Newsletter 02/2022 edited by Loredano Pollegioni

Do you have news concerning the D-amino acids field to announce? Is there a relevant published paper to mention? Write us and take the advantage of this bimonthly Newsletter.

The Editor’s pick selection of the most intriguing papers is highlighted in yellow.

D-AAs AND PHYSIOLOGY:

• Chiral resolution of plasma amino acids reveals enantiomer-selective associations with organ functions

Suzuki M, Shimizu-Hirota R, Mita M, Hamase K, Sasabe J
Amino Acids. (2022) 54(3):421-432. doi: 10.1007/s00726-022-03140-w. 

Here, Sasabe and his group analyze the enantiomers levels of all proteinogenic amino acids (AAs) in plasma and urine of healthy humans, using a two-dimensional HPLC system and investigate their correlation with other biochemical parameters. They detect four D-AAs (namely, D-asparagine, D-alanine, D-serine, and D-proline) at low concentrations in plasma. These D-AAs show a significanlty higher fractional excretion than the L-counterparts, being their content much higher in urine. Authors also report distinct patterns of correlations between D- or L-AAs and parameters of clinical biochemistry, likely due to their distinct origins, dynamics, and metabolism. Chiral analyses of body fluids AAs therefore, reveals totally diferent associations of the enantiomers with organ functions.

• Astrocytes Render Memory Flexible by Releasing D-Serine and Regulating NMDA Receptor Tone in the Hippocampus

Koh W, Park M, Chun YE, Lee J, Shim HS, Park MG, Kim S, Sa M, Joo J, Kang H, Oh SJ, Woo J, Chun H, Lee SE, Hong J, Feng J, Li Y, Ryu H, Cho J, Lee CJ
Biol Psychiatry (2022) 91(8):740-752. doi: 10.1016/j.biopsych.2021.10.012.

This interesting study provides a comprehensive understanding of how norepinephrine, NMDAR tone and memory formation are associated. It follows previous line of evidence suggesting that the release of glutamate from astrocytes via the Ca2+-activated, glutamate-permeable anion channel BEST1, might contribute to hippocampal NMDAR tone and LTD, and identifies D-serine as a novel permeant molecule passing through this channel. Authors show that astrocytes are crucially involved in reversal learning and flexible memory formation by co-releasing D-serine and glutamate through BEST1, on activation of the norepinephrine–a1- adrenoreceptor pathway, and this yields to an enhanced NMDAR tone and heterosynaptic LTD during initial memory acquisition. The reported findings significantly contribute to the understanding of astrocytic roles in memory formation alongside providing potential therapeutic targets for impaired cognitive flexibility in psychiatric disorders.

• Astroglial gliotransmitters released via Cx43 hemichannels regulate NMDAR-dependent transmission and short-term fear memory in the basolateral amygdala

Linsambarth S, Carvajal FJ, Moraga-Amaro R, Mendez L, Tamburini G, Jimenez I, Verdugo DA, Gómez GI, Jury N, Martínez P, van Zundert B, Varela-Nallar L, Retamal MA, Martin C, Altenberg GA, Fiori MC, Cerpa W, Orellana JA, Stehberg J
FASEB J. (2022) 36(2):e22134. doi: 10.1096/fj.202100798RR.

In this intriguing work, authors propose that gap junction Cx43 hemichannels mediate the release of glutamate and D-serine from astrocytes and this led to the the activation of post-synaptic NMDAR at the basolateral amygdala (BLA) during training for fear conditioning, allowing the formation of short-term and subsequent long-term memory. They find that the selective inhibition of Cx43 hemichannels in BLA slices, by the synthetic cell-permeable mimetic peptide TAT-Cx43L2, induce a reduction in post-synaptic NMDAR-mediated currents, an effect that is prevented by the co-administration with a mixture of glutamate and D-serine (but not each separately). Moreover, they provide the first direct evidence of the permeation of D-serine through purified Cx43 hemichannels reconstituted in liposomes.

• D-aspartate and N-methyl-D-aspartate promote proliferative activity in mouse spermatocyte GC-2 cells

Falvo S, Santillo A, Chieffi Baccari G, Cioffi F, Di Fiore MM
Reprod Biol. (2022) 22(1):100601. doi: 10.1016/j.repbio.2021.100601.

In this paper Di Fiore group explore the mechanisms underlying the role of D-aspartate (D-Asp) and N-methyl-D-aspartate (NMDA) on spermatogenesis. They investigate the effect of D-Asp and NMDA treatments on cell proliferation, as well as on mitochondrial functionality (essential for the meiotic activity) in a mouse spermatocyte-derived cell line (GC-2). In GC-2 spermatocytes, both D-Asp and NMDA stimulate the AMPAR/ERK/Akt pathway activity (playing a relevant role in spermatogonial proliferation) and induce the expressions of PCNA, p-H3, and SYCP3 proteins. Authors also observed that mitochondrial biogenesis as well as mitochondrial fusion are increased in treated cells, while mitochondria fission is inhibited, suggesting the involvement of D-Asp and NMDA in the metabolic shift occurring during meiosis. Altogether, the reported findings indicate an active role of these amino acids in germ cell maturation.

• Free D-Amino Acids in Salivary Gland in Rat

Yoshikawa M.,Kan T., Shirose K., Watanabe M., Matsuda M., Ito K., Kawaguchi M.
Biology 2022, 11(3), 390; https://doi.org/10.3390/biology11030390.

Several free D-amino acids have been observed in saliva, but their origin and the enzymes involved in their metabolism and catabolism remain to be clarified. Here, large amounts of D-Asp and small amounts of D-Ser and D-Ala were detected in all three major salivary glands in rat. Protein levels of D-amino acid oxidase and D-aspartate oxidase, the enzymes responsible for their catabolism, were determined in all three types of salivary gland; the level of serine racemase, the D-Ser synthetic enzyme, in parotid glands amounted to approximately 40% of that observed in the cerebral cortex; the NMDA receptor subunit NR1 and NR2D were also detected in all three major salivary glands. These results suggest that D-AAs play a physiological role in a range of endocrine and exocrine function in salivary glands.

• D-Cysteine supplementation partially protects against ferroptosis induced by xCT dysfunction via increasing the availability of glutathione

HommaT, Osaki T,  Kobayashi S, Sato H, Fujii J
Journal of Clinical Biochemistry and Nutrition, Article ID 21-143, https://doi.org/10.3164/jcbn.21-143.

In this study, the anti-ferroptotic properties of D-cysteine (D-Cys) are investigated. This form of cell death is induced in Hepa 1-6 cells by the inhibition of the Cys transporter xCT by erastin, and is substantially suppressed by D-Cys, which is shown to promote the cellular uptake of L-Cys and the subsequent synthesis of GSH.

D-AAs AND PATHOLOGIES: 

• Machine Learning algorithm unveils glutamatergic alterations in the post-mortem schizophrenia brain

De Rosa, A., Fontana, A., Nuzzo, T., Garofalo M., Di Maio A., Punzo D., Copetti M., Bertolino A., Errico F., Rampino A., de Bartolomeis A., Usiello A. 
Schizophr 8, 8 (2022). https://doi.org/10.1038/s41537-022-00231-1

The authors studied in the post-mortem dorsolateral prefrontal cortex and hippocampus of schizophrenia patients and non-psychiatric controls, the levels of L-Glu, D-Ser, Gly, L-Asp and D-Asp, as well as the mRNA and protein levels of pre- and post-synaptic key molecules involved in the glutamatergic synapse functioning, such as glutamate receptors, their interacting scaffolding proteins, plasma membrane and vesicular glutamate transporters, enzymes involved either in glutamate-dependent GABA neurotransmitter synthesis or in post-synaptic NMDA receptor-mediated signaling and the pre-synaptic marker Synapsin-1. None of the investigated molecules was differently represented in the post-mortem DLPFC and hippocampus of schizophrenic compared with controls. Nonetheless, multivariable hypothesis-driven analyses revealed that the presence of schizophrenia was significantly affected by variations in neuroactive amino acid levels and glutamate-related synaptic elements. A Machine Learning hypothesis-free unveiled other discriminative clusters of molecules, one in the DLPFC and another in the hippocampus. The authors reported molecular signatures encompassing elements of the glutamate synapse able to discriminate healthy controls from patients with schizophrenia.

• D-Amino Acids as a Biomarker in Schizophrenia

Taniguchi K, Sawamura H, Ikeda Y, Tsuji A, Kitagishi Y, Matsuda S. 
Diseases (2022) 10(1):9. doi: 10.3390/diseases10010009.

In this review authors go over the roles of D-serine and D-aspartate in brain health and/or neuropsychiatric disorders with a focus on schizophrenia. The bidirectional communication between the nervous systems and intestinal functions through the gut–brain axis via the production of D-amino acids and the role of the microbiome in affecting brain development and functioning is also discussed.

• A novel D-amino acid peptide with therapeutic potential (ISAD1) inhibits aggregation of neurotoxic disease-relevant mutant Tau and prevents Tau toxicity in vitro

Aillaud I, Kaniyappan S, Chandupatla RR, Ramirez LM, Alkhashrom S, Eichler J, Horn AHC, Zweckstetter M, Mandelkow E, Sticht H, Funke SA
Alzheimers Res Ther. (2022) 14(1):15. doi: 10.1186/s13195-022-00959-z

Aillaud and collaborators reporte about the development of novel therapeutic D-amino acid peptides to be used as inhibitors of Tau pathological fibrillization. They select a novel Tau binding L-peptide by phage display and synthesize its D-amino acid version ISAD1 (as well as its retro inversed form, ISAD1rev). ISAD1 is efficiently up-taken by cultured cells and prevents cytotoxicity either when the pro-aggregant TauRDΔK is ectopically expressed or Tau fibrils are added to the culture medium. The D-peptide binds to Tau in the aggregation-prone PHF6 region and inhibits fibrillization, not only of the full-length Tau (TauFL), but also of disease-relevant Tau mutants. Moreover, it induces the formation of large high molecular weight TauFL and TauRDΔK oligomers that lack proper thiofavin-positive β-sheet conformation, thus promoting of-pathway assembly of Tau and preventing its toxicity. The latter findings make it a promising therapeutic to prevent Tau pathology in AD and other Tau-associated diseases.

• Inhibition of glial D-serine release rescues synaptic damage after brain injury

Tapanes SA, Arizanovska D, Díaz MM, Folorunso OO, Harvey T, Brown SE, Radzishevsky I, Close LN, Jagid JR, Graciolli Cordeiro J, Wolosker H, Balu DT, Liebl DJ. 
Glia. (2022) doi: 10.1002/glia.24161

It is known that D-serine (produced and release from neurons in physiological conditions) is mainly synthesized by glial cells under inflammatory conditions. Here authors propose a novel and key role for D-serine in the pathological dysfunctions after traumatic brain injury (TBI). They show that injury-induced microgliosis and astrogliosis mediate synaptic damage and memory impairment after TBI through enhanced D-serine synthesis and release. Most interestingly, both D-serine biosynthetic enzyme (serine racemase) and transporter Slc1a4 (ASCT1) are upregulated in the cortex of human TBI patients, suggesting that an increased expression of serine racemase may be strongly associated with gliosis. Since the ablation of D-serine synthesis or transporter mediated release, in either reactive glial cell type, is sufficient to prevent TBI-induced synaptic damage and memory deficits, the authors conclude that controlling D-serine metabolic pathway in these cells represents a potential therapeutic strategy to normalize synaptic pathology and memory deficits following TBI.

ENZYMES ACTIVE ON D-AAs:

• Identification and characterization of a serine racemase in the silkworm Bombyx mori 

Tanaka Y, Yoshimura T, Hakamata M, Saito C, Sumitani M, Sezutsu H, Hemmi H, Ito T J Biochem. (2022) mvac026. doi: 10.1093/jb/mvac026

In this paper Tanaka and collaborators reported about the identification of a new type of pyridoxal 5′-phosphate (PLP)-dependent serine racemase (SR) in B. mori, catalyzing the racemization and dehydration of both serine isomers. The authors investigate the silkworm SR (BmSR) structural and functional properties showing that it possesses distinctive features compared to the mammalian one. They enzyme expression and activity are primarily detected in the fat body and this explain the marked increase in D-serine (up to 50% of total serine) during the insect development, from the laval to the pupal stage.

• Novel transaminases from thermophiles: from discovery to application

Cárdenas-Fernández M, Sinclair O, Ward JM. 
Microb Biotechnol. (2022) 15(1):305-317. doi: 10.1111/1751-7915.13940

Transaminases (TAs) are widely distributed PLP-dependent enzymes largely utilized in the synthesis of fine chemicals and pharmaceuticals. This work explore the possible application of thermophilic TAs, which have been poorly described to date. To this aim, 94 putative TAs are successfully cloned from nine thermophilic microorganisms upon a genome mining approach and recombinatly expressed in E. coli using a chemical chaperone media containing D-sorbitol. Among them pQR2590, a steroselective class IV TA from G. stearothermophilus accepts D-serine only as a substrate (optimum temperature of 60 °C) and a very high specific activity.

D-AAs &  BIOTECHNOLOGICAL APPLICATIONS:

• Carbon dots confined in N-doped carbon as peroxidase-like nanozyme for detection of gastric cancer relevant D-amino acids 

Zhe Li, Wendong Liu, Pengjuan Ni, Chenghui Zhang, Bo Wang, Guangbin Duan, Chuanxia Chen, Yuanyuan Jiang, Yizhong Lu
Chemical Engineering Journal (2022) Volume 428, 131396 https://doi.org/10.1016/j.cej.2021.131396

In this work the authors set up a nanozyme-based colorimetric assay to evaluate the concentrations of D-proline and D-alanine in human saliva and quickly distinguish healthy individuals from patients with early gastric cancer, in which the levels of these two amino acids are significantly higher. The novelty of the proposed method consists in coupling D-amino acid oxidase catalysed reaction to carbon dots confined in N-doped carbon (CDs@NC) encapsulated with Zeolitic imidazolate framework (ZIF-8) filled with glucose (a highly active peroxidase-like nanozyme), for the dectection of H2O2 production in the presence of the dye 3,3′,5,5′-tetramethylbenzidine (TMB). Authors claim that this assay will make the diagnosis of early gastric cancer analysis simpler and quicker.

• Enantioselective recognition of L/D-amino acids in the chiral nanochannels of a metal-organic framework

Xiaohui Niu, Simeng Yan, Jinliang Chen, Hongxia Li, Kunjie Wang
Electrochimica Acta (2022) Volume 405,139809
https://doi.org/10.1016/j.electacta.2021.139809

Here the construction of an electrochemical sensing interface based on the chiral metal-organic frameworks (MOF) synthesized via in-situ substitution of 2-methylimidazole on ZIF-8 by D-histidine and the introduction of Ketjen Black (KB) is reported. Authors show that the KB/D-His-ZIF-8 composite material not only has excellent electrochemical property, but also has good enantio-discrimination ability for amino acids in voltammetry experiments.

• Simultaneous Preparation of (S)-2-Aminobutane and D-Alanine or D-Homoalanine via Biocatalytic Transamination at High Substrate Concentration

Jianjiong Li, Shanshan Yu, Yingang Wang, Peiyuan Yao, Qiaqing Wu, Dunming Zhu
Org. Process Res. Dev. 2022, https://doi.org/10.1021/acs.oprd.1c00408

(S)-2-Aminobutane, D-Ala, and D-homoalanine are important intermediates for the production of various active pharmaceutical ingredients and food additives. Here, an ω-transaminase (ω-TA) from Sinirhodobacter hungdaonensis (ShdTA) was identified. It catalyzed the kinetic resolution of 800 mM racemic 2-aminobutane using pyruvate as the amino acceptor, leading to the simultaneous isolation of enantiopure (S)-2-aminobutane and D-Ala in 46% and 90% yield, respectively. In addition, (S)-2-aminobutane (98% ee) and D-homoalanine (99% ee) were isolated in 45% and 93% yield, respectively, in the kinetic resolution of 400 mM racemic 2-aminobutane coupled with deamination of L-threonine by threonine deaminase.

• A dual action of D-amino acids on anti-biofilm activity and moisture-protection of inhalable ciprofloxacin powders

Chang RYK, Li M, Chow MYT, Ke WR, Tai W, Chan HK
Eur J Pharm Biopharm. (2022) 17:S0939-6411(22)00051-0. doi:10.1016/j.ejpb.2022.03.003

Pseudomonas aeruginosa is a highly problematic Gram-negative bacterium and a notorious biofilm producer that causes severe and recalcitrant infections. Recently, studying molecules with biofilm dispersant activities, among which D-amino acids, has gained an increasing interest. In this paper, Chang and colleagues assessed the combined use of the antibiotic ciprofloxacin and hydrophobic D-amino acids (namely D-methionine and D-triptophan) to produce inhalable powders, with enhanced antibiofilm activity.

• Selective chiroptical sensing of D/L-cysteine

Kariapper FS, Thanzeel FY, Zandi LS, Wolf C
Org Biomol Chem. (2022) Mar 28. doi: 10.1039/d2ob00198e

Here the setup of a specific and selective assay for free cysteine enantiomers, based on a chromophoric bifunctional UV/CD probe (o-nitrophenacyl bromide), that allows chiroptical sensing in solution, is reported. Authors are confident that this sensing method can easily be adapted by academic and industrial laboratories and is amenable to automated high-throughput systems.

• Polymer Conjugates of Antimicrobial Peptides (AMPs) with D-Amino Acids (D-aa): State of the Art and Future Opportunities

Bellotto, O.; Semeraro, S.; Bandiera, A.; Tramer, F.; Pavan, N.; Marchesan, S.
Pharmaceutics 2022, 14, 446. https://doi.org/10.3390/ pharmaceutics14020446 

This review reports on the last 5 years’ progress in the area of antimicrobial peptides (AMPs) that contain D-amino acids, focusing on their role and the advantages that may arise from their introduction into AMPs. Covalent conjugation with polymers represents an interesting strategy to modulate the pharmacokinetic profile of AMPs, to enhance their biocompatibility profile and to avoid biofilm formation on their surface.

The D-amino acids International Research Center “DAAIR“ has been established in Gerenzano (Varese, Italy) in 2019 with the aim to support and perform scientific research projects and activities on the field of D-amino acids. The Center, located inside the Fondazione Istituto Insubrico Ricerca per la Vita, is aimed to represent a pole of excellence at international level for dissemination and research involving the D-amino acids (Director Silvia Sacchi).

The guiding principle is support the research projects aimed to investigate the involvement of D-amino acids in main physiological processes, from bacteria to humans. The ultimate goal is to actively participate to the elucidation of the mechanisms by which the D-amino acids perform specific functions, and to identify their presence and concentration in different organisms and compartments, also with regards to well-established functional states, with particular emphasis to pathological states. Understand the involvement of D-amino acids in important diseases as a way to set up novel therapeutic strategies.

Contacts: info@d-aminoacids.com;
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www.d-aminoacids.com