Vitamin B6 Metabolism Determines T Cell Anti-Tumor Responses.

Targeting T cell metabolism is an established method of immunomodulation. Following activation, T cells engage distinct metabolic programs leading to the uptake and processing of nutrients that determine cell proliferation and differentiation. Redirection of T cell fate by modulation of these metabolic programs has been shown to boost or suppress immune responses in vitro and in vivo. Using publicly available T cell transcriptomic and proteomic datasets we identified vitamin B6-dependent transaminases as key metabolic enzymes driving T cell activation and differentiation. Inhibition of vitamin B6 metabolism using the pyridoxal 5'-phosphate (PLP) inhibitor, aminoxyacetic acid (AOA), suppresses CD8+ T cell proliferation and effector differentiation in a dose-dependent manner. We show that pyridoxal phosphate phosphatase (PDXP), a negative regulator of intracellular vitamin B6 levels, is under the control of the hypoxia-inducible transcription factor (HIF1), a central driver of T cell metabolism. Furthermore, by adoptive transfer of CD8 T cells into a C57BL/6 mouse melanoma model, we demonstrate the requirement for vitamin B6-dependent enzyme activity in mediating effective anti-tumor responses. Our findings show that vitamin B6 metabolism is required for CD8+ T cell proliferation and effector differentiation in vitro and in vivo. Targeting vitamin B6 metabolism may therefore serve as an immunodulatory strategy to improve anti-tumor immunotherapy.

Oral exposure to the anti-pyridoxine compound 1-amino D-proline further perturbs homocysteine metabolism through the transsulfuration pathway in moderately vitamin B6 deficient rats.

Pyridoxal 5'-phosphate (PLP; a B6 vitamer) serves as an important cofactor in a myriad of metabolic reactions, including the transsulfuration (TS) pathway, which converts homocysteine (Hcy) to cysteine. While overt vitamin B6 deficiency is rare, moderate deficiency is common and may be exacerbated by anti-pyridoxine factors in the food supply. To this end, we developed a model of moderate B6 deficiency and a study was conducted to examine the in vivo effect of 1-amino D-proline (1ADP), an anti-pyridoxine factor found in flaxseed, on indices of Hcy metabolism through the TS pathway in moderately B6 deficient rats. Male weaning rats received a semi-purified diet containing either 7 mg/kg (control; CD) or 0.7 mg/kg (moderately deficient; MD) diet of pyridoxine·hydrochloride (PN∙HCl), each with 1 of 4 levels of 1ADP, viz. 0, 0.1, 1 and 10 mg/kg diet for 5 weeks. Perturbations in vitamin B6 biomarkers were more pronounced in the MD group. Plasma PLP was significantly reduced, while plasma Hcy (8-fold) and cystathionine (11-fold) were increased in rats consuming the highest amount of 1ADP in the MD group. The activities of hepatic cystathionine ß-synthase and cystathionine γ-lyase enzymes were significantly reduced in rats consuming the highest 1ADP compared to the lowest, for both levels of PN∙HCl. Dilation of hepatic central veins and sinusoids, mild steatosis and increased liver triglycerides were present in MD rats consuming the highest 1ADP level. The current data provide evidence that the consumption of an anti-pyridoxine factor linked to flaxseed may pose a risk for subjects who are moderate/severe vitamin B6 deficient.

Role of MC-1 alone and in combination with tissue plasminogen activator in focal ischemic brain injury in rats.

OBJECT: Pyridoxal-5-phosphate (PLP), the biologically active form of pyridoxine, can rescue neurons from death in vitro and in vivo. In the present project, the authors have studied whether MC-1, an analog of PLP, alone or in combination with the thrombolytic agent tissue plasminogen activator (tPA), can protect the brains of rats injured by ischemia. METHODS: Ischemic brain injury was induced in rats by injecting a preformed blood clot in the middle cerebral artery (MCA). Neurological deficits and infarct volumes caused by the embolus were measured to evaluate the effects of MC-1 on the ischemic injury. Systemic blood pressure and local brain blood flow were also monitored. Administration of different doses of MC-1 1 hour after embolization significantly reduced the infarct volume and improved functional recovery. Injection of MC-1 (40 mg/kg) at 3 or 6 hours after embolization also reduced the volume of the infarct significantly and improved functional recovery. Combined treatment with MC-1 and tPA was also neuroprotective, although it was not superior to treatment involving either MC-1 or tPA alone. Treatment with MC-1 did not result in significant changes in either systemic blood pressure or local blood flow in the ischemic brain. CONCLUSIONS: These data support the hypothesis that in the focal embolic stroke model in rats MC-1 is a neuroprotective agent. The neuroprotection this compound provides still exists when MC-1 administration is delayed up to 6 hours after ischemic injury.

A GDP-fucose-protected, pyridoxal-5'-Phosphate/NaBH(4)-sensitive lys residue common to human alpha1-->3Fucosyltransferases corresponds to Lys(300) in FucT-IV.

Human alpha1-->3/4fucosyltransferases (FucTs) contain a common essential pyridoxal-5'-phosphate(PLP)/NaBH(4) reactive, GDP-fucose-protectable Lys. For identification, site-directed mutants at lysines of FucT-IV and -VII were prepared and tested. Non conserved lysine mutants K119Y and K394Q were similar to wild-type FucT-IV. However, mutants of conserved lysines K228R and K300R were distinct. The specific activity of K228R was 2- to 3-fold lower but retained K(m) values for donor and acceptor substrates as wild-type FucT-IV. The specific activity of K300R was reduced over 400-fold with an apparent K(m) for GDP-fucose over 200 microM. FucT-VII mutants K169R and K240R (equivalent to K228R and K300R for FucT-IV, respectively) were inactive. No change in PLP/NaBH(4) sensitivity occurred with K119Y, K228R, and K394Q compared to wild-type FucT-IV. These and previous results (A. L. Sherwood, A. T. Nguyen, J. M. Whitaker, B. A. Macher, M. R. Stroud, and E. H. Holmes, J. Biol. Chem. 273, 25256-25260, 1998) demonstrate that of three conserved lysines in FucT-IV, two (Lys(228) and Lys(283)) are not involved in substrate binding but perhaps in catalysis. The third site, Lys(300), is involved in GDP-fucose binding and PLP/NaBH(4) inactivation.

The cosubstrate NADP(H) protects lysine 601 in the porcine NADPH-cytochrome P-450 reductase against pyridoxylation.

Lys601 in NADPH-cytochrome P-450 reductase is modified by reductive alkylation with pyridoxal 5'-phosphate (pyridoxylation). Lys601 is protected against modification by the cosubstrate NADP(H).

Changes in serum copper and zinc during treatment with anticancer drugs interfering with pyridoxal phosphate.

Hexamethylmelamine, pentamethylmelamine and procarbazine are anticancer drugs known to interfere with pyridoxal phosphate. This paper presents results on copper and zinc serum levels during the treatment with each of these drugs used as single agents. Six NZW rabbits weighing 2.7-4.5 kg were used in these experiments. Hexamethylmelamine and procarbazine were administered by gastric gavage and pentamethylmelamine by intravenous route at the daily doses of 100 mg, 30 mg and 50 mg/kg of body weight respectively for up to four days. Blood samples were collected in metal free tubes at fasting state before and during the treatment. Student's paired t-test was used for statistical analysis. The pretreatment serum copper concentration significantly (p = 0.05) increased and conversely the serum zinc concentration significantly (p = 0.05) decreased during each drug treatment. Consequently the copper/zinc ration significantly increased from 0.32, 0.33 and 0.27 to 1.16, 0.63 and 1.13 for hexamethylmelamine, pentamethylmelamine and procarbazine respectively. These results indicate, that daily administration of three anticancer drugs interfering with pyridoxal phosphate causes changes in serum copper and zinc levels with inversed relationship between both changes.

Pyridoxal phosphate protects against an irreversible temperature-dependent inactivation of hepatic delta-aminolevulinic acid synthase.

The stability of hepatic delta-aminolevulinic acid synthase (ALAS), the first and rate-limiting enzyme of the heme biosynthetic pathway, was investigated. Incubation of the mitochondrial matrix fraction obtained from either control or allylisopropylacetamide-induced rats at 37 degrees C in Tris-Cl, pH 7.4, EDTA, and dithiothreitol resulted in a rapid decrease in ALAS activity such that 50-70% of the activity was lost after 30 min. Similar decreases in ALAS activity were observed when a cytosolic fraction from the induced animals was incubated at 37 degrees C. Addition of 0.1 mM pyridoxal-P, the cofactor of ALAS, to the preincubation medium completely prevented the observed loss of activity; however, dialysis of the inactive matrix fraction against several changes of buffer containing pyridoxal-P did not restore activity, suggesting that the inactivation was irreversible. These decreases in ALAS activity in the absence of pyridoxal-P were temperature dependent, as a 55% loss of ALAS activity was observed after a 60-min incubation at 30 degrees C, while the enzyme was completely stable when preincubated at 22 degrees C for 60 min. This inactivation of ALAS does not appear to involve proteolytic digestion, as addition of a wide spectrum of protease inhibitors to the preincubation medium in the absence of pyridoxal-P did not protect against the inactivation. The suggestion is made that the cofactor, pyridoxal-P, may dissociate from the enzyme during the preincubation and, consequently, the apoenzyme may be irreversibly inactivated at temperatures above 22 degrees C.

Anticonvulsant activity of muscimol and gamma-aminobutyric acid against pyridoxal phosphate-induced epileptic seizures.

The intracerebroventricular injection of pyridoxal phosphate (PLP, 0.125-1.25 mumol/rat) causes epileptic seizures (4 min leads to 1 min) that are preventable or reversible by GABA (1 mumol/rat), by muscimol (0.025 mumol/rat), or by diazepam (1.75 mumol/rat). At the peak of PLP-induced convulsions, the activities of GAD and GABA-T in 14 regions of rat brain remained unaltered, whereas the concentrations of PLP remained elevated. The PLP-induced convulsion was blocked by DABA (10 mumol/rat) but was not altered by beta-alanine (50 mumol/rat). The previous in vitro studies have shown that PLP increases the uptake of [3H]GABA into synaptosomes and inhibits the binding of [3H]GABA to synaptic membranes. These data suggest that PLP-induced convulsion is due to reduced availability of GABA to its recognition sites, rather than to alteration in the activity of GABA metabolizing enzymes, or unavailability of PLP as a coenzyme for GAD and GABA-T. Since the duration of PLP-induced epileptic seizures is short and can be prevented by GABA agonists, PLP may be used as a tool to study the nature of GABA-mediated neuroinhibition and the properties of GABA receptor sites.

Physicochemical evidence for the existence of two pyridoxal 5'-phosphate binding sites on glutamate dehydrogenase and characterization of their functional role.

Kinetic studies of pyridoxal 5'-phosphate binding to glutamate dehydrogenase (EC 1.4.1.3) has provided evidence for two specific binding sites, chemically identified as Lys 126 and Lys 333. Use of protecting ligands permitted the selective modification of only one of these lysines, and showed that (1) Lys 333 modification results in depolymerisation of the enzyme into active hexamers; (2) Lys 126-modified enzyme was 92% inactivated. The residual activity was desensitized to GTP. The inactivation process was cooperative, maximum inactivation occurring as soon as half of the Lys 126 were modified.

Reversible modification of pig heart mitochondrial malate dehydrogenase by pyridoxal 5'-phosphate.

1. Pig heart mitochondrial malate dehydrogenase incubated with pyridoxal 5'-phosphate at pH 8.0 and 25 degrees C gradually loses activity. Such inactivation can be largely reversed by dialysis or by addition of L-lysine or L-cysteine, and can be made permanent by NaBH4 reduction. 2. Modification of malate dehydrogenase with pyridoxal 5'-phosphate at 35 degrees C involves two phases, an initial inactivation which is reversible and a slower irreversible second stage. 3. The initial reaction between pyridoxal 5'-phosphate and malate dehydrogenase appears to involve reversible formation of a Schiff base with the epsilon-amino group of a lysine residue. 4. Inactivation of malate dehydrogenase by pyridoxal 5'-phosphate at 10 degrees C involves only the reversible reaction. 5. At 10 degrees C repeated cycles of treatment with pyridoxal 5'-phosphate and NaBH4 reduction lead to a stepwise decline in residual activity. 6. Apparent Km values for malate and NAD+ are unaltered in the partially inactivated enzyme. 7. NAD+ and NADH give only partial protection against pyridoxal 5'-phosphate inactivation. Substrates give no effect.