bioRxiv preprint doi: https://doi.org/10.1101/2020.06.15.151860; this version posted June 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Dietary and pharmacological induction of serine synthesis genes Alexei Vazquez1,2,* 1Cancer Research UK Beatson Institute, Glasgow, UK 2Institute of Cancer Sciences, University of Glasgow, Glasgow, UK Email: [email protected] Abstract gene coding for 3-phosphoglycerate dehydrogenase (Phgdh), the first enzyme of serine synthesis, causes There is an increasing interest in the pathway of L-serine neurodevelopmental abnormalities and it is embryonic synthesis and its. Although L-serine and downstream lethal 4 in mice. products can be obtained from the diet, serine deficiency has been documented in neurological disorders, macular The manifestation of serine deficiency in the human degeneration and aging. This evidence calls for strategies population call for strategies to increase the availability of to induce serine synthesis. Here I address this problem serine. Dietary serine supplementation seems the obvious taking advantage of the wealth of data deposited in the strategy 5. However, for a number of reasons, serine gene expression omnibus database. I uncover that low supplementation is not an effective approach in general 5. protein and ketogenic diets increase the expression of Dietary serine is rapidly incorporated into proteins or serine synthesis genes in the liver and the brain relative to broken down to glycine and formate 6. Mammalian control diets. I discover oestrogen medications, the organisms do not have a store for amino acids other than antifolate methotrexate and serine synthesis inhibitors as protein 7, but proteins are made to perform specific classes of compounds inducing the expression of serine functions and they are degraded as a last resort. synthesis genes in the liver. Future work is required to The induction of serine synthesis from the glycolytic investigate the use of these interventions for the intermediate 3-phosphoglycerate is potentially a longer management of serine deficiency disorders. lasting intervention. Here I investigate different strategies Introduction to induce the expression of genes encoding for the serine synthesis enzymes. L-Serine is a building block of proteins and phospholipids (Fig. 1). L-serine is converted to D-serine in the brain by serine racemase. Serine is also broken down to glycine and formate, providing additional building blocks for the synthesis of purines and glutathione. D-serine and glycine are co-agonists of the N-methyl-D-aspartate (NMDA) receptor together with glutamate, linking serine metabolism to neurological signalling. L-Serine is a nonessential amino acid and mammalians organisms satisfy their serine demand from dietary protein, the serine synthesis from glycine and formate and the serine synthesis from the glycolytic intermediate 3- phosphoglycerate (Fig. 1). Deficiencies in these supply routes may compromise the physiological functions dependent on serine. Serine deficiency has been implicated in physiological disorders in humans and animal models. Inborn errors of Figure 1. Serine metabolism. serine biosynthesis cause neurological disorders 1. Low Schematic representation of serine metabolism and its blood serine is a risk factor for macular telangiectasia type interaction with central metabolism. 1C stands for 2 and peripheral neuropathy 2. Defective metabolism of one-carbon. serine to glycine and formate impairs the activation of naïve T cells in aging mice 3. Homozygous deletion the 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.15.151860; this version posted June 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Materials and Methods The significant induction or repression of a given gene signature on a given sample was quantified using Gene Set I have previously introduced a gene expression analysis to Enrichment Analysis (GSEA) 9 as previously described 10. identify candidate compounds inducing a specified gene GSEA results in a positive and a negative score quantifying signature in a specified tissue 8. For sake of self- the induction or repression of the gene signature, consistency, I transcribe sections of that methodology together with their associated statistical significance here. (here 100,000 permutations of the genes assignments to Gene signatures probes). A sample was defined positive (red) for a The gene signature for serine synthesis contains the genes signature whenever it manifested a significant positive Phgdh, Psat1 and Psph, encoding for the enzymes 3- score (statistical significance £0.05), negative phosphoglycerate dehydrogenase, phosphoserine (blue) whenever it manifested a significant negative score transaminase and phosphoserine phosphatase, (statistical significance £0.05) and no significant change respectively. The remaining gene signatures were (black) otherwise. obtained from public repositories or literature reports Positive hits and they are listed in the Supplementary Table 1. In the input dataset there are multiple compounds tested Gene expression profiles under multiple conditions (dose and duration of The gene expression profiles were downloaded from treatment), and each compound/condition was tested in Gene Expression Omnibus. The accession numbers are triplicates. A compound tested at a specific condition was GSE57800 (organism: Sprague Dawley rat, gender: male, deemed positive if it significantly induced the serine tissue: heart, control: water vehicle, time: 3 days), synthesis gene signature in the 3 replicates tested. A GSE57805 (organism: primary Sprague Dawley rat compound was deemed a hit if it has a significant hepatocytes, control: DMSO vehicle, time: 1 day), enrichment of positives across the conditions where it GSE57811 (organism: Sprague Dawley rat, gender: male, was tested, given the total number of conditions tested tissue: kidney, control: water vehicle, time: 3 days), and the total number of conditions scoring positive in the GSE57815 (organism: Sprague Dawley rat, gender: male, whole dataset, using a hypergeometric distribution tissue: liver, control: water vehicle, time: 0.25 days) and (Supplementary Table 2). GSE57816 (organism: Sprague Dawley rat, gender: male, tissue: thigh muscle, control: water vehicle, time: 3 days) for the pharmacological interventions; GSE144207 Results (organism: brown rat, gender: male, tissue: liver, control: Dietary interventions control diet, time: 4 weeks) for the low protein diet, The synthesis of serine from 3-phosphoglycerate GSE7699 (organism: C57BL/6 mice, gender: male, tissue: proceeds through three enzymatic steps catalysed by 3- liver, control: chow diet, time: 6 weeks) and GSE115342 phosphoglycerate dehydrogenase (PHGDH), (organism: B6.Cg-Lepob/J mice, gender: female, tissue: phosphoserine transaminase (PSAT1) and phosphoserine cerebral cortex, control: regular chow, time: 7 weeks) for phosphatase (PSPH). The genes encoding for these the ketogenic diet; GSE51885 (organism: C57BL/6 mice, enzymes are under the transcriptional regulation of ATF4, gender: male, tissue: liver, control: current LAR diet, time: the master regulator of the amino acid stress response 11. 3 weeks) for the mixed diets and GSE120347 (organism: Experiments with cell cultures demonstrate the induction human neuroblastoma cell line BE(2)-C, control: DMSO, of the ATF4 transcriptional activity upon deprivation of time: 2 days) for the PHGDH inhibitor. In all cases gene any of the 21 proteinogenic amino acids 12. It is worth expression was quantified using microarrays and the RMA asking if the same is true in the context of whole-body signal (in log2 scale) was used as the gene expression metabolism. To address this question, I took advantage of readout. For each dataset, the average of the log2 a study reporting liver gene expression profiles of mice expression across controls was subtracted from all subject to a low protein diet (GSE144207, unpublished). samples. As a readout I will use gene signatures for the annotated Gene set enrichment analysis targets of ATF4, the genes encoding for the serine 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.15.151860; this version posted June 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Figure 2. Impact of dietary interventions on the expression of serine synthesis genes. A) Liver gene signatures of mice subject to a low protein diet. B) Liver gene signatures in rats subject to a ketogenic diet. C) Liver and D) cerebral cortex gene signatures in another cohort of mice subject to a ketogenic diet. E) Liver gene signatures in mice subject to different diets. Columns represent samples clustered by intervention and rows represent gene signatures. <== Points to the serine synthesis gene signature. Red indicates a significant gene signature induction relative to the average in controls, blue a significant repression and black no significant change. Keto: ketogenic diet, Fruc: high fructose