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Phosphoglucomutase (EC 5.4.2.2., PGM), a key enzyme of glycogenolysis and glycogenesis, catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate, whereas phosphomannomutase (EC 5.4.2.8., PMM) transfers the phosphate group from the 1′ to the 6′, or from the 6′ to the 1′ position in mannose phosphate. However, in the hyperthermophilic archaeon Thermococcus kodakarensis, a single gene, Tk1108, encodes a protein with both PGM and PMM activities. Here, we report biophysical analysis and the 2.45 Å resolution cryo-EM structure of this novel enzyme. Our results demonstrate a specific arrangement of the four subunits in the quaternary structure, displaying a distinct catalytic cleft required for the bifunctional activity at extremely high temperatures. To the best of our knowledge, this is the first biophysical characterization and cryo-EM structure elucidation of a thermostable, bifunctional PGM/PMM.

To elucidate the starch synthesis pathway and the role of this reserve in rice pollen, we characterized mutations in the plastidic phosphoglucomutase, OspPGM, and the plastidic large subunit of ADP-glucose (ADP-Glc) pyrophosphorylase, OsAGPL4. Both genes were up-regulated in maturing pollen, a stage when starch begins to accumulate. Progeny analysis of self-pollinated heterozygous lines carrying the OspPGM mutant alleles, osppgm-1 and osppgm-2, or the OsAGPL4 mutant allele, osagpl4-1, as well as reciprocal crosses between the wild type (WT) and heterozygotes revealed that loss of OspPGM or OsAGPL4 caused male sterility, with the former condition rescued by the introduction of the WT OspPGM gene. While iodine staining and transmission electron microscopy analyses of pollen grains from homozygous osppgm-1 lines produced by anther culture confirmed the starch null phenotype, pollen from homozygous osagpl4 mutant lines, osagpl4-2 and osagpl4-3, generated by the CRISPR/Cas system, accumulated small amounts of starch which were sufficient to produce viable seed. Such osagpl4 mutant pollen, however, was unable to compete against WT pollen successfully, validating the important role of this reserve in fertilization. Our results demonstrate that starch is mainly polymerized from ADP-Glc synthesized from plastidic hexose phosphates in rice pollen and that starch is an essential requirement for successful fertilization in rice.

Two brothers with an undefined congenital disorder of glycosylation were found to have phosphoglucomutase 1 deficiency, which has previously been described as a glycogen storage disorder. Supplementation with galactose improves protein glycosylation in this disease. Protein N-glycosylation is a ubiquitous process in all organ systems. During N-glycosylation, glycan precursors are assembled from monosaccharide units and then covalently attached to asparagine residues in the nascent peptide chain of a protein (Figure 1). The protein-bound glycans undergo further processing to generate mature glycoproteins. Genetic defects in protein N-glycosylation, designated as congenital disorders of glycosylation, lead to multisystem disorders. Mutations of genes involved in N-glycosylation may affect either the biosynthesis of the glycan precursor (congenital disorder of glycosylation type I [CDG-I]) or the processing of the glycan after its attachment to the protein (congenital disorder of glycosylation type . . .

Triple-negative breast cancer (TNBC) is characterized by a pronounced hypoxic tumor microenvironment, with cancer-associated fibroblasts (CAFs) serving as the predominant cellular component and playing crucial roles in regulating tumor progression. However, the mechanism by which CAFs affect the biological behavior of tumor cells in hypoxic environment remain elusive. This study employed a bead-based multiplex immunoassay to analyze a panel of cytokines/chemokines and identified colony stimulating factor 3 (CSF3) as a significantly elevated component in the secretome of hypoxic CAFs. We found that CSF3 promoted the invasive behavior of TNBC cells by activating the downstream signaling pathway of its receptor, CSF3R. RNA sequencing analysis further revealed that phosphoglucomutase 2-like 1 (PGM2L1) is a downstream target of the CSF3/CSF3R signaling, enhancing the glycolysis pathway and providing energy to support the malignant phenotype of breast cancer. In vivo, we further confirmed that CSF3 promotes TNBC progression by targeting PGM2L1. These findings suggest that targeting CSF3/CSF3R may represent a potential therapeutic approach for TNBC.

• Rice (Oryza sativa) tiller angle is a key component for achieving ideal plant architecture and higher grain yield. However, the molecular mechanism underlying rice tiller angle remains elusive. • We characterized a novel rice tiller angle mutant lazy2 (la2) and isolated the causative gene LA2 through map-based cloning. Biochemical, molecular and genetic studies were conducted to elucidate the LA2-involved tiller angle regulatory mechanism. • The la2 mutant shows large tiller angle with impaired shoot gravitropism and defective asymmetric distribution of auxin. We found that starch granules in amyloplasts are completely lost in the gravity-sensing leaf sheath base cells of la2, whereas the seed development is not affected. LA2 encodes a novel chloroplastic protein that can interact with the starch biosynthetic enzyme Oryza sativa plastidic phosphoglucomutase (OspPGM) to regulate starch biosynthesis in rice shoot gravity-sensing cells. Genetic analysis showed that LA2 regulates shoot gravitropism and tiller angle by acting upstream of LA1 to mediate lateral auxin transport. • Our studies revealed that LA2 acts as a novel regulator of rice tiller angle by specifically regulating starch biosynthesis in gravity-sensing cells, and established the framework of the starch-statolith-dependent rice tiller angle regulatory pathway, providing new insights into the rice tiller angle regulatory network.

The placenta plays an essential role facilitating nutrient, gas and waste exchange between the maternal and fetal systems for optimal fetal growth. When placental development is impaired and the placenta dysfunctional, serious pregnancy complications such as fetal growth restriction and preeclampsia may arise. Previously, phosphoglucomutase-5 (PGM5) transcripts were found to be highly elevated in the blood of patients whose pregnancies were complicated by fetal growth restriction and preeclampsia. As both conditions feature placental insufficiency, here we aimed to characterise PGM5 levels in the healthy and dysfunctional placenta. PGM5 expression was detectable in all placental samples across gestation, in cases of preterm preeclampsia, fetal growth restriction and controls. PGM5 mRNA expression was significantly downregulated in the pathological placentas compared to controls, but PGM5 protein production was not dysregulated. Isolated cytotrophoblast and placental explant tissue exposed to hypoxia (modelling placental dysfunction) demonstrated significantly increased PGM5 expression, but again did not change protein levels. Silencing PGM5 expression under hypoxic conditions in primary cytotrophoblast did not alter anti-angiogenic sFLT-1 secretion but increased expression of multiple genes associated with cell growth, apoptosis and oxidative stress, whilst also increasing cell viability. Expression of PGM5 in all placental samples assessed suggests that PGM5 has functions in the placenta. However, further investigation could be performed to explore the discrepancies in protein and mRNA expression, as well as the precise function of PGM5 in the placenta, and whether altered PGM5 levels may be important for placental development.

Glycans serve as important regulators of antibody activities and half-lives. IgE is the most heavily glycosylated antibody, but in comparison to other antibodies little is known about its glycan structure function relationships. We therefore describe the site specific IgE glycosylation from a patient with a novel hyper IgE syndrome linked to mutations in PGM3, which is an enzyme involved in synthesizing UDP-GlcNAc, a sugar donor widely required for glycosylation. A two-step method was developed to prepare two IgE samples from less than 1 mL of serum collected from a patient with PGM3 mutation and a patient with atopic dermatitis as a control subject. Then, a glycoproteomic strategy was used to study the site-specific glycosylation. No glycosylation was found at Asn264, whilst high mannose glycans were only detected at Asn275, tri-antennary glycans were exclusively observed at Asn99 and Asn252, and non-fucosylated complex glycans were detected at Asn99. The results showed similar glycosylation profiles between the two IgE samples. These observations, together with previous knowledge of IgE glycosylation, imply that IgE glycosylation is similarly regulated among healthy control, allergy and PGM3 related hyper IgE syndrome.

Phosphoglucomutase 1 (PGM1) is a key enzyme for the regulation of energy metabolism from glycogen and glycolysis, as it catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate. PGM1 deficiency is an autosomal recessive disorder characterized by a highly heterogenous clinical spectrum, including hypoglycemia, cleft palate, liver dysfunction, growth delay, exercise intolerance, and dilated cardiomyopathy. Abnormal protein glycosylation has been observed in this disease. Oral supplementation with D-galactose efficiently restores protein glycosylation by replenishing the lacking pool of UDP-galactose, and rescues some symptoms, such as hypoglycemia, hepatopathy, and growth delay. However, D-galactose effects on skeletal muscle and heart symptoms remain unclear. In this study, we established an in vitro muscle model for PGM1 deficiency to investigate the role of PGM1 and the effect of D-galactose on nucleotide sugars and energy metabolism. Genome-editing of C2C12 myoblasts via CRISPR/Cas9 resulted in Pgm1 (mouse homologue of human PGM1, according to updated nomenclature) knockout clones, which showed impaired maturation to myotubes. No difference was found for steady-state levels of nucleotide sugars, while dynamic flux analysis based on 13C6-galactose suggested a block in the use of galactose for energy production in knockout myoblasts. Subsequent analyses revealed a lower basal respiration and mitochondrial ATP production capacity in the knockout myoblasts and myotubes, which were not restored by D-galactose. In conclusion, an in vitro mouse muscle cell model has been established to study the muscle-specific metabolic mechanisms in PGM1 deficiency, which suggested that galactose was unable to restore the reduced energy production capacity.

The obligate intracellular bacteria Chlamydia trachomatis store glycogen in the lumen of the vacuoles in which they grow. Glycogen catabolism generates glucose-1-phosphate (Glc1P), while the bacteria can take up only glucose-6-phosphate (Glc6P). We tested whether the conversion of Glc1P into Glc6P could be catalyzed by a phosphoglucomutase (PGM) of host or bacterial origin. We found no evidence for the presence of the host PGM in the vacuole. Two C. trachomatis proteins, CT295 and CT815, are potential PGMs. By reconstituting the reaction using purified proteins, and by complementing PGM deficient fibroblasts, we demonstrated that only CT295 displayed robust PGM activity. Intriguingly, we showed that glycogen accumulation in the lumen of the vacuole of a subset of Chlamydia species ( C. trachomatis , C. muridarum , C. suis ) correlated with the presence, in CT295 orthologs, of a secretion signal recognized by the type three secretion (T3S) machinery of Shigella . C. caviae and C. pneumoniae do not accumulate glycogen, and their CT295 orthologs lack T3S signals. In conclusion, we established that the conversion of Glc1P into Glc6P was accomplished by a bacterial PGM, through the acquisition of a T3S signal in a “housekeeping” protein. Acquisition of this signal likely contributed to shaping glycogen metabolism within Chlamydiaceae .

This study for the first time investigated the role of metabolic enzyme phosphoglucomutase A ( pgmA ) in Mycobacterium tuberculosis (Mtb), revealing its crucial functions as a toggle switch between biosynthesis and growth. This work highlights the importance of pgmA in maintaining the metabolic flexibility of Mtb during the nutritional switch. The presence of pgmA is critical for the production of membrane-associated glycolipid, which helps maintain the cell wall integrity under various growth and stress conditions. This adaptability is pivotal for generating starvation-induced antibiotic tolerance in Mtb. In addition to the clinical context, these findings provide a mechanistic foundation for understanding adaptive strategies by Mtb to harsh environments and the development of drug-tolerant bacilli.