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Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe NE-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)–linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine–4-epimerase (GALE) like conventional GalNAc–based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotidesugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan–specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, “bump-and-hole” (BH)–GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.

Schizophrenia is characterized by reduced hippocampal volume, decreased dendritic spine density, altered neuroplasticity signaling pathways, and cognitive deficits associated with impaired hippocampal function. We sought to determine whether this diverse pathology could be linked to NMDA receptor (NMDAR) hypofunction, and thus used the serine racemase-null mutant mouse (SR ⁻/⁻), which has less than 10% of normal brain d -serine, an NMDAR coagonist. We found that d -serine was necessary for the maintenance of long-term potentiation in the adult hippocampal dentate gyrus and for full NMDAR activity on granule cells. SR ⁻/⁻ mice had reduced dendritic spines and hippocampal volume. These morphological changes were paralleled by diminished BDNF/Akt/mammalian target of rapamycin (mTOR) signaling and impaired performance on a trace-conditioning memory task. Chronic d -serine treatment normalized the electrophysiological, neurochemical, and cognitive deficits in SR ⁻/⁻ mice. These results demonstrate that NMDAR hypofunction can reproduce the numerous hippocampal deficits associated with schizophrenia, which can be reversed by chronic peripheral d -serine treatment.

Accumulating evidence from both genetic and clinico-pharmacological studies suggests that D -serine, an endogenous coagonist to the NMDA subtype glutamate receptor, may be implicated in schizophrenia (SZ). Although an association of genes for D -serine degradation, such as D -amino acid oxidase and G72, has been reported, a role for D -serine in SZ has been unclear. In this study, we identify and characterize protein interacting with C-kinase (PICK1) as a protein interactor of the D -serine synthesizing enzyme, serine racemase (SR). The binding of endogenous PICK1 and SR requires the PDZ domain of PICK1. The gene coding for PICK1 is located at chromosome 22q13, a region frequently linked to SZ. In a case–control association study using well-characterized Japanese subjects, we observe an association of the PICK1 gene with SZ, which is more prominent in disorganized SZ. Our findings implicating PICK1 as a susceptibility gene for SZ are consistent with a role for D -serine in the disease.

Benzylisoquinoline alkaloids (BIAs) are a diverse class of medicinal plant natural products. Nearly 500 dimeric bisbenzylisoquinoline alkaloids (bisBIAs), produced by the coupling of two BIA monomers, have been characterized and display a range of pharmacological properties, including anti-inflammatory, antitumor, and antiarrhythmic activities. In recent years, microbial platforms have been engineered to produce several classes of BIAs, which are rare or difficult to obtain from natural plant hosts, including protoberberines, morphinans, and phthalideisoquinolines. However, the heterologous biosyntheses of bisBIAs have thus far been largely unexplored. Here, we describe the engineering of yeast strains that produce the Type I bisBIAs guattegaumerine and berbamunine de novo. Through strain engineering, protein engineering, and optimization of growth conditions, a 10,000-fold improvement in the production of guattegaumerine, the major bisBIA pathway product, was observed. By replacing the cytochrome P450 used in the final coupling reaction with a chimeric variant, the product profile was inverted to instead produce solely berbamunine. Our highest titer engineered yeast strains produced 108 and 25 mg/L of guattegaumerine and berbamunine, respectively. Finally, the inclusion of two additional putative BIA biosynthesis enzymes, SiCNMT2 and NnOMT5, into our bisBIA biosynthetic strains enabled the production of two derivatives of bisBIA pathway intermediates de novo: magnocurarine and armepavine. The de novo heterologous biosyntheses of bisBIAs presented here provide the foundation for the production of additional medicinal bis- BIAs in yeast.

Chemotherapy is the first-tier treatment regime for gastric cancer (GC) patients at advance stages. Mesenchymal stem cell (MSC) cam affect drug-resistance of GC cells in tumor microenvironment, but the detailed mechanism remains poorly understood. Present study aimed to investigate the regulation of MSC-induced long non-coding RNA (lncRNA) in GC. Dysregulated lncRNAs in GC were analyzed based on GEO data. Stemness and drug-resistance of GC cells were detected by sphere formation, colony formation, CCK-8, and flow cytometry analyses. MicroRNA (miRNA)-related pathways were analyzed by online KEGG analysis tool DAVID6.8. Molecular interactions were determined by luciferase reporter assay, pulldown, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), and co-immunoprecipitation (CoIP). Results revealed that MSC co-culture improved stemness and drug-resistance of GC cells. LncRNA histocompatibility leukocyte antigen complex P5 (HCP5) was induced in GC cells by MSC co-culture, contributing to stemness and drug-resistance. Mechanistically, HCP5 sequestered miR-3619-5p and upregulated PPARG coactivator 1 alpha (PPARGC1A), increasing transcription complex Peroxisome proliferator activated receptor (PPAR) coactivator‐1α (PGC1α)/CEBPB and transcriptionally inducing carnitine palmitoyltransferase 1 (CPT1), which prompted the fatty acid oxidation (FAO) in GC cells. In conclusion, MSC-induced lncRNA HCP5 drove FAO through miR-3619-5p/AMPK/PGC1α/CEBPB axis to promote stemness and chemo-resistance of GC, indicating that targeting HCP5 was a novel approach to enhancing the efficacy of chemotherapy in GC.

Nonribosomal peptide synthetases (NRPSs) are modular assembly-line megaenzymes that synthesize diverse metabolites with wide-ranging biological activities. The structural dynamics of synthetic elongation has remained unclear. Here, we present cryo-EM structures of PchE, an NRPS elongation module, in distinct conformations. The domain organization reveals a unique “H”-shaped head-to-tail dimeric architecture. The capture of both aryl and peptidyl carrier protein-tethered substrates and intermediates inside the heterocyclization domain and l -cysteinyl adenylate in the adenylation domain illustrates the catalytic and recognition residues. The multilevel structural transitions guided by the adenylation C-terminal subdomain in combination with the inserted epimerase and the conformational changes of the heterocyclization tunnel are controlled by two residues. Moreover, we visualized the direct structural dynamics of the full catalytic cycle from thiolation to epimerization. This study establishes the catalytic trajectory of PchE and sheds light on the rational re-engineering of domain-inserted dimeric NRPSs for the production of novel pharmaceutical agents. The catalytic domains in nonribosomal peptide synthetases (NRPSs) are responsible for a choreography of events that elongates substrates into natural products. Here, the authors present cryo-EM structures of a siderophore-producing dimeric NRPS elongation module in multiple distinct conformations, which provides insight into the mechanisms of catalytic trajectory.

Hypercholesterolemia, the driving force of atherosclerosis, accelerates the expansion and mobilization of hematopoietic stem and progenitor cells (HSPCs). The molecular determinants connecting hypercholesterolemia with hematopoiesis are unclear. Here, we report that a somite-derived prohematopoietic cue, AIBP, orchestrates HSPC emergence from the hemogenic endothelium, a type of specialized endothelium manifesting hematopoietic potential. Mechanistically, AIBP-mediated cholesterol efflux activates endothelial Srebp2, the master transcription factor for cholesterol biosynthesis, which in turn transactivates Notch and promotes HSPC emergence. Srebp2 inhibition impairs hypercholesterolemia-induced HSPC expansion. Srebp2 activation and Notch up-regulation are associated with HSPC expansion in hypercholesterolemic human subjects. Genome-wide chromatin immunoprecipitation followed by sequencing (ChIP-seq), RNA sequencing (RNA-seq), and assay for transposase-accessible chromatin using sequencing (ATAC-seq) indicate that Srebp2 transregulates Notch pathway genes required for hematopoiesis. Our studies outline an AIBP-regulated Srebp2-dependent paradigm for HSPC emergence in development and HPSC expansion in atherosclerotic cardiovascular disease.

Background: Cellobiose 2-epimerases (CEs) are promising enzymes that catalyze the conversion of lactose to the bioactive disaccharides epilactose and lactulose. Low-temperature (≤8 °C) lactose bioconversion is essential in dairy applications to prevent microbial contamination. Unfortunately, most CEs are not particularly active at these low temperatures. Therefore, this work aims to discover and characterize novel cold-active CEs. Results Two novel mesophilic CEs from Teredinibacter haidensis ( Th CE) and Cellvibrio japonicus ( Cj CE) were characterized, which showed both epi- and isomerization activity additionally at low temperatures. Th CE showed a maximum epimerization activity (595.4 ± 4.4 nkat/mg protein) at pH 7.5 and 35 °C, while Cj CE achieved maximum epimerization activity (887.4 ± 3.9 nkat/mg protein) at pH 9.5 and 40 °C. In addition, Cj CE maintained an >80% activity across a wide pH (6–9.5) and temperature range (30–45 °C). Both CEs exhibited significant activity and stability at 8 °C. Specifically, Th CE and Cj CE retained around 20 and 40% of their epimerization activity at 8 °C and showed half-lives of 96.3 and 72.2 days, respectively. Moreover, Cj CE was also found to be capable of catalyzing the isomerization reaction at 8 °C. The isomerization activity of Th CE and Cj CE increased ~370-fold (70.8 ± 2.7 nkat/mg protein) and ~230-fold (66.1 ± 0.3 nkat/mg protein), respectively, at lactose concentrations up to 600 mM (35 °C for Th CE and 40 °C for Cj CE). This demonstrates how important it is to conduct investigations of CEs for lactose isomerization to lactulose up to the solubility limit of lactose. Conclusions This study identified two new CEs that exhibit interesting catalytic properties for possible application in the generation of disaccharides with prebiotic properties from lactose at low temperatures.

Natural D-serine (D-Ser) has been detected in animals more than two decades ago, but little is known about the physiological functions of D-Ser. Here we reveal sleep regulation by endogenous D-Ser. Sleep was decreased in mutants defective in D-Ser synthesis or its receptor the N-methyl-D-aspartic receptor 1 (NMDAR1), but increased in mutants defective in D-Ser degradation. D-Ser but not L-Ser rescued the phenotype of mutants lacking serine racemase (SR), the key enzyme for D-Ser synthesis. Pharmacological and triple gene knockout experiments indicate that D-Ser functions upstream of NMDAR1. Expression of SR was detected in both the nervous system and the intestines. Strikingly, reintroduction of SR into specific intestinal epithelial cells rescued the sleep phenotype of sr mutants. Our results have established a novel physiological function for endogenous D-Ser and a surprising role for intestinal cells. The physiological function of endogenous D-serine remains a mystery. Here the authors show that endogenous D-serine plays an important role in regulating sleep and that, while the D-serine synthesizing enzyme serine racemase (SR) is expressed both in the nervous system and the intestines, the SR in the intestine is shown to be functionally sufficient for sleep regulation.

The lasso peptide MS-271 is a ribosomally synthesized and post-translationally modified peptide (RiPP) consisting of 21 amino acids with D-tryptophan at the C -terminus, and is derived from the precursor peptide MslA. MslH, encoded in the MS-271 biosynthetic gene cluster ( msl ), catalyzes the epimerization at the Cα center of the MslA C -terminal Trp21, leading to epi -MslA. The detailed catalytic process, including the catalytic site and cofactors, has remained enigmatic. Herein, based on X-ray crystallographic studies in association with MslA core peptide analogues, we show that MslH is a metallo-dependent peptide epimerase with a calcineurin-like fold. The crystal structure analysis, followed by site-directed mutagenesis, docking simulation, and ICP-MS studies demonstrate that MslH employs acid/base chemistry to facilitate the reversible epimerization of the C- terminal Trp21 of MslA, by utilizing two pairs of His/Asp catalytic residues that are electrostatically tethered to a six-coordination motif with a Ca(II) ion via water molecules. MslH, encoded in the MS-271 biosynthetic gene cluster, catalyzes the epimerization at the Cα center of the MslA C-terminal Trp21, however, the detailed catalytic process was unknown. Here, the authors report MslH is a metallo-dependent peptide epimerase with a calcineurin-like fold.