Homocysteine-methionine cycle is a metabolic sensor system controlling methylation-regulated pathological signaling

Abstract

Homocysteine-Methionine (HM) cycle produces universal methyl group donor S-adenosylmethione (SAM), methyltransferase inhibitor S-adenosylhomocysteine (SAH) and homocysteine (Hcy). Hyperhomocysteinemia (HHcy) is established as an independent risk factor for cardiovascular disease (CVD) and other degenerative disease. We selected 115 genes in the extended HM cycle (31 metabolic enzymes and 84 methyltransferases), examined their protein subcellular location/partner protein, investigated their mRNA levels and mapped their corresponding histone methylation status in 35 disease conditions via mining a set of public databases and intensive literature research. We have 6 major findings. 1) All HM metabolic enzymes are located only in the cytosol except for cystathionine-β-synthase (CBS), which was identified in both cytosol and nucleus. 2) Eight disease conditions encountered only histone hypomethylation on 8 histone residues (H3R2/K4/R8/K9/K27/K36/K79 and H4R3). Nine disease conditions had only histone hypermethylation on 8 histone residues (H3R2/K4/K9/K27/K36/K79 and H4R3/K20). 3) We classified 9 disease types with differential HM cycle expression pattern. Eleven disease conditions presented most 4 HM cycle pathway suppression. 4) Three disease conditions had all 4 HM cycle pathway suppression and only histone hypomethylation on H3R2/K4/R8/K9/K36 and H4R3. 5) Eleven HM cycle metabolic enzymes interact with 955 proteins. 6) Five paired HM cycle proteins interact with each other. We conclude that HM cycle is a key metabolic sensor system which mediates receptor-independent metabolism-associated danger signal recognition and modulates SAM/SAH-dependent methylation in disease conditions and that hypomethylation on frequently modified histone residues is a key mechanism for metabolic disorders, autoimmune disease and CVD. We propose that HM metabolism takes place in the cytosol, that nuclear methylation equilibration requires a nuclear-cytosol transfer of SAM/SAH/Hcy, and that Hcy clearance is essential for genetic protection.

Keywords: Homocysteine-methionine cycle; Metabolic sensor; SAM/SAH-dependent methylation.

Fig. 1

HM cycle senses metabolic risk factors and modulates SAM/SAH-dependent methylation and pathogenic signaling. A. Extended HM cycle, gene selection & database mining strategy. We selected 115 genes in this cycle from Human metabolome database (https://www.hmdb.ca/). The HM cycle includes 3 major pathways. ① Demethylation pathway: Met is converted to SAM, which becomes SAH after donating the methyl group for cellular methylation. SAH is then converted to Hcy, a dual directional reaction, interacting with adenosine metabolism. ② Remethylation pathway: Hcy is converted back to Met by regaining the methyl group from folate cycle or choline/betaine metabolism. ③ Transsulfuration pathway: Hcy is converted to Cys. The extended HM cycle includes additional pathways, such as ④ 84 MTs, ⑤ folate cycle, ⑥ choline/betaine metabolism, ⑦ Hcy synthesis and ⑧ adenosine metabolism. These 115 genes’ mRNA levels in human disease conditions were analyzed by mining NIH-NCBI GEO database (https://www.ncbi.nlm.nih.gov/geo/), protein subcellular localization and interacting partner were identified by using information in Human Protein Atlas (https://www.proteinatlas.org/), compartment subcellular location database (https://compartments.jensenlab.org) and NIH-NCBI gene database (https://www.ncbi.nlm.nih.gov/gene), respectively. B. Cytosol-nucleus SAM, SAH and Hcy transfer hypothesis. All essential enzymes in the HM metabolic cycle are located in the cytosol, only 21 out of 84 MTs (25%) in the cytosol but the majority of MTs (72.6%) in the nucleus. Some of the HM cycle metabolic processes are not active in the nucleus because their metabolic enzymes are not identified there and indicated by dash lines. For example, enzymes for SAM synthesis, SAH clearance and Hcy synthesis are missing in the nucleus. C. CBS facilitates Hcy clearance in the nucleus. Since only one Hcy clearance enzyme exists in the nucleus, we propose that CBS facilitates Hcy clearance in the nucleus. Abbreviations: Ade, adenosine; Hcy, homocysteine; SAM, S-Adenosylmethionine; SAH, S-Adenosylhomocysteine; Met, Methionine; MTs, Methyltransferase; CBS, Cystathionine-β-synthase; HT, homocysteine thiolactone. Abbreviations for gene and enzyme names are explained in Supplementary Table 1.

Fig. 2

Identification of HM cycle gene expression changes and SAM/SAH-responsive signal pathways in 7 disease categories. We examined gene expression of 115 enzymes in extended HM cycle in microarray datasets of 35 disease conditions in NIH-NCBI GEO database (https://www.ncbi.nlm.nih.gov/geo/). These 35 disease conditions were classified in 7 categories. Signal pathways regulated by altered gene expression were analyzed using Ingenuity Pathway Analysis (IPA, http://www.ingenuity.com/), and termed as SAM/SAH-responsive signal pathways. Significantly up- or down-regulated genes and pathways are presented in Venn diagrams and listed. A. HM cycle gene mRNA level changes: 7 disease categories are presented as (1) 5 metabolic diseases; (2) 6 metabolite treatments; (3) 4 vascular diseases; (4) 4 aging diseases; (5) 5 autoimmune diseases; (6) 5 digestive cancers; (7) 6 other cancers. B. HM cycle gene expression-determined SAM/SAH-responsive signal pathway changes: The same 7 disease categories, as in panel A, are presented. Abbreviations for metabolites: Nt, Nucleotides; 5’-IMP, Inosine 5’-phosphate; NA, Noradrenaline; AD, Adrenaline; Met, Methionine; Ade, Adenosine; Gly, Glycine; Cys, Cysteine; Hcy, Homocysteine; Lys, Lysine; Ser, Serine; SAM, S-adenosyl-L-methionine. Abbreviations for diseases: HFH, Heterozygote family hypercholesterolemia; T2DM, Type 2 diabetes mellitus; TC, T cell; βC, β cell; def., deficiency; Glu, glucose; Ox-LDL, Oxidized low-density lipoprotein; Ox-PAPC, Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; VB12, Vitamin B 12; IS, Ischemic stroke; AS, Atherosclerosis; AOD, Aortic occlusive disease; CAD, Coronary artery disease; HGPS, Hutchinson-Gilford progeria syndrome; PD, Parkinson’s disease; AD, Alzheimer’s disease; OSIMOF, Old sepsis induced multiple organ failure; RA, Rheumatoid arthritis; SLE, Systemic lupus erythematosus; CC, Colon Cancer; ESCC, Esophageal Squamous Cell Carcinomas; GA, Gastric Adenocarcinoma; IC, Intrahepatic Cholangiocarcinoma; PDA, Pancreatic Ductal Adenocarcinom; OC, Ovarian carcinoma; PC, Prostate cancer; BA, Breast adenocarcinoma, NSCLC, Non-small cell lung cancer; BC, Bladder cancer; CCRCC, Clear cell renal cell carcinoma. Abbreviations for gene and enzyme names are explained in Supplementary Table 1.

Fig. 2

Identification of HM cycle gene expression changes and SAM/SAH-responsive signal pathways in 7 disease categories. We examined gene expression of 115 enzymes in extended HM cycle in microarray datasets of 35 disease conditions in NIH-NCBI GEO database (https://www.ncbi.nlm.nih.gov/geo/). These 35 disease conditions were classified in 7 categories. Signal pathways regulated by altered gene expression were analyzed using Ingenuity Pathway Analysis (IPA, http://www.ingenuity.com/), and termed as SAM/SAH-responsive signal pathways. Significantly up- or down-regulated genes and pathways are presented in Venn diagrams and listed. A. HM cycle gene mRNA level changes: 7 disease categories are presented as (1) 5 metabolic diseases; (2) 6 metabolite treatments; (3) 4 vascular diseases; (4) 4 aging diseases; (5) 5 autoimmune diseases; (6) 5 digestive cancers; (7) 6 other cancers. B. HM cycle gene expression-determined SAM/SAH-responsive signal pathway changes: The same 7 disease categories, as in panel A, are presented. Abbreviations for metabolites: Nt, Nucleotides; 5’-IMP, Inosine 5’-phosphate; NA, Noradrenaline; AD, Adrenaline; Met, Methionine; Ade, Adenosine; Gly, Glycine; Cys, Cysteine; Hcy, Homocysteine; Lys, Lysine; Ser, Serine; SAM, S-adenosyl-L-methionine. Abbreviations for diseases: HFH, Heterozygote family hypercholesterolemia; T2DM, Type 2 diabetes mellitus; TC, T cell; βC, β cell; def., deficiency; Glu, glucose; Ox-LDL, Oxidized low-density lipoprotein; Ox-PAPC, Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; VB12, Vitamin B 12; IS, Ischemic stroke; AS, Atherosclerosis; AOD, Aortic occlusive disease; CAD, Coronary artery disease; HGPS, Hutchinson-Gilford progeria syndrome; PD, Parkinson’s disease; AD, Alzheimer’s disease; OSIMOF, Old sepsis induced multiple organ failure; RA, Rheumatoid arthritis; SLE, Systemic lupus erythematosus; CC, Colon Cancer; ESCC, Esophageal Squamous Cell Carcinomas; GA, Gastric Adenocarcinoma; IC, Intrahepatic Cholangiocarcinoma; PDA, Pancreatic Ductal Adenocarcinom; OC, Ovarian carcinoma; PC, Prostate cancer; BA, Breast adenocarcinoma, NSCLC, Non-small cell lung cancer; BC, Bladder cancer; CCRCC, Clear cell renal cell carcinoma. Abbreviations for gene and enzyme names are explained in Supplementary Table 1.

Fig. 3

HM cycle gene expression-determined SAM/SAH-dependent H3/H4 histone methylations changes in disease, and disease classification. We examined extended HM cycle gene expression of 35 disease conditions microarray data in NIH-NCBI GEO database (https://www.ncbi.nlm.nih.gov/geo/), and determined histone methylations changes based on corresponding MT. We defined induced MT gene as hypermethylation and reduced MT gene as hypomethylation on their corresponding histone methylation site (Supplementary Table 2A). A. H3 methylation changes. H3 histone methylation are changed at H3-R2, K4, R8, K9, R17, R26, K27, K36, K79 in human disease. Disease conditions are presented by code. Percentage indicates the frequency of each modification in total methylation changes modified. Codes for disease with individual hypermethylation changes are placed above the histone bar, whereas for that with individual hypomethylation changes below the histone bar. B. H4 methylation changes. H4 histone methylation are changed at H4-R3 and K20 in human disease. C. Disease conditions. Numeric coded 29 human disease conditions are explained. Numeric codes and disease names are underlined with bold letter (9) for conditions identified as histone hypermethylation only, or highlighted in italic bold letter (8) for conditions identified as histone hypomethylation only. D. Disease classification based on HM cycle pathway gene expression pattern. To examine the association between HM cycle metabolites with human disease, we classified human disease into 9 types based on differential gene expression pattern in HM cycle pathway. Eleven diseases with mostly reduced genes (T2DM-βC, OSIMOF, SLE, HFH, Osteoarthritis, PDA, AOD, BC, IC, GA, CCRCC), 6 with mostly induced genes (PD, CC, HGPS, BA, ESC, CAD), and 3 with mixed gene expression changes (Psoriasis, NSCLN, OC) in HM cycle pathway. Arrows for reduced genes are highlighted with grey shade. Abbreviations: R, Arginine; K, lysine. Abbreviations for diseases: T2DM (βC), Type 2 diabetes mellitus (β cell); OSIMOF, Old sepsis induced multiple organ failure; SLE, systemic lupus erythematosus; HFH, heterozygote family hypercholesterolemia; PDA, pancreatic ductal adenocarcinoma; AOD, aortic occlusive disease; BC, bladder cancer; IC, intrahepatic cholangiocarcinoma; GA, gastric adenocarcinoma; CCRCC, clear cell renal cell carcinoma; PD, Parkinson’s disease; CC, colon cancer; HGPS, Hutchinson-Gilford progeria syndrome; BA, breast adenocarcinoma; ESCC, Esophageal squamous cell carcinomas; CAD, coronary artery disease; NSCLN, non-small cell lung cancer; OC, Ovarian carcinoma. Abbreviations for gene and enzyme names are explained in Supplementary Table 1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Working model, novel hypotheses, discoveries and conclusions. A. Working model. We identified HM cycle as a metabolic sensor system. Genes in extended HM cycle can be regulated via receptor-independent MADS recognition which leads to three responses: (1) HM cycle pathway suppression, (2) SAM/SAH-dependent histone MTs suppression, and (3) 38 SAM/SAH-responsive signal pathway regulation, which are associated with 11, 8 and 35 disease conditions, respectively. Three human disease conditions, SLE, T2DM (βC) and AOD, overlapped in all three responses and marked by histone hypomethylation at H3R2, H3K4, H3R8, H3K9, H3K36, or H4R3 site. B. Classic model of receptor-dependent pathogenic signaling. This classical model is featured by ligand-receptor specific molecular recognition. C. Novel model of receptor-independent MADS-mediated methylation-regulated pathogenic signaling. This model describes receptor-independent MADS recognition, and emphasizes metabolic risk factor/sensor-mediated pathogenic signaling and HM cycle-regulated SAM/SAH-dependent methylation. D. Discoveries. We listed 6 major discoveries. E. Novel hypotheses and conclusions. Summary of 6 novel hypotheses generated from this study. Abbreviations: HM, homocysteine-methionine; MADS, metabolism-associated danger signal; MT, methyltransferase; CBS, Cystathionine-β-synthase; Arg, Arginine; Lys, Lysine; SLE, Systemic lupus erythematosus; T2DM-βC, Type 2 diabetes mellitus β cell; AOD, Aortic occlusive disease; CVD, Cardiovascular disease; NCBI, National Center of Biotechnology Information; GEO, Gene Expression Omnibus; Hcy, homocysteine; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; H3/H4, Histone 3/Histone 4; his, histone; His-Lys, Histone Lysine; His-Arg, Histone Arginine; hyper-me, hypermethylation; hypo-me, hypomethylation; Glu, glucose. DAMP, damage-associated molecular pattern; PAMP, pathogen-associated molecular pattern; PRR, pathogen recognition receptor; HHcy, hyperhomocysteinemia; HG, hyperglycemia, HL, hyperlipidemia; Ar, arsenite; Ca, catechol; Di, diphthamide; Ha, histamine; NAM, nicotinamide; NAS, N-acetylserotonin; PE, phosphatidylethanolamine; PEOH, phenylethanolamine; Tr, tryptamine; Th, thiopurine; Co, coenzyme. Abbreviations for disease are explained in Fig. 2 legend.

Fig. 4

Working model, novel hypotheses, discoveries and conclusions. A. Working model. We identified HM cycle as a metabolic sensor system. Genes in extended HM cycle can be regulated via receptor-independent MADS recognition which leads to three responses: (1) HM cycle pathway suppression, (2) SAM/SAH-dependent histone MTs suppression, and (3) 38 SAM/SAH-responsive signal pathway regulation, which are associated with 11, 8 and 35 disease conditions, respectively. Three human disease conditions, SLE, T2DM (βC) and AOD, overlapped in all three responses and marked by histone hypomethylation at H3R2, H3K4, H3R8, H3K9, H3K36, or H4R3 site. B. Classic model of receptor-dependent pathogenic signaling. This classical model is featured by ligand-receptor specific molecular recognition. C. Novel model of receptor-independent MADS-mediated methylation-regulated pathogenic signaling. This model describes receptor-independent MADS recognition, and emphasizes metabolic risk factor/sensor-mediated pathogenic signaling and HM cycle-regulated SAM/SAH-dependent methylation. D. Discoveries. We listed 6 major discoveries. E. Novel hypotheses and conclusions. Summary of 6 novel hypotheses generated from this study. Abbreviations: HM, homocysteine-methionine; MADS, metabolism-associated danger signal; MT, methyltransferase; CBS, Cystathionine-β-synthase; Arg, Arginine; Lys, Lysine; SLE, Systemic lupus erythematosus; T2DM-βC, Type 2 diabetes mellitus β cell; AOD, Aortic occlusive disease; CVD, Cardiovascular disease; NCBI, National Center of Biotechnology Information; GEO, Gene Expression Omnibus; Hcy, homocysteine; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; H3/H4, Histone 3/Histone 4; his, histone; His-Lys, Histone Lysine; His-Arg, Histone Arginine; hyper-me, hypermethylation; hypo-me, hypomethylation; Glu, glucose. DAMP, damage-associated molecular pattern; PAMP, pathogen-associated molecular pattern; PRR, pathogen recognition receptor; HHcy, hyperhomocysteinemia; HG, hyperglycemia, HL, hyperlipidemia; Ar, arsenite; Ca, catechol; Di, diphthamide; Ha, histamine; NAM, nicotinamide; NAS, N-acetylserotonin; PE, phosphatidylethanolamine; PEOH, phenylethanolamine; Tr, tryptamine; Th, thiopurine; Co, coenzyme. Abbreviations for disease are explained in Fig. 2 legend.