Hydrogen sulfide-mediated persulfidation regulates homocysteine metabolism and enhances ferroptosis in non-small cell lung cancer.

Hydrogen sulfide (H2S), a metabolite of the transsulfuration pathway, has been implicated in ferroptosis, a unique form of cell death caused by lipid peroxidation. While the exact mechanisms controlling ferroptosis remain unclear, our study reveals that H2S sensitizes human non-small cell lung cancer (NSCLC) cells to this process, particularly when cysteine levels are low. Combining H2S with cystine depletion significantly enhances the effectiveness of ferroptosis-based cancer therapy. Mechanistically, H2S persulfidates the 195th cysteine on S-adenosyl homocysteine hydrolase (SAHH), reducing its enzymatic activity. This leads to decreased homocysteine levels, subsequently lowering cysteine and glutathione concentrations under cystine depletion conditions. These changes ultimately increase the vulnerability of NSCLC cells to ferroptosis. Our findings establish H2S as a key regulator of homocysteine metabolism and a critical factor in determining NSCLC cell susceptibility to ferroptosis. These results highlight the potential of H2S-based therapies to improve the efficacy of ferroptosis-targeted cancer treatments for NSCLC.

Hydrogen sulfide: A whiff of trouble for cancer cell survival.

Hydrogen sulfide (H2S) can regulate biological processes by post-translational persulfidation of proteins at select cysteine residues. In this issue of Molecular Cell, Zheng et al.1 identify the enzyme SAHH as an H2S substrate, which upon persulfidation disrupts homocysteine metabolism and sensitizes lung cancer cells to ferroptosis.

Thiol reductive stress activates the hypoxia response pathway.

Owing to their capability to disrupt the oxidative protein folding environment in the endoplasmic reticulum (ER), thiol antioxidants, such as dithiothreitol (DTT), are used as ER-specific stressors. We recently showed that thiol antioxidants modulate the methionine-homocysteine cycle by upregulating an S-adenosylmethionine-dependent methyltransferase, rips-1, in Caenorhabditis elegans. However, the changes in cellular physiology induced by thiol stress that modulate the methionine-homocysteine cycle remain uncharacterized. Here, using forward genetic screens in C. elegans, we discover that thiol stress enhances rips-1 expression via the hypoxia response pathway. We demonstrate that thiol stress activates the hypoxia response pathway. The activation of the hypoxia response pathway by thiol stress is conserved in human cells. The hypoxia response pathway enhances thiol toxicity via rips-1 expression and confers protection against thiol toxicity via rips-1-independent mechanisms. Finally, we show that DTT might activate the hypoxia response pathway by producing hydrogen sulfide. Our studies reveal an intriguing interaction between thiol-mediated reductive stress and the hypoxia response pathway and challenge the current model that thiol antioxidant DTT disrupts only the ER milieu in the cell.

Homocysteine Modification in Protein Structure/Function and Human Disease.

Epidemiological studies established that elevated homocysteine, an important intermediate in folate, vitamin B12, and one carbon metabolism, is associated with poor health, including heart and brain diseases. Earlier studies show that patients with severe hyperhomocysteinemia, first identified in the 1960s, exhibit neurological and cardiovascular abnormalities and premature death due to vascular complications. Although homocysteine is considered to be a nonprotein amino acid, studies over the past 2 decades have led to discoveries of protein-related homocysteine metabolism and mechanisms by which homocysteine can become a component of proteins. Homocysteine-containing proteins lose their biological function and acquire cytotoxic, proinflammatory, proatherothrombotic, and proneuropathic properties, which can account for the various disease phenotypes associated with hyperhomocysteinemia. This review describes mechanisms by which hyperhomocysteinemia affects cellular proteostasis, provides a comprehensive account of the biological chemistry of homocysteine-containing proteins, and discusses pathophysiological consequences and clinical implications of their formation.

Homocysteine-induced sustained GluN2A NMDA receptor stimulation leads to mitochondrial ROS generation and neurotoxicity.

Homocysteine, a sulfur-containing amino acid derived from methionine metabolism, is a known agonist of N-methyl-D-aspartate receptor (NMDAR) and is involved in neurotoxicity. Our previous findings showed that neuronal exposure to elevated homocysteine levels leads to sustained low-level increase in intracellular Ca2+, which is dependent on GluN2A subunit-containing NMDAR (GluN2A-NMDAR) stimulation. These studies further showed a role of ERK MAPK in homocysteine-GluN2A-NMDAR-mediated neuronal death. However, the intracellular mechanisms associated with such sustained GluN2A-NMDAR stimulation and subsequent Ca2+ influx have remained unexplored. Using live-cell imaging with Fluo3-AM and biochemical approaches, we show that homocysteine-GluN2A NMDAR-induced initial Ca2+ influx triggers sequential phosphorylation and subsequent activation of the proline rich tyrosine kinase 2 (Pyk2) and Src family kinases, which in turn phosphorylates GluN2A-Tyr1325 residue of GluN2A-NMDARs to maintain channel activity. The continuity of this cycle of events leads to sustained Ca2+ influx through GluN2A-NMDAR. Our findings also show that lack of activation of the regulatory tyrosine phosphatase STEP, which can limit Pyk2 and Src family kinase activity further contributes to the maintenance of this cycle. Additional studies using live-cell imaging of neurons expressing a redox-sensitive GFP targeted to the mitochondrial matrix show that treatment with homocysteine leads to a progressive increase in mitochondrial reactive oxygen species generation, which is dependent on GluN2A-NMDAR-mediated sustained ERK MAPK activation. This later finding demonstrates a novel role of GluN2A-NMDAR in homocysteine-induced mitochondrial ROS generation and highlights the role of ERK MAPK as the intermediary signaling pathway between GluN2A-NMDAR stimulation and mitochondrial reactive oxygen species generation.

Homocysteine metabolites impair the PHF8/H4K20me1/mTOR/autophagy pathway by upregulating the expression of histone demethylase PHF8-targeting microRNAs in human vascular endothelial cells and mice.

The inability to efficiently metabolize homocysteine (Hcy) due to nutritional and genetic deficiencies, leads to hyperhomocysteinemia (HHcy) and endothelial dysfunction, a hallmark of atherosclerosis which underpins cardiovascular disease (CVD). PHF8 is a histone demethylase that demethylates H4K20me1, which affects the mammalian target of rapamycin (mTOR) signaling and autophagy, processes that play important roles in CVD. PHF8 is regulated by microRNA (miR) such as miR-22-3p and miR-1229-3p. Biochemically, HHcy is characterized by elevated levels of Hcy, Hcy-thiolactone and N-Hcy-protein. Here, we examined the effects of these metabolites on miR-22-3p, miR-1229-3p, and their target PHF8, as well as on the downstream consequences of these effects on H4K20me1, mTOR-, and autophagy-related proteins and mRNAs expression in human umbilical vein endothelial cells (HUVEC). We found that treatments with N-Hcy-protein, Hcy-thiolactone, or Hcy upregulated miR-22-3p and miR-1229-3p, attenuated PHF8 expression, upregulated H4K20me1, mTOR, and phospho-mTOR. Autophagy-related proteins (BECN1, ATG5, ATG7, lipidated LC3-II, and LC3-II/LC3-I ratio) were significantly downregulated by at least one of these metabolites. We also found similar changes in the expression of miR-22-3p, Phf8, mTOR- and autophagy-related proteins/mRNAs in vivo in hearts of Cbs-/- mice, which show severe HHcy and endothelial dysfunction. Treatments with inhibitors of miR-22-3p or miR-1229-3p abrogated the effects of Hcy-thiolactone, N-Hcy-protein, and Hcy on miR expression and on PHF8, H4K20me1, mTOR-, and autophagy-related proteins/mRNAs in HUVEC. Taken together, these findings show that Hcy metabolites upregulate miR-22-3p and miR-1229-3p expression, which then dysregulate the PHF8/H4K20me1/mTOR/autophagy pathway, important for vascular homeostasis.

Dysregulation of hepatic one-carbon metabolism in classical homocystinuria: Implications of redox-sensitive DHFR repression and tetrahydrofolate depletion for pathogenesis and treatment.

Cystathionine beta-synthase-deficient homocystinuria (HCU) is a life-threatening disorder of sulfur metabolism. HCU can be treated by using betaine to lower tissue and plasma levels of homocysteine (Hcy). Here, we show that mice with severely elevated Hcy and potentially deficient in the folate species tetrahydrofolate (THF) exhibit a very limited response to betaine indicating that THF plays a critical role in treatment efficacy. Analysis of a mouse model of HCU revealed a 10-fold increase in hepatic levels of 5-methyl -THF and a 30-fold accumulation of formiminoglutamic acid, consistent with a paucity of THF. Neither of these metabolite accumulations were reversed or ameliorated by betaine treatment. Hepatic expression of the THF-generating enzyme dihydrofolate reductase (DHFR) was significantly repressed in HCU mice and expression was not increased by betaine treatment but appears to be sensitive to cellular redox status. Expression of the DHFR reaction partner thymidylate synthase was also repressed and metabolomic analysis detected widespread alteration of hepatic histidine and glutamine metabolism. Many individuals with HCU exhibit endothelial dysfunction. DHFR plays a key role in nitric oxide (NO) generation due to its role in regenerating oxidized tetrahydrobiopterin, and we observed a significant decrease in plasma NOx (NO2 + NO3) levels in HCU mice. Additional impairment of NO generation may also come from the HCU-mediated induction of the 20-hydroxyeicosatetraenoic acid generating cytochrome CYP4A. Collectively, our data shows that HCU induces dysfunctional one-carbon metabolism with the potential to both impair betaine treatment and contribute to multiple aspects of pathogenesis in this disease.

Ciliary neurotrophic factor promotes the development of homocysteine-induced vascular endothelial injury through inflammation mediated by the JAK2/STAT3 signaling pathway.

Elevated homocysteine (Hcy) levels have been recognized as significant risk factor for cardiovascular and cerebrovascular diseases, closely related to endothelial injury. While expression of Ciliary Neurotrophic Factor (CNTF) significantly increases during Hcy-induced vascular endothelial cell injury, the precise molecular pathways through which CNTF operates remain to be clarified. To induce vascular endothelial cell injury, human umbilical vein endothelial cells (HUVECs) were treated with Hcy. Cell viability and apoptosis in HUVECs were assessed using the CCK-8 assay and flow cytometry. Western blot analysis determined the expression levels of the JAK2-STAT3 pathway, inflammation-related factors (IL-1ß, NLRP3, ICAM-1, VCAM-1), and apoptosis-related factors (cleaved Caspase-3 and Bax). Immunofluorescence staining and western blotting were employed to examine CD31 and α-SMA expression. Knockdown of CNTF was achieved using lentiviral interference, and its effects on inflammation and cell injury were evaluated. Chromatin immunoprecipitation (ChIP) and dual luciferase reporter analysis were conducted to investigate the interaction between the MAFK and CNTF promoters. Our results indicated that Hcy induced high expression of CNTF and activated the JAK2-STAT3 signaling pathway, thereby upregulating factors associated with inflammation and cell apoptosis. Inhibiting CNTF alleviated Hcy-induced inflammation and cell injury. MAFK was identified as a transcription factor promoting CNTF transcription, and its overexpression exacerbated inflammation and cell injury in Hcy-treated HUVECs through the CNTF-JAK2-STAT3 axis, which could be reversed by knocking down CNTF. Activation of MAFK leads to CNTF upregulation, which activates the JAK2-STAT3 signaling pathway, regulating inflammation and inducing injury in Hcy-exposed vascular endothelial cells. Targeting CNTF or its upstream regulator MAFK may represent potential therapeutic strategies for mitigating endothelial dysfunction associated with hyperhomocysteinemia and cardiovascular diseases.

Homocysteine concentration in coronary artery disease and severity of coronary lesions.

Our previous study reckons that the impact of the rs1801133 variant of 5,10-methylenetetrahydrofolate reductase (MTHFR) on coronary artery disease (CAD) is possibly mediated by cardiometabolic disorder. This study is performed to verify this hypothesis. Four hundred and thirty CAD patients and 216 CAD-free individuals were enrolled in this case-control study. The rs1801133 variant was genotyped by PCR-RFLP. Severity of coronary lesions was evaluated by number of stenotic coronary vessels and extent of coronary stenosis. The rs1801133 T allele significantly increased homocysteine levels in patients with CAD and CAD-free individuals. Individuals with the T allele of rs1801133 had an increased risk of developing CAD. In contrast, individuals with the TT genotype of rs1801133 were at high risk of multiple vessel lesions. The carriers of CT genotype had higher levels of systolic blood pressure (SBP), low-density lipoprotein cholesterol (LDL-C), and high-sensitivity C-reactive protein (hs-CRP), and lower levels of apolipoprotein A1 (APOA1) than those with CC genotype in male patients with CAD. The receiver operating characteristic (ROC) curve and precision-recall (PR) curve indicated that hyperhomocysteinemia was sensitive to predict the severity of CAD. Multivariate logistic regression revealed that homocysteine, rs1801133, age, smoking, weight, body mass index (BMI), lipoprotein(a) [Lp(a)], and hs-CRP were independent risk factors for CAD. The increased risk of CAD and severity of coronary lesions associated with rs1801133 in the Chinese Han population were attributed, at least partly, to high homocysteine levels. Hyperhomocysteinemia had a high predictive value for severe CAD or multiple vessel lesions.

Disease-causing cystathionine ß-synthase linker mutations impair allosteric regulation.

Cystathionine ß-synthase (CBS) catalyzes the committing step in the transsulfuration pathway, which is important for clearing homocysteine and furnishing cysteine. The transsulfuration pathway also generates H2S, a signaling molecule. CBS is a modular protein with a heme and pyridoxal phosphate-binding catalytic core, which is separated by a linker region from the C-terminal regulatory domain that binds S-adenosylmethionine (AdoMet), an allosteric activator. Recent cryo-EM structures reveal that CBS exists in a fibrillar form and undergoes a dramatic architectural rearrangement between the basal and AdoMet-bound states. CBS is the single most common locus of mutations associated with homocystinuria, and, in this study, we have characterized three clinical variants (K384E/N and M391I), which reside in the linker region. The native fibrillar form is destabilized in the variants, and differences in their limited proteolytic fingerprints also reveal conformational alterations. The crystal structure of the truncated K384N variant, lacking the regulatory domain, reveals that the overall fold of the catalytic core is unperturbed. M391I CBS exhibits a modest (1.4-fold) decrease while the K384E/N variants exhibit a significant (∼8-fold) decrease in basal activity, which is either unresponsive to or inhibited by AdoMet. Pre-steady state kinetic analyses reveal that the K384E/N substitutions exhibit pleiotropic effects and that the differences between them are expressed in the second half reaction, that is, homocysteine binding and reaction with the aminoacrylate intermediate. Together, these studies point to an important role for the linker in stabilizing the higher-order oligomeric structure of CBS and enabling AdoMet-dependent regulation.

One-carbon metabolism biomarkers and upper gastrointestinal cancer in the Golestan Cohort Study.

Incidence of esophageal and gastric cancer has been linked to low B-vitamin status. We conducted matched nested case-control studies of incident esophageal squamous cell carcinoma (ESCC; 340 case-control pairs) and gastric cancer (GC; 352 case-control pairs) within the Golestan Cohort Study. The primary exposure was plasma biomarkers: riboflavin and flavin mononucleotide (FMN) (vitamin B2), pyridoxal phosphate (PLP) (B6), cobalamin (B12), para-aminobenzoylglutamate (pABG) (folate), and total homocysteine (tHcy); and indicators for deficiency: 3-hydroxykyurenine-ratio (HK-r for vitamin B6) and methylmalonic acid (MMA for B12). We estimated odds ratios (ORs) and 95% confidence intervals (CIs) using conditional logistic regression adjusting for matching factors and potential confounders. High proportions of participants had low B-vitamin and high tHcy levels. None of the measured vitamin B levels was associated with the risk of ESCC and GC, but elevated level of MMA was marginally associated with ESCC (OR = 1.42, 95% CI = 0.99-2.04) and associated with GC (OR = 1.53, 95% CI = 1.05-2.22). Risk of GC was higher for the highest versus lowest quartile of HK-r (OR = 1.95, 95%CI = 1.19-3.21) and for elevated versus non-elevated HK-r level (OR = 1.59, 95% CI = 1.13-2.25). Risk of ESCC (OR = 2.81, 95% CI = 1.54-5.13) and gastric cancer (OR = 2.09, 95%CI = 1.17-3.73) was higher for the highest versus lowest quartile of tHcy. In conclusion, insufficient vitamin B12 was associated with higher risk of ESCC and GC, and insufficient vitamin B6 status was associated with higher risk of GC in this population with prevalent low plasma B-vitamin status. Higher level of tHcy, a global indicator of OCM function, was associated with higher risk of ESCC and GC.

Homocysteine-potentiated Kelch-like ECH-associated protein 1 promotes senescence of neuroblastoma 2a cells via inhibiting ubiquitination of ß-catenin.

Elevated serum homocysteine (Hcy) level is a risk factor for Alzheimer's disease (AD) and accelerates cell aging. However, the mechanism by which Hcy induces neuronal senescence remains largely unknown. In this study, we observed that Hcy significantly promoted senescence in neuroblastoma 2a (N2a) cells with elevated ß-catenin and Kelch-like ECH-associated protein 1 (KEAP1) levels. Intriguingly, Hcy promoted the interaction between KEAP1 and the Wilms tumor gene on the X chromosome (WTX) while hampering the ß-catenin-WTX interaction. Mechanistically, Hcy attenuated the methylation level of the KEAP1 promoter CpG island and activated KEAP1 transcription. However, a slow degradation rate rather than transcriptional activation contributed to the high level of ß-catenin. Hcy-upregulated KEAP1 competed with ß-catenin to bind to WTX. Knockdown of both ß-catenin and KEAP1 attenuated Hcy-induced senescence in N2a cells. Our data highlight a crucial role of the KEAP1-ß-catenin pathway in Hcy-induced neuronal-like senescence and uncover a promising target for AD treatment.

Discovery and Identification of Three Homocysteine Metabolites by Chemical Derivatization and Mass Spectrometry Fragmentation.

Discovery and identification of a new endogenous metabolite are typically hindered by requirements of large sample volumes and multistage purifications to guide synthesis of the standard. Presented here is a metabolomics platform that uses chemical tagging and tandem mass spectrometry to determine structure, direct synthesis, and confirm identity. Three new homocysteine metabolites are reported: N-succinyl homocysteine, 2-methyl-1,3-thiazinane-4-carboxylic acid (MTCA), and homolanthinone.

Equivalent Response Strategy for Sensing Total Biothiols in Human Serums and Living Cells Using a Hemicyanine-Based Self-Immolative Probe.

Biothiols including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are crucial in maintaining the redox balance in the body, and the metabolism and transportation of biothiols rely on the coreaction of diverse proteins and enzymes. The abnormal concentrations and metabolism of biothiols are closely associated with many diseases. However, due to the same active reaction site of the sulfydryl group in biothiols, it is inevitable to bear a confused signal of mutual influence on both nonselective detection and discriminate detection, which presents a serious challenge of accurately sensing or imaging the three biothiols. By assigning an α,ß-unsaturated ketone moiety as a Michael acceptor to trigger thiols to complete the irreversible equivalent domino response processes of nucleophilic addition, olefinic bond migration, and self-immolation, a targeted strategy was rationally pointed out, and herein, a hemicyanine-based probe CyOCy was prepared as a proof of strategy demonstration. The new probe could be equivalently lit up by Cys, Hcy, GSH, and even biothiol combinations (Cys/Hcy, Cys/GSH, Hcy/GSH, or Cys/Hcy/GSH) with unified linear ranges, detection limits, and response times. The probe CyOCy has been successfully used for the accurate quantification of total biothiols in the serum samples of healthy persons and coronary heart disease patients. In addition, the probe has been applied for cell screening, exogenous biothiol imaging, and monitoring drug-induced biothiol fluctuations. The purposive thinking of this work may provide an effective avenue for the accurate sensing of multicomponent samples.

Simultaneous SERS Sensing of Cysteine and Homocysteine in Blood Based on the CBT-Cys Click Reaction: Toward Precisive Diagnosis of Schizophrenia.

At present, there is a lack of sufficiently specific laboratory diagnostic indicators for schizophrenia. Serum homocysteine (Hcy) levels have been found to be related to schizophrenia. Cysteine (Cys) is a demethylation product in the metabolism of Hcy, and they always coexist with highly similar structures in vivo. There are few reports on the use of Cys as a diagnostic biomarker for schizophrenia in collaboration with Hcy, mainly because the rapid, economical, accurate, and high-throughput simultaneous detection of Cys and Hcy in serum is highly challenging. Herein, a click reaction-based surface-enhanced Raman spectroscopy (SERS) sensor was developed for simultaneous and selective detection of Cys and Hcy. Through the efficient and specific CBT-Cys click reaction between the probe containing cyan benzothiazole and Cys/Hcy, the tiny methylene difference between the molecular structures of Cys and Hcy was converted into the difference between the ring skeletons of the corresponding products that could be identified by plasmonic silver nanoparticle enhanced molecular fingerprint spectroscopy to realize discriminative detection. Furthermore, the SERS sensor was successfully applied to the detection in related patient serum samples, and it was found that the combined analysis of Cys and Hcy can improve the diagnostic accuracy of schizophrenia compared to a single indicator.

Development of a chemiluminescent immunoassay based on magnetic solid phase for quantification of homocysteine in human serum.

BACKGROUND: Homocysteine (HCY) is a sulfur-containing amino acid that is an independent or important risk factor for the occurrence of many chronic diseases and is one of the most important indicators for determining health risks. However, existing HCY detection methods do not meet the requirements of clinical diagnosis. Therefore, there is an urgent need to establish new detection methods to meet the needs of clinical detection. RESULTS: In this study, we used the principle of competitive method to establish a new method for the determination of HCY in human serum using a chemiluminescent enzyme immunoassay in conjunction with a chemiluminescent assay instrument that uses magnetic microparticles as the solid phase of the immunoreaction. The established method achieved satisfactory results in terms of minimum detection limit, specificity, accuracy, and clinical application. The limit of detection was 0.03 ng/mL. The intra-assay coefficient of variation (CV) was 1.94-5.05%, the inter-assay CV was 2.29-6.88%, and the recovery rate was 88.60-93.27%. Cross-reactivity with L-cysteine ranged from 0.0100 to 0.0200 µmol/L, and cross-reactivity with glutathione ranged from 0.0100 to 0.200 µmol/L, all of which were less than the limit of detection (LoD) of this method. The linear factor R of this method was greater than 0.99. CONCLUSIONS: In summary, the developed method showed a good correlation with the product from Abbott. A total of 996 clinical patients with cardiovascular diseases were evaluated using the method developed in this study.

Serum interleukin-17 A and homocysteine levels in children with autism.

BACKGROUND: Autism Spectrum Disorder (ASD) is a neurodevelopmental condition that typically emerges early in childhood. This study aimed to explore the potential link between serum levels of vitamin B12 and homocysteine (Hcy) and the severity of ASD symptoms in children. METHODS: In this study, 50 children diagnosed with ASD comprised the observation group, while 50 healthy children constituted the control group. Serum levels of IL-17 A, Hcy, folate, and vitamin B12 were compared between the study group and control group, as well as among children with different degrees of ASD severity. The correlation between the Childhood Autism Rating Scale (CARS) score and serum levels of IL-17 A, Hcy, folate, and vitamin B12 was examined. Additionally, the relationship between serum IL-17 A and Hcy levels and their association with the severity ASD were explored. RESULTS: Compared to the control group, the observation group demonstrated elevated serum Hcy and IL-17 A levels alongside decreased folate and vitamin B12 levels. Individuals with severe ASD exhibited higher Hcy and IL-17 A levels but lower folate and vitamin B12 levels compared to those with mild to moderate ASD. The CARS score showed negative correlations with serum folate and vitamin B12 levels and positive correlations with serum IL-17 A and Hcy levels in ASD patients. Additionally, serum Hcy and IL-17 A levels were correlated with ASD severity. CONCLUSION: Children diagnosed with ASD presented with reduced serum vitamin B12 levels and increased levels of Hcy, potentially contributing to the onset and severity of ASD.

Metabolic engineering of CHO cells towards cysteine prototrophy and systems analysis of the ensuing phenotype.

Chinese hamster ovary (CHO) cells require cysteine for growth and productivity in fed-batch cultures. In intensified processes, supplementation of cysteine at high concentrations is a challenge due to its limited solubility and instability in solution. Methionine can be converted to cysteine (CYS) but key enzymes, cystathionine beta-synthase (Cbs) and cystathionine gamma-lyase (Cth), are not active in CHO cells resulting in accumulation of an intermediate, homocysteine (HCY), in cell culture milieu. In this study, Cbs and Cth were overexpressed in CHO cells to confer cysteine prototrophy, i.e., the ability to grow in a cysteine free environment. These pools (CbCt) needed homocysteine and beta-mercaptoethanol (ßME) to grow in CYS-free medium. To increase intracellular homocysteine levels, Gnmt was overexpressed in CbCt pools. The resultant cell pools (GnCbCt), post adaptation in CYS-free medium with decreasing residual HCY and ßME levels, were able to proliferate in the HCY-free, ßME-free and CYS-free environment. Interestingly, CbCt pools were also able to be adapted to grow in HCY-free and CYS-free conditions, albeit at significantly higher doubling times than GnCbCt cells, but couldn't completely adapt to ßME-free conditions. Further, single cell clones derived from the GnCbCt cell pool had a wide range in expression levels of Cbs, Cth and Gnmt and, when cultivated in CYS-free fed-batch conditions, performed similarly to the wild type (WT) cell line cultivated in CYS supplemented fed-batch culture. Intracellular metabolomic analysis showed that HCY and glutathione (GSH) levels were lower in the CbCt pool in CYS-free conditions but were restored closer to WT levels in the GnCbCt cells cultivated in CYS-free conditions. Transcriptomic analysis showed that GnCbCt cells upregulated several genes encoding transporters as well as methionine catabolism and transsulfuration pathway enzymes that support these cells to biosynthesize cysteine effectively. Further, 'omics analysis suggested CbCt pool was under ferroptotic stress in CYS-free conditions, which, when inhibited, enhanced the growth and viability of these cells in CYS-free conditions.

Growth requirement for methionine in human melanoma-derived cell lines with different levels of MMACHC expression and methylation.

Methionine dependence, the inability to grow in culture when methionine in the medium is replaced by its metabolic precursor homocysteine, occurs in many tumor cell lines. In most affected lines, the cause of methionine dependence is not known. An exception is the melanoma-derived cell line MeWo-LC1, in which hypermethylation of the MMACHC gene is associated with decreased MMACHC expression. Decreased expression results in decreased provision of the methylcobalamin cofactor required for activity of methionine synthase and thus decreased conversion of homocysteine to methionine. Analysis of data in the Cancer Cell Line Encyclopedia Archive demonstrated that MMACHC hypermethylation and decreased MMACHC expression occurred more frequently in melanoma cell lines when compared to other tumor cell lines. We further investigated methionine dependence and aspects of MMACHC function in a panel of six melanoma lines, including both melanoma lines with known methionine dependence status (MeWo, which is methionine independent, and A375, which is methionine dependent). We found that the previously unclassified melanoma lines HMCB, Colo829 and SH-4 were methionine dependent, while SK-Mel-28 was methionine independent. However, despite varying levels of MMACHC methylation and expression, none of the tested lines had decreased methylcobalamin and adenosylcobalamin synthesis as seen in MeWo-LC1, and the functions of both cobalamin-dependent enzymes methionine synthase and methylmalonyl-CoA mutase were intact. Thus, while melanoma lines were characterized by relatively high levels of MMACHC methylation and low expression, the defect in metabolism observed in MeWo-LC1 was unique, and decreased MMACHC expression was not a cause of methionine dependence in the other melanoma lines.

The MMACHC variant c.158T>C: Mild clinical and biochemical phenotypes and marked hydroxocobalamin response in cblC patients.

Mutations in MMACHC cause cobalamin C disease (cblC, OMIM 277400), the commonest inborn error of vitamin B12 metabolism. In cblC, deficient activation of cobalamin results in methylcobalamin and adenosylcobalamin deficiency, elevating methylmalonic acid (MMA) and total plasma homocysteine (tHcy). We retrospectively reviewed the medical files of seven cblC patients: three compound heterozygotes for the MMACHC (NM_015506.3) missense variant c.158T>C p.(Leu53Pro) in trans with the common pathogenic mutation c.271dupA (p.(Arg91Lysfs*14), "compounds"), and four c.271dupA homozygotes ("homozygotes"). Compounds receiving hydroxocobalamin intramuscular injection monotherapy had age-appropriate psychomotor performance and normal ophthalmological examinations. In contrast, c.271dupA homozygotes showed marked psychomotor retardation, retinopathy and feeding problems despite penta-therapy (hydroxocobalamin, betaine, folinic acid, l-carnitine and acetylsalicylic acid). Pretreatment levels of plasma and urine MMA and tHcy were higher in c.271dupA homozygotes than in compounds. Under treatment, levels of the compounds approached or entered the reference range but not those of c.271dupA homozygotes (tHcy: compounds 9.8-32.9 µM, homozygotes 41.6-106.8 (normal (N) < 14); plasma MMA: compounds 0.14-0.81 µM, homozygotes, 10.4-61 (N < 0.4); urine MMA: compounds 1.75-48 mmol/mol creatinine, homozygotes 143-493 (N < 10)). Patient skin fibroblasts all had low cobalamin uptake, but this was milder in compound cells. Also, the distribution pattern of cobalamin species was qualitatively different between cells from compounds and from homozygotes. Compared to the classic cblC phenotype presented by c.271dupA homozygous patients, c.[158T>C];[271dupA] compounds had mild clinical and biochemical phenotypes and responded strikingly to hydroxocobalamin monotherapy.