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D‐3‐phosphoglycerate dehydrogenase (PHGDH) catalyzes the NAD+‐dependent conversion of D‐3‐phospho‐glycerate to 3‐phosphohydroxypyruvate, the first step in the phosphorylated pathway for L‐serine (L‐Ser) biosynthesis. L‐Ser plays different relevant metabolic roles in eukaryotic cells: alterations in L‐Ser metabolism have been linked to serious neurological disorders. The human PHGDH (hPHGDH), showing a homotetrameric state in solution, is made of four domains, among which there are two regulatory domains at the C‐terminus: the aspartate kinase‐chorismate mutase‐tyrA prephenate dehydrogenase (ACT) and allosteric substrate‐binding (ASB) domains. The structure of hPHGDH was solved only for a truncated, dimeric form harboring the N‐terminal end containing the substrate and the cofactor binding domains. A model ensemble of the tetrameric hPHGDH was generated using AlphaFold coupled with molecular dynamics refinement. By analyzing the inter‐subunit interactions at the tetrameric interface, the residues F418, L478, P479, R454, and Y495 were selected and their role was studied by the alanine‐scanning mutagenesis approach. The F418A variant modifies the putative ASB, slightly alters the activity, the fraction of protein in the tetrameric state, and the protein stability; it seems relevant in dimers' recognition to yield the tetrameric oligomer. On the contrary, the R454A, L478A, P479A, and Y495A variants (ACT domain) determine a loss of the tetrameric assembly, resulting in low stability and misfolding, triggering the aggregation and hampering the activity. The predicted tetrameric interface seems mediated by residues at the ACT domain, and the tetramer formation seems crucial for proper folding of hPHGDH, which, in turn, is essential for both stability and functionality. [ABSTRACT FROM AUTHOR]

In the brain, the non‐essential amino acid L‐serine is produced through the phosphorylated pathway (PP) starting from the glycolytic intermediate 3‐phosphoglycerate: among the different roles played by this amino acid, it can be converted into D‐serine and glycine, the two main co‐agonists of NMDA receptors. In humans, the enzymes of the PP, namely phosphoglycerate dehydrogenase (hPHGDH, which catalyzes the first and rate‐limiting step of this pathway), 3‐phosphoserine aminotransferase, and 3‐phosphoserine phosphatase are likely organized in the cytosol as a metabolic assembly (a "serinosome"). The hPHGDH deficiency is a pathological condition biochemically characterized by reduced levels of L‐serine in plasma and cerebrospinal fluid and clinically identified by severe neurological impairment. Here, three single‐point variants responsible for hPHGDH deficiency and Neu‐Laxova syndrome have been studied. Their biochemical characterization shows that V261M, V425M, and V490M substitutions alter either the kinetic (both maximal activity and Km for 3‐phosphoglycerate in the physiological direction) and the structural properties (secondary, tertiary, and quaternary structure, favoring aggregation) of hPHGDH. All the three variants have been successfully ectopically expressed in U251 cells, thus the pathological effect is not due to hindered expression level. At the cellular level, mistargeting and aggregation phenomena have been observed in cells transiently expressing the pathological protein variants, as well as a reduced L‐serine cellular level. Previous studies demonstrated that the pharmacological supplementation of L‐serine in hPHGDH deficiencies could ameliorate some of the related symptoms: our results now suggest the use of additional and alternative therapeutic approaches. [ABSTRACT FROM AUTHOR]

The human flavoenzyme D‐aspartate oxidase (hDASPO) controls the level of D‐aspartate in the brain, a molecule acting as an agonist of NMDA receptors and modulator of AMPA and mGlu5 receptors. hDASPO‐induced D‐aspartate degradation prevents age‐dependent deterioration of brain functions and is related to psychiatric disorders such as schizophrenia and autism. Notwithstanding this crucial role, less is known about hDASPO regulation. Here, we report that hDASPO is nitrosylated in vitro, while no evidence of sulfhydration and phosphorylation is apparent: nitrosylation affects the activity of the human flavoenzyme to a limited extent. Furthermore, hDASPO interacts with the primate‐specific protein pLG72 (a well‐known negative chaperone of D‐amino acid oxidase, the enzyme deputed to D‐serine degradation in the human brain), yielding a ~114 kDa complex, with a micromolar dissociation constant, promoting the flavoenzyme inactivation. At the cellular level, pLG72 and hDASPO generate a cytosolic complex: the expression of pLG72 negatively affects the hDASPO level by reducing its half‐life. We propose that pLG72 binding may represent a protective mechanism aimed at avoiding cytotoxicity due to H2O2 produced by the hDASPO enzymatic degradation of D‐aspartate, especially before the final targeting to peroxisomes. [ABSTRACT FROM AUTHOR]

Astrocytes are essential players in development and functions, being particularly relevant as regulators of brain energy metabolism, ionic homeostasis and synaptic transmission. They are also the major source of l‐serine in the brain, which is synthesized from the glycolytic intermediate 3‐phosphoglycerate through the phosphorylated pathway. l‐Serine is the precursor of the two main co‐agonists of the N‐methyl‐d‐aspartate receptor, glycine and d‐serine. Strikingly, dysfunctions in both l‐ and d‐serine metabolism are associated with neurological and psychiatric disorders. Here, we exploited a differentiation protocol, based on the generation of human mature astrocytes from neural stem cells, and investigated the modification of the proteomic and metabolomic profile during the differentiation process. We show that differentiated astrocytes are more similar to mature rather than to reactive ones, and that axogenesis and pyrimidine metabolism increase up to 30 days along with the folate cycle and sphingolipid metabolism. Consistent with the proliferation and cellular maturation processes that are taking place, also the intracellular levels of l‐serine, glycine, threonine, l‐ and d‐aspartate (which level is unexpectedly higher than that of d‐serine) show the same biosynthetic time course. A significant utilization of l‐serine from the medium is apparent while glycine is first consumed and then released with a peak at 30 days, parallel to its intracellular level. These results underline how metabolism changes during astrocyte differentiation, highlight that d‐serine synthesis is restricted in differentiated astrocytes and provide a valuable model for developing potential novel therapeutic approaches to address brain diseases, especially the ones related to serine metabolism alterations. [ABSTRACT FROM AUTHOR]

De novol‐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. [ABSTRACT FROM AUTHOR]

Human d‐aspartate oxidase (hDASPO) is a FAD‐dependent enzyme responsible for the degradation of d‐aspartate (d‐Asp). In the mammalian central nervous system, d‐Asp behaves as a classical neurotransmitter, it is thought to be involved in neural development, brain morphology and behavior, and appears to be involved in several pathological states, such as schizophrenia and Alzheimer's disease. Apparently, the human DDO gene produces alternative transcripts encoding for three putative hDASPO isoforms, constituted by 341 (the 'canonical' form), 369, and 282 amino acids. Despite the increasing interest in hDASPO and its physiological role, little is known about these different isoforms. Here, the additional N‐terminal peptide present in the hDASPO_369 isoform only has been identified in hippocampus of Alzheimer's disease female patients, while peptides corresponding to the remaining part of the protein were present in samples from male and female healthy controls and Alzheimer's disease patients. The hDASPO_369 isoform was largely expressed in E. coli as insoluble protein, hampering with its biochemical characterization. Furthermore, we generated U87 human glioblastoma cell clones stably expressing hDASPO_341 and, for the first time, hDASPO_369 isoforms; the latter protein showed a lower expression compared with the canonical isoform. Both protein isoforms are active (showing similar kinetic properties), localize to the peroxisomes, are very stable (a half‐life of approximately 100 h has been estimated), and are primarily degraded through the ubiquitin–proteasome system. These studies shed light on the properties of hDASPO isoforms with the final aim to clarify the mechanisms controlling brain levels of the neuromodulator d‐Asp. [ABSTRACT FROM AUTHOR]