Significance: Phgdh is a crucial enzyme in the astrocytic synthesis of l -serine from glucose. Our study uncovers the role of glial-neuronal cross-talk in regulating NMDAR synaptic activation through the astrocytic export of Phgdh-derived l -serine to generate the coagonists d -serine and glycine. We clarify the relative roles of d -serine and glycine by showing that like d -serine, Phgdh-derived glycine mediates NMDAR synaptic activation in the mature hippocampus. We show that glycine is a significant regulator of d -serine synthesis and release, revealing a cross-talk between glycine and d -serine metabolism that fine tunes NMDAR activation. Our data reveal a multifaceted mechanism regulating NMDAR activity, which ultimately depends on a Phgdh-dependent serine shuttle.

Human serine racemase is a pyridoxal 5′-phosphate (PLP)-dependent dimeric enzyme that catalyzes the reversible racemization of L-serine and D-serine and their dehydration to pyruvate and ammonia. As D-serine is the co-agonist of the N-methyl-D-aspartate receptors for glutamate, the most abundant excitatory neurotransmitter in the brain, the structure, dynamics, function, regulation and cellular localization of serine racemase have been investigated in detail. Serine racemase belongs to the fold-type II of the PLP-dependent enzyme family and structural models from several orthologs are available. The comparison of structures of serine racemase co-crystallized with or without ligands indicates the presence of at least one open and one closed conformation, suggesting that conformational flexibility plays a relevant role in enzyme regulation. ATP, Mg²⁺, Ca²⁺, anions, NADH and protein interactors, as well as the post-translational modifications nitrosylation and phosphorylation, finely tune the racemase and dehydratase activities and their relative reaction rates. Further information on serine racemase structure and dynamics resulted from the search for inhibitors with potential therapeutic applications. The cumulative knowledge on human serine racemase allowed obtaining insights into its conformational landscape and into the mechanisms of cross-talk between the effector binding sites and the active site.

Besides their long-known critical role in embryonic growth and in cancer development and progression, erythropoietin-producing hepatocellular carcinoma type B (EphB) receptor tyrosine kinases and their ephrin-B ligands are involved in the modulation of immune responses and in remodeling and maintaining the integrity of the intestinal epithelial layer. These processes are critically involved in the pathogenesis of inflammatory-based disorders of the gut, like inflammatory bowel diseases (IBDs). Accordingly, our aim was to investigate the role of the EphB/ephrin-B system in intestinal inflammation by assessing the local and systemic effects produced by its pharmacological manipulation in 2,4,6-trinitrobenzenesulfonic acid (TNBS)- (Th1-dependent model) and dextran sulphate sodium (DSS)- (innate response model) induced colitis in mice. To this purpose, we administered chimeric Fc-conjugated proteins, allegedly able to uni-directionally activate either forward (ephrin-B1-Fc) or reverse (EphB1-Fc) signaling, and the soluble monomeric EphB4 extracellular domain protein, that, simultaneously interfering with both signaling pathways, acts as EphB/ephrin-B antagonist.The blockade of the EphB/ephrin-B forward signaling by EphB4 and EphB1-Fc was ineffective against DSS-induced colitis while it evoked remarkable beneficial effects against TNBS colitis: it counteracted all the evaluated inflammatory responses and the changes elicited on splenic T lymphocytes subpopulations, without preventing the appearance of a splice variant of ephrin-B2 gene elicited by the haptenating agent in the colon. Interestingly, EphB4, preferentially displacing EphB4/ephrin-B2 interaction over EphB1/ephrin-B1 binding, was able to promote Tumor Necrosis Factor alpha (TNFα) release by splenic mononuclear cells in vitro. On the whole, the collected results point to a potential role of the EphB/ephrin-B system as a pharmacological target in intestinal inflammatory disorders and suggest that the therapeutic efficacy of its blockade seemingly works through the modulation of immune responses, independent of the changes at the transcriptional and translational level of EphB4 and ephrin-B2 genes.

De novo l‐serine biosynthesis in the mammalian astrocytes proceeds via a linear, three‐step pathway (the phosphorylated pathway) catalysed by 3‐phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT) and phosphoserine phosphatase (PSP). The first reaction, catalysed by PHGDH and using the glycolytic intermediate 3‐phosphoglycerate, is strongly shifted towards the reagents, and coupling to the following step by PSAT is required to push the equilibrium towards l‐serine formation; the last step, catalysed by PSP, is virtually irreversible and inhibited by the final product l‐serine. Very little is known about the regulation of the human phosphorylated pathway and the ability of the three enzymes to organise in a complex with potential regulatory functions. Here, the complex formation was investigated in differentiated human astrocytes, by proximity ligation assay, and in vitro on the human recombinant enzymes. The results indicate that the three enzymes co‐localise in cytoplasmic clusters that more stably engage PSAT and PSP. Although in vitro analyses based on native PAGE, size exclusion chromatography and cross‐linking experiments do not show the formation of a stable complex, kinetic studies of the reconstituted pathway using physiological enzyme and substrate concentrations support cluster formation and indicate that PHGDH catalyses the rate‐limiting step while PSP reaction is the driving force for the whole pathway. The enzyme agglomerate assembly of the phosphorylated pathway (the putative ‘serinosome’) delivers a relevant level of sophistication to the control of l‐serine biosynthesis in human cells, a process strictly related to the modulation of the brain levels of d‐serine and glycine, the main co‐agonists of N‐methyl‐d‐aspartate receptors and various pathological states.

Organisms from all kingdoms of life synthesize L‐serine (L‐Ser) from 3‐phosphoglycerate through the phosphorylated pathway, a three‐step diversion of glycolysis. Phosphoserine aminotransferase (PSAT) catalyzes the intermediate step, the pyridoxal 5′‐phosphate‐dependent transamination of 3‐phosphohydroxypyruvate and L‐glutamate to O‐phosphoserine (OPS) and α‐ketoglutarate. PSAT is particularly relevant in the central nervous system of mammals because L‐Ser is the metabolic precursor of D‐serine, cysteine, phospholipids, and nucleotides. Several mutations in the human psat gene have been linked to serine deficiency disorders, characterized by severe neurological symptoms. Furthermore, PSAT is overexpressed in many tumors and this overexpression has been associated with poor clinical outcomes. Here, we report the detailed functional and structural characterization of the recombinant human PSAT. The reaction catalyzed by PSAT is reversible, with an equilibrium constant of about 10, and the enzyme is very efficient, with a k_cat/K_m of 5.9 × 10⁶ M⁻¹ s⁻¹, thus contributing in driving the pathway towards the products despite the extremely unfavorable first step catalyzed by 3‐phosphoglycerate dehydrogenase. The 3D X‐ray crystal structure of PSAT was solved in the substrate‐free as well as in the OPS‐bound forms. Both structures contain eight protein molecules in the asymmetric unit, arranged in four dimers, with a bound cofactor in each subunit. In the substrate‐free form, the active site of PSAT contains a sulfate ion that, in the substrate‐bound form, is replaced by the phosphate group of OPS. Interestingly, fast crystal soaking used to produce the substrate‐bound form allowed the trapping of different intermediates along the catalytic cycle.