16:0 activates its function, while acylation with C18:0 (which turns into C18:1) inactivates its function.
As a result, this work changed the way we view C18:0 from simply being a metabolite to being an important dietary signaling molecule that links nutritional uptake to cellular physiology. This is clinically relevant because dietary C18:0 is known to reduce cancer and cardiovascular risk. By discovering how C18:0 regulates cell signaling, this provides a molecular starting point for understanding how C18:0 affects normal development and disease.
To study C18:0 signaling and fatty acid modifications of proteins the aims of the project were to:
1) identify as complete a set as possible of proteins that are stearoylated in human and Drosophila cells, thereby characterizing the cellular 'stearylome',
2) study how stearoylation affects the molecular function of these target proteins, and thereby cellular growth and metabolism, and
3) study how stearoylation is added, and possibly removed, from target proteins.
Overall, the project was successful. We discovered that dozens of proteins can be post-translationally modified with lipids, and that in almost all cases single cysteine residues on proteins can be modified with a variety of lipids including C16:0 and C18:0. By studying in depth the functional consequences on some proteins, we discovered that the identity of the lipid that modifies a protein can determine the consequence of the modification on the protein's function. For instance, for GNAI proteins, we found that acylation with C16:0 activates its function, while acylation with C18:0 (which turns into C18:1) inactivates its function.
As a result, this work changed the way we view C18:0 from simply being a metabolite to being an important dietary signaling molecule that links nutritional uptake to cellular physiology. This is clinically relevant because dietary C18:0 is known to reduce cancer and cardiovascular risk. By discovering how C18:0 regulates cell signaling, this provides a molecular starting point for understanding how C18:0 affects normal development and disease.
The aim of the project was to bring together two fields - the field of nutrient sensing and the field of protein post-translational modifications - to uncover a mechanism how nutrients can regulate cell signaling and animal physiology. Prior to the ERC project, we discovered that one protein, the Transferrin Receptor (TfR1) can be differentially post-translationally modified by two different lipids - either palmitic acid (C16:0) or stearic acid (C18:0) - and that the identity of the fatty acid modifying TfR1 affects the ability of TfR1 to signal into the cell. The aim of the ERC project was to generalize this single observation and to see whether differential acylation of proteins with different lipids is a broadly-applicable mechanism for how lipids affect cell signaling.
Protein palmitoylation has been known since several decades as a protein post-translational mechanism whereby palmitic acid is attached to cysteine residues of proteins. More recent mass spectrometry data have revealed that also other fatty acids can be attached to proteins, and indeed in half of all cases, the lipid modifying a protein is a fatty acid other than palmitic acid. It was unclear, however, whether different proteins get acylated with different fatty acids, or whether individual protein species can be acylated with different fatty acids. So the first goal of the ERC project was to understand whether differential acylation of single proteins is a general phenomenon. We employed a proteomics approach to identify the human proteins that are palmitoylated or 'stearoylated' (ie modified with C18:0) and discovered that all proteins that are stearoylated can also be palmitoylated in vivo. We went on to show that in all cases that we tested, the stearoylation or palmitoylation occurs on the same cysteine residues of the protein, meaning that there is competition in vivo for these two modifications. Finally, we showed that exposure of cells to either C16:0 or C18:0 will shift the balance of protein palmitoylation or stearoylation, meaning that the metabolic environment of a cell will influence the post-translational modifications of proteins in the cell. This work was published in the Journal of Biological Chemistry (Nuskova et al. 2023). Surprisingly, this mechanism works not only in cell culture but also in vivo. We found that when people ingest stearic acid, this has differential effects on signaling compared to ingestion of palmitic acid (Senyilmaz-Tiebe Nature Communications 2018). We also published a review describing this novel concept of nutrient sensing whereby metabolite levels affect the stoichiometry of post-translational modifications on proteins in Developmental Cell (Figlia et al. 2020).
The second goal of the ERC project was to identify cases where differential acylation of proteins affects their function, and to understand how. We tested a number of proteins. In some cases, we did not find a differential function for stearoylated versus palmitoylation. For instance, we found that a component of the mTORC1 signaling pathway, LAMTOR1, is palmitoylated or stearoylated, however this did not affect mTORC1 localization or signaling (Task 2.2). In some cases, however, we found a clear effect. For instance, we discovered that the GNAI proteins, which mediate signaling downstream of receptor tyrosine kinases such as EGFR, are either stearoylated or palmitoylated on Cys 3. If they are palmitoylated, they enter specific subdomains of the plasma membrane called detergent-resistant-membranes (DRMs), thereby entering into proximity with EGFR and potentiating oncogenic signaling downstream of EGFR. Instead, if they are stearoylated on Cys 3, surprisingly, we found that this lipid becomes desaturated to C18:1, causing the GNAI proteins to exit DRMs, dampening EGFR signaling. Thus, this work which was published in Nature Communications (Nuskova et al. 2021) not only showed that differential acylation of a protein with different lipids can have differential effects on its activity, but it also revealed the detailed molecular mechanism how this works.
Finally, the goal of the third work package was to study how stearoylation is added and removed from proteins. We identified several different ZDHHC acyl-transferases as being responsible for stearoylating TfR1 and GNAI proteins. What was interesting about this finding was that multiple different acyltransferases can transfer either C16:0 or C18:0 onto proteins, meaning that they are quite promiscuous. Instead, the degree of acylation of proteins with C16:0 versus C18:0 depends on the relative ratio of these two metabolites in cells (Nuskova et al. JBC 2023). Hence, this is a mechanism for metabolite sensing. Finally, regarding removal of stearoylation, we found that this is catalyzed by acyl protein thioesterase 1 (APT1), as it can be inhibited with palmostatin B.
In sum, the objectives of this project were achieved.
Protein palmitoylation has been known since several decades as a protein post-translational mechanism whereby palmitic acid is attached to cysteine residues of proteins. More recent mass spectrometry data have revealed that also other fatty acids can be attached to proteins, and indeed in half of all cases, the lipid modifying a protein is a fatty acid other than palmitic acid. It was unclear, however, whether different proteins get acylated with different fatty acids, or whether individual protein species can be acylated with different fatty acids. So the first goal of the ERC project was to understand whether differential acylation of single proteins is a general phenomenon. We employed a proteomics approach to identify the human proteins that are palmitoylated or 'stearoylated' (ie modified with C18:0) and discovered that all proteins that are stearoylated can also be palmitoylated in vivo. We went on to show that in all cases that we tested, the stearoylation or palmitoylation occurs on the same cysteine residues of the protein, meaning that there is competition in vivo for these two modifications. Finally, we showed that exposure of cells to either C16:0 or C18:0 will shift the balance of protein palmitoylation or stearoylation, meaning that the metabolic environment of a cell will influence the post-translational modifications of proteins in the cell. This work was published in the Journal of Biological Chemistry (Nuskova et al. 2023). Surprisingly, this mechanism works not only in cell culture but also in vivo. We found that when people ingest stearic acid, this has differential effects on signaling compared to ingestion of palmitic acid (Senyilmaz-Tiebe Nature Communications 2018). We also published a review describing this novel concept of nutrient sensing whereby metabolite levels affect the stoichiometry of post-translational modifications on proteins in Developmental Cell (Figlia et al. 2020).
The second goal of the ERC project was to identify cases where differential acylation of proteins affects their function, and to understand how. We tested a number of proteins. In some cases, we did not find a differential function for stearoylated versus palmitoylation. For instance, we found that a component of the mTORC1 signaling pathway, LAMTOR1, is palmitoylated or stearoylated, however this did not affect mTORC1 localization or signaling (Task 2.2). In some cases, however, we found a clear effect. For instance, we discovered that the GNAI proteins, which mediate signaling downstream of receptor tyrosine kinases such as EGFR, are either stearoylated or palmitoylated on Cys 3. If they are palmitoylated, they enter specific subdomains of the plasma membrane called detergent-resistant-membranes (DRMs), thereby entering into proximity with EGFR and potentiating oncogenic signaling downstream of EGFR. Instead, if they are stearoylated on Cys 3, surprisingly, we found that this lipid becomes desaturated to C18:1, causing the GNAI proteins to exit DRMs, dampening EGFR signaling. Thus, this work which was published in Nature Communications (Nuskova et al. 2021) not only showed that differential acylation of a protein with different lipids can have differential effects on its activity, but it also revealed the detailed molecular mechanism how this works.
Finally, the goal of the third work package was to study how stearoylation is added and removed from proteins. We identified several different ZDHHC acyl-transferases as being responsible for stearoylating TfR1 and GNAI proteins. What was interesting about this finding was that multiple different acyltransferases can transfer either C16:0 or C18:0 onto proteins, meaning that they are quite promiscuous. Instead, the degree of acylation of proteins with C16:0 versus C18:0 depends on the relative ratio of these two metabolites in cells (Nuskova et al. JBC 2023). Hence, this is a mechanism for metabolite sensing. Finally, regarding removal of stearoylation, we found that this is catalyzed by acyl protein thioesterase 1 (APT1), as it can be inhibited with palmostatin B.
In sum, the objectives of this project were achieved.
The discovery that stearic acid ingestion in humans can affect mitochondrial morphology in vivo lays the groundwork for the physiological relevance of all the rest of the research in this ERC project.
Our results on the effect of stearic acid on growth-factor signaling via GNAI proteins and the characterization of the stearoyltransferase ZDHHC6 will open new insights on the complex metabolism and function of protein fatty acid modifications.
Our results on the effect of stearic acid on growth-factor signaling via GNAI proteins and the characterization of the stearoyltransferase ZDHHC6 will open new insights on the complex metabolism and function of protein fatty acid modifications.