Tumor methionine metabolism drives T-cell exhaustion in hepatocellular carcinoma.

T-cell exhaustion denotes a hypofunctional state of T lymphocytes commonly found in cancer, but how tumor cells drive T-cell exhaustion remains elusive. Here, we find T-cell exhaustion linked to overall survival in 675 hepatocellular carcinoma (HCC) patients with diverse ethnicities and etiologies. Integrative omics analyses uncover oncogenic reprograming of HCC methionine recycling with elevated 5-methylthioadenosine (MTA) and S-adenosylmethionine (SAM) to be tightly linked to T-cell exhaustion. SAM and MTA induce T-cell dysfunction in vitro. Moreover, CRISPR-Cas9-mediated deletion of MAT2A, a key SAM producing enzyme, results in an inhibition of T-cell dysfunction and HCC growth in mice. Thus, reprogramming of tumor methionine metabolism may be a viable therapeutic strategy to improve HCC immunity.

Gene-centric approach identifies new and known loci for FVIII activity and VWF antigen levels in European Americans and African Americans.

Coagulation factor VIII and von Willebrand factor (VWF) are key proteins in procoagulant activation. Higher FVIII coagulant activity (FVIII :C) and VWF antigen (VWF :Ag) are risk factors for cardiovascular disease and venous thromboembolism. Beyond associations with ABO blood group, genetic determinants of FVIII and VWF are not well understood, especially in non European-American populations. We performed a genetic association study of FVIII :C and VWF:Ag that assessed 50,000 gene-centric single nucleotide polymorphisms (SNPs) in 18,556 European Americans (EAs) and 5,047 African Americans (AAs) from five population-based cohorts. Previously unreported associations for FVIII :C were identified in both AAs and EAs with KNG1 (most significantly associated SNP rs710446, Ile581Thr, Ile581Thr, P = 5.10 × 10(-7) in EAs and P = 3.88 × 10(-3) in AAs) and VWF rs7962217 (Gly2705Arg,P = 6.30 × 10(-9) in EAs and P = 2.98 × 10(-2) in AAs. Significant associations for FVIII :C were also observed with F8/TMLHE region SNP rs12557310 in EAs (P = 8.02 × 10(-10) ), with VWF rs1800380 in AAs (P = 5.62 × 10(-11) ), and with MAT1A rs2236568 in AAs (P51.69 × 10(-6) ). We replicated previously reported associations of FVIII :C and VWF :Ag with the ABO blood group, VWF rs1063856(Thr789Ala), rs216321 (Ala852Gln), and VWF rs2229446 (Arg2185Gln). Findings from this study expand our understanding of genetic influences for FVIII :C and VWF :Ag in both EAs and AAs.

Hypermethioninaemia due to methionine adenosyltransferase I/III (MAT I/III) deficiency: diagnosis in an expanded neonatal screening programme.

The Expanded Newborn Screening Program (MS/MS) in the region of Galicia (NW Spain) was initiated in 2000 and includes the measurement of methionine levels in dried blood spots. Between June 2000 and June 2007, 140 818 newborns were analysed, and six cases of persistent hypermethioninaemia were detected: one homocystinuria due to cystathionine ß-synthase (CßS) deficiency, and five methionine adenosyltransferase I/III (MAT I/III) deficiencies. The five cases of MAT I/III deficiency represent an incidence of 1/28 163 newborns. In these five patients, methionine levels in dried blood spots ranged from 50 to 147 µmol/L. At confirmation of the persistence of the hypermethioninaemia in a subsequent plasma sample, plasma methionine concentrations were moderately elevated in 4 of the 5 patients (mean 256 µmol/L), while total homocysteine (tHcy) was normal; the remaining patient showed plasma methionine of 573 µmol/L and tHcy of 22.8 µmol/L. All five patients were heterozygous for the same dominant mutation, R264H in the MAT1A gene. With a diet not exceeding recommended protein requirements for their age, all patients maintained methionine levels below 300 µmol/L. Currently, with a mean of 2.5 years since diagnosis, the patients are asymptomatic and show developmental quotients within the normal range. Our results show a rather high frequency of hypermethioninaemia due to MAT I/III deficiency in the Galician neonatal population, indicating a need for further studies to evaluate the impact of persistent isolated hypermethioninaemia in neonatal screening programmes.

Methionine adenosyltransferase activity in erythrocytes and spinal cord of patients with sporadic amyotrophic lateral sclerosis.

The role of transmethylation mechanisms in the etiology of amyotrophic lateral sclerosis (ALS) is hitherto unexplored. The activity of L-methionine S-adenosyltransferase (MAT), a regulatory enzyme of S-adenosylmethionine biosynthesis, was investigated in erythrocytes of 21 patients with ALS, spinal cord specimens of 7 ALS patients, and matched controls. In ALS patients the activity of MAT in erythrocytes was sex-dependent. In comparison with controls, the male group presented a 33% higher V(max) (P < 0.05) and a 41% decrease in the affinity of MAT for methionine (K(m), P < 0.05). The type of ALS onset (limb or bulbar), age, or duration of the disease did not influence erythrocyte MAT activity. In the spinal cord, the activity of MAT was homogeneously distributed through dorsal horn, ventral horn, and white matter. Comparisons between data from controls and ALS patients and analysis of sex effect showed no significant differences. The kinetic difference of erythrocyte MAT in the male group of ALS patients might be interesting to explore since it is well known that there is a male predominance of 1.5 to 2. 5:1 in ALS.

Erythrocyte and brain methionine adenosyltransferase activities in patients with schizophrenia.

The activity of methionine adenosyltransferase (MAT) was investigated in erythrocytes and postmortem brain specimens (cortex gyrus frontalis, hippocampus and thalamus) of patients with schizophrenia treated with neuroleptics. In comparison with the control group, abnormally low values of MAT Vmax and an increased MAT affinity towards methionine (lower Km values) were found in erythrocytes. In the brain, a regionally selective decrease of MAT Km was found in cortex gyrus frontalis but the Vmax values were however, unchanged. In the regions of cortex gyrus frontalis and hippocampus, but not in thalamus, the values of Vmax and Km were inversely correlated with the duration of schizophrenia. In rats treated for 28 days with the typical neuroleptic haloperidol and the atypical clozapine, a significant increase of MAT activity was found in the corpus striatum. There is the possibility that the changes observed in MAT activity in patients with schizophrenia are attributed to the neuroleptic medication.

Influence of osmotic stress on protein methylation in resealed erythrocytes.

Resealed erythrocytes are a useful means of targeting exogenous proteins or extending their activity. We tested human resealed erythrocytes as a model system for studying protein methyl esterification, a reaction involved in the processing of spontaneously deamidated/isomerized polypeptides. Our results show that resealed erythrocytes are still active in the metabolic processes that lead to the formation of methyl-esterified proteins. The methylation pattern of endogenous membrane proteins appeared to be similar to that of normal erythrocytes, with bands 2.1, 3, 4.1 and 4.2 as the major methyl acceptors. We detected methyl esterification of ovalbumin, as an exogenous substrate trapped within resealed erythrocytes. Methyl incorporation was almost completely inhibited by simultaneously loading red cells with adenosine and homocysteine thiolactone, in vivo precursors of the transmethylation inhibitor S-adenosylhomocysteine. We investigated the effects of repeated resealing procedures on methyl acceptability of endogenous membrane proteins. We found that methyl-incorporation levels increased, despite an apparent conserved protein composition of the membrane. This result suggests that osmotic stress to the membrane may be responsible for increased protein methylation due to the appearance of new sites or an increased accessibility of existing sites.

Quantification of erythrocyte S-adenosyl-L-methionine levels and its application in enzyme studies.

A highly selective high-performance liquid chromatographic method for the quantification of human erythrocyte S-adenosyl-L-methionine levels is described. A strong cation-exchange sorbent with propylsulphonic acid functional groups was used to extract S-adenosyl-L-methionine and S-adenosylethionine (internal standard) from erythrocytes. Quantification of erythrocyte S-adenosyl-L-methionine levels was achieved by using reversed-phase high-performance liquid chromatography and ultraviolet detection at 254 nm. This method was adapted to measure methionine-adenosyltransferase activity in erythrocytes, which enables us to study the possible role of altered methylation in different diseases.

Clinical correlations of one-carbon metabolism abnormalities.

1. Ninety psychiatric inpatients with a DSM III diagnosis of schizophrenia, mania, or major depression were studied. 2. Upon admission/transfer to the Clinical Studies Unit, and prior to discharge, measurements of symptom severity (BPRS, Ham-D, Young's Mania Scale) and blood samples were obtained. 3. Erythrocytes from these paired (admission and discharge) blood samples were assayed for methionine adenosyltransferase (MAT) activity and phosphatidylcholine (PC) content. 4. Comparisons were made between the changes in MAT Vmax, or % PC, and changes in symptom severity. 5. For the majority of the patients (79.3% of the schizophrenics; 84.6% of the depressives; and 93.8% of the manics), clinical improvement was associated with a "normalization" of enzyme activity. The association between changes in % PC and clinical response did not achieve significant correlation.

Abnormalities of one-carbon metabolism in psychiatric disorders: study of methionine adenosyltransferase kinetics and lipid composition of erythrocyte membranes.

Two independent lines of inquiry have implicated some disturbance of one-carbon cycle metabolism in affective disorders. Folic acid deficiency commonly leads to depression, and S-adenosylmethionine has been reported to have antidepressant properties. Methionine adenosyltransferase has been reported to be underactive in depression and schizophrenia and overactive in mania. This study reports the effects on erythrocyte methionine adenosyltransferase (MAT) kinetics (Vmax) of a 2-week treatment in a population of patients housed on a psychiatric research ward. The drug-free schizophrenic patients and depressives had, upon admission, low Vmax values, and the drug-free manic patients had high Vmax values on admission. After 2 weeks of appropriate treatment, the values for all three patient samples showed significant normalization (i.e., the levels rose in schizophrenics and depressives and fell in manics). We have further shown that pretreatment low levels of erythrocyte membrane phosphatidylcholine in depressives and high levels in manics show statistically significant normalization following 2 weeks of pharmacotherapy. The significance of these results is discussed.

Medication effects on one-carbon metabolism in schizophrenia, mania, and major depression.

Erythrocyte methionine adenosyltransferase (MAT) activity (Vmax) and phosphatidylcholine (PC) levels previously have been found increased in manic patients and decreased in depressive and schizophrenic patients. To evaluate whether these abnormalities were the result of medication effects, erythrocyte MAT activity (Vmax) was assayed for paired samples from 29 schizophrenic, 16 manic, and 12 depressive patients, an erythrocyte PC levels were obtained for paired samples from 13 schizophrenic, seven manic, and seven depressive patients. Patients were medication free for at least 3 weeks. Vmax was significantly increased in schizophrenic and depressive patients (p less than 0.01; p less than 0.01) and significantly decreased (p less than 0.01) in manic patients after 2 weeks of psychotropic medication. Similar trends were found in PC levels. The findings of those one-carbon metabolism tests following medication are generally opposite to those reported to be related to specific disorders and tend toward normalization. Moreover, in vitro preincubation of erythrocytes of three normal subjects with the most commonly used neuroleptics had no consistent effects of MAT Vmax. These findings confirm previous studies that showed similarities in one-carbon metabolism of schizophrenic and depressed patients as opposed to manic patients and suggest that medications tend to correct or minimize rather than induce such abnormalities.

Enzymatic basis for the calcium-induced decrease of membrane protein methyl esterification in intact erythrocytes. Evidence for an impairment of S-adenosylmethionine synthesis.

The effect of Ca2+ loading, induced by the ionophore A23187, on methyl esterification of membrane proteins (i.e. bands 2.1, 3, 4.1 and 4.5) has been investigated in intact human erythrocytes. When the cells were incubated with L-[methyl-3H]methionine, 40 microM CaCl2 and 10 microM A23187 induce a 50% inhibition of membrane protein methyl esterification. This effect is selectively due to the increased intracellular Ca2+ concentration, as it is antagonized by 10 mM EGTA, and other divalent cations such as Mn2+ do not exert any inhibition. In order to clarify the mechanism(s) of the reported inhibition, the various events involved in the methyl esterification process in vivo were analyzed. L-Methionine uptake as well as protein methylase II activity are not directly affected by altered intracellular Ca2+ concentrations. Conversely in the Ca2+-loaded erythrocytes the conversion of [3H]methionine into [3H]AdoMet, catalyzed by AdoMet synthetase, decreases up to 25%. When the undialyzed erythrocyte cytosolic fraction is assayed in vitro for AdoMet synthetase the activity of the enzyme from the CaCl2/A23187-treated erythrocytes is significantly lower than the control, up to 5 mM ATP. This result suggests that in the Ca2+-loaded erythrocytes the ATP intracellular concentration is significantly lowered. The direct evaluation of ATP intracellular concentration, by HPLC, confirms a significant drop of ATP level, as a consequence of the Ca2+ loading. The removal of Ca2+ from the cells quantitatively restores both the AdoMet synthesis and the methyl esterification levels. The possible role of altered ATP intracellular concentrations as a regulatory factor in the AdoMet-dependent reactions as well as in post-translational protein methylation related to the ageing process is also discussed.

One-carbon metabolism disturbances in affective disorders. A preliminary report.

Methionine adenosyltransferase (MAT) activity (Vmax) and the relative amount of phosphatidylcholine (% PC) were measured in erythrocytes of up to 30 DSM-III diagnosed manic, 17 unipolar depressed patients, and 28 normal controls. Manic subjects had significantly higher and depressed subjects significantly lower MAT Vmax than normals. The relative amount of PC was in the low range for the depressives, and in the high range for the manics. Depressive patients present, in these tests, similar abnormalities to those seen previously in schizophrenic patients. Clinical and diagnostic implications of these findings are discussed.

S-adenosyl-L-methionine synthetase from human erythrocytes: role in the regulation of cellular S-adenosylmethionine levels.

The properties of human erythrocyte S-adenosyl-L-methionine synthetase (ATP:L-methionine S-adenosyltransferase, EC 2.5.1.6) were studied with respect to the role of S-adenosylmethionine in transmethylation reactions. Kinetic values obtained with both a cytosolic and a 350-fold purified preparation of enzyme were compared with measured intracellular concentrations of substrates and products. This analysis revealed that effective regulation of enzyme activity and product concentration can occur through feedback inhibition by S-adenosylmethionine (Ki = 2.0-2.9 microM; the endogenous concentration is 3.5 microM). This enzyme can be distinguished from S-adenosylmethionine synthetases found in other tissues and appears to be specialized for its role in erythrocyte methyl group metabolism, especially with regard to protein carboxyl methyl-transfer reactions.

Intermittent hypermethioninaemia associated with normal hepatic methionine adenosyltransferase activity: report of a case.

A 5-month-old infant was found to have hypermethioninaemia (0.8 mumol/ml) that has persisted intermittently (0.02-1.3 mumol/ml) over a period of 3 years. She presented with developmental delay and failure to thrive associated with gross abuse and neglect. Histological examination of the liver revealed inflammation of the portal triads. The activity of hepatic L-methionine-S-adenosyltransferase (EC 2.5.1.6) was normal. Whether the biochemical findings were the cause or the result of the hepatic damage is uncertain, but the minimal histological findings in the liver suggest a primary biochemical defect.

Kinetic evidence for decreased methionine adenosyltransferase activity in erythrocytes from schizophrenics.

We have observed significantly lower kinetic parameters (KM and Vmax) for methionine adenosyltransferase activity in erythrocytes obtained from early onset schizophrenics when compared to samples from normal subjects. These differences are apparently not due to differences in S-adenosylmethionine (SAM) utilization. These results offer an explanation for the conflicting reports of previous investigators and support the concept that undermethylation may characterize some forms of schizophrenia. Methylation is involved in multiple aspects of metabolism and although similar differences in the MAT enzyme in the brain have not been reported, such a deficit could have profound effects on the nervous system. Decreased availability of SAM could decrease catecholamine metabolism or rates of phospholipid methylation.