Astrocyte-derived lactate aggravates brain injury of ischemic stroke in mice by promoting the formation of protein lactylation.

Aim: Although lactate supplementation at the reperfusion stage of ischemic stroke has been shown to offer neuroprotection, whether the role of accumulated lactate at the ischemia phase is neuroprotection or not remains largely unknown. Thus, in this study, we aimed to investigate the roles and mechanisms of accumulated brain lactate at the ischemia stage in regulating brain injury of ischemic stroke. Methods and Results: Pharmacological inhibition of lactate production by either inhibiting LDHA or glycolysis markedly attenuated the mouse brain injury of ischemic stroke. In contrast, additional lactate supplement further aggravates brain injury, which may be closely related to the induction of neuronal death and A1 astrocytes. The contributing roles of increased lactate at the ischemic stage may be related to the promotive formation of protein lysine lactylation (Kla), while the post-treatment of lactate at the reperfusion stage did not influence the brain protein Kla levels with neuroprotection. Increased protein Kla levels were found mainly in neurons by the HPLC-MS/MS analysis and immunofluorescent staining. Then, pharmacological inhibition of lactate production or blocking the lactate shuttle to neurons showed markedly decreased protein Kla levels in the ischemic brains. Additionally, Ldha specific knockout in astrocytes (Aldh1l1 CreERT2; Ldha fl/fl mice, cKO) mice with MCAO were constructed and the results showed that the protein Kla level was decreased accompanied by a decrease in the volume of cerebral infarction in cKO mice compared to the control groups. Furthermore, blocking the protein Kla formation by inhibiting the writer p300 with its antagonist A-485 significantly alleviates neuronal death and glial activation of cerebral ischemia with a reduction in the protein Kla level, resulting in extending reperfusion window and improving functional recovery for ischemic stroke. Conclusion: Collectively, increased brain lactate derived from astrocytes aggravates ischemic brain injury by promoting the protein Kla formation, suggesting that inhibiting lactate production or the formation of protein Kla at the ischemia stage presents new therapeutic targets for the treatment of ischemic stroke.

Efficient synthesis of γ-glutamyl compounds by co-expression of γ-glutamylmethylamide synthetase and polyphosphate kinase in engineered Escherichia coli.

γ-Glutamyl compounds have unveiled their importance as active substances or precursors of pharmaceuticals. In this research, an approach for enzymatic synthesis of γ-glutamyl compounds was developed using γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays and polyphosphate kinase (PPK) from Corynebacterium glutamicum. GMAS and PPK were co-recombined in pETDuet-1 plasmid and co-expressed in E. coli BL21 (DE3), and the enzymatic properties of GMAS and PPK were investigated, respectively. Under the catalysis of the co-expression system, L-theanine was synthesized with 89.8% conversion when the substrate molar ratio of sodium glutamate and ethylamine (1:1.4) and only 2 mM ATP were used. A total of 14 γ-glutamyl compounds were synthesized by this one-pot method and purified by cation exchange resin and isoelectric point crystallization with a yield range from 22.3 to 72.7%. This study provided an efficient approach for the synthesis of γ-glutamyl compounds by GMAS and PPK co-expression system.

A novel non-blue laccase from Bacillus amyloliquefaciens: secretory expression and characterization.

Laccases are copper-containing enzymes which possess a promising potential in many industrial and environmental applications. Here we describe the cloning, extracellular expression and characterization of a novel non-blue laccase from Bacillus amyloliquefaciens in Pichia pastoris. The recombinant enzyme was secreted into the culture supernatant with high activity. It lacks the absorption band at 610 nm typical for blue laccases. However, electron paramagnetic resonance (EPR) spectrum proved the existence of type 1 copper center that was not detectable in the UV-visible spectrum. Metal content analysis revealed that the enzyme contains two copper ions, one iron ion and one zinc ion per protein molecular, suggesting that it is a novel non-blue laccase. The pH and temperature optima of the recombinant laccase were 6.6 and 60°C, respectively, and it was stable at pH 9.0 for 10 days. The enzyme activity was slightly activated by NaCl with concentration up to 200 mM. The purified laccase showed high efficiency in decolorizing reactive black 5 and indigo carmine, achieving more than 93% decolorization after 1h. The extreme robustness of the recombinant B. amyloliquefaciens laccase offers several advantages over most fungal laccases in various industrial applications.