Genetic Analysis of Two Novel GPI Variants Disrupting H Bonds and Localization Characteristics of 55 Gene Variants Associated with Glucose-6-phosphate Isomerase Deficiency.

OBJECTIVE: Glucose-6-phosphate isomerase (GPI) deficiency is a rare hereditary nonspherocytic hemolytic anemia caused by GPI gene variants. This disorder exhibits wide heterogeneity in its clinical manifestations and molecular characteristics, often posing challenges for precise diagnoses using conventional methods. To this end, this study aimed to identify the novel variants responsible for GPI deficiency in a Chinese family. METHODS: The clinical manifestations of the patient were summarized and analyzed for GPI deficiency phenotype diagnosis. Novel compound heterozygous variants of the GPI gene, c.174C>A (p.Asn58Lys) and c.1538G>T (p.Trp513Leu), were identified using whole-exome and Sanger sequencing. The AlphaFold program and Chimera software were used to analyze the effects of compound heterozygous variants on GPI structure. RESULTS: By characterizing 53 GPI missense/nonsense variants from previous literature and two novel missense variants identified in this study, we found that most variants were located in exons 3, 4, 12, and 18, with a few localized in exons 8, 9, and 14. This study identified novel compound heterozygous variants associated with GPI deficiency. These pathogenic variants disrupt hydrogen bonds formed by highly conserved GPI amino acids. CONCLUSION: Early family-based sequencing analyses, especially for patients with congenital anemia, can help increase diagnostic accuracy for GPI deficiency, improve child healthcare, and enable genetic counseling.

Glucose phosphate isomerase deficiency demasked by whole-genome sequencing: a case report.

BACKGROUND: Glucose-6-phosphate isomerase deficiency is a rare genetic disorder causing hereditary nonspherocytic hemolytic anemia. It is the second most common glycolytic enzymopathy in red blood cells. About 90 cases are reported worldwide, with symptoms including chronic hemolytic anemia, jaundice, splenomegaly, gallstones, cholecystitis, and in severe cases, neurological impairments, hydrops fetalis, and neonatal death. CASE PRESENTATION: This paper details the case of the first Danish patient diagnosed with glucose-6-phosphate isomerase deficiency. The patient, a 27-year-old white female, suffered from lifelong anemia of unknown origin for decades. Diagnosis was established through whole-genome sequencing, which identified two GPI missense variants: the previously documented variant p.(Thr224Met) and a newly discovered variant p.(Tyr341Cys). The pathogenicity of these variants was verified enzymatically. CONCLUSIONS: Whole-genome sequencing stands as a potent tool for identifying hereditary anemias, ensuring optimal management strategies.

Production of D-glucaric acid with phosphoglucose isomerase-deficient Saccharomyces cerevisiae.

D-Glucaric acid is a potential biobased platform chemical. Previously mainly Escherichia coli, but also the yeast Saccharomyces cerevisiae, and Pichia pastoris, have been engineered for conversion of D-glucose to D-glucaric acid via myo-inositol. One reason for low yields from the yeast strains is the strong flux towards glycolysis. Thus, to decrease the flux of D-glucose to biomass, and to increase D-glucaric acid yield, the four step D-glucaric acid pathway was introduced into a phosphoglucose isomerase deficient (Pgi1p-deficient) Saccharomyces cerevisiae strain. High D-glucose concentrations are toxic to the Pgi1p-deficient strains, so various feeding strategies and use of polymeric substrates were studied. Uniformly labelled 13C-glucose confirmed conversion of D-glucose to D-glucaric acid. In batch bioreactor cultures with pulsed D-fructose and ethanol provision 1.3 g D-glucaric acid L-1 was produced. The D-glucaric acid titer (0.71 g D-glucaric acid L-1) was lower in nitrogen limited conditions, but the yield, 0.23 g D-glucaric acid [g D-glucose consumed]-1, was among the highest that has so far been reported from yeast. Accumulation of myo-inositol indicated that myo-inositol oxygenase activity was limiting, and that there would be potential to even higher yield. The Pgi1p-deficiency in S. cerevisiae provides an approach that in combination with other reported modifications and bioprocess strategies would promote the development of high yield D-glucaric acid yeast strains.

Deglycation activity of the Escherichia coli glycolytic enzyme phosphoglucose isomerase.

Glycation is a spontaneous chemical reaction, which affects the structure and function of proteins under normal physiological conditions. Therefore, organisms have evolved diverse mechanisms to combat glycation. In this study, we show that the Escherichia coli glycolytic enzyme phosphoglucose isomerase (Pgi) exhibits deglycation activity. We found that E. coli Pgi catalyzes the breakdown of glucose 6-phosphate (G6P)-derived Amadori products (APs) in chicken lysozyme. The affinity of Pgi to the glycated lysozyme (Km, 1.1 mM) was ten times lower than the affinity to its native substrate, fructose 6-phosphate (Km, 0.1 mM). However, the high kinetic constants of the enzyme with the glycated lysozyme (kcat, 396 s-1 and kcat/Km, 3.6 × 105 M-1 s-1) indicated that the Pgi amadoriase activity may have physiological implications. Indeed, when using total E. coli protein (20 mg/mL) as a substrate in the deglycation reaction, we observed a release of G6P from the bacterial protein at a Pgi specific activity of 33 µmol/min/mg. Further, we detected 11.4 % lower APs concentration in protein extracts from Pgi-proficient vs. deficient cells (p = 0.0006) under conditions where the G6P concentration in Pgi-proficient cells was four times higher than in Pgi-deficient cells (p = 0.0001). Altogether, these data point to physiological relevance of the Pgi deglycation activity.

RHBDF1 deficiency suppresses melanoma glycolysis and enhances efficacy of immunotherapy by facilitating glucose-6-phosphate isomerase degradation via TRIM32.

Melanoma is a dangerous form of skin cancer, making it important to investigate new mechanisms and approaches to enhance the effectiveness of treatment. Here, we establish a positive correlation between the human rhomboid family-1 (RHBDF1) protein and melanoma malignancy. We demonstrate that the melanoma RHBDF1 decrease dramatically inhibits tumor growth and the development of lung metastases, which may be related to the impaired glycolysis. We show that RHBDF1 function is essential to the maintenance of high levels of glycolytic enzymes, especially glucose-6-phosphate isomerase (GPI). Additionally, we discover that the E3 ubiquitin ligase tripartite motif-containing 32 (TRIM32) mediates the K27/K63-linked ubiquitination of GPI and the ensuing lysosomal degradation process. We prove that the multi-transmembrane domain of RHBDF1 is in competition with GPI, preventing the latter from interacting with NCL1-HT2A-LIN41 (NHL) domain of TRIM32. We also note that the mouse RHBDF1's R747 and Y799 are crucial for competitive binding and GPI protection. Artificially silencing the Rhbdf1 gene in a mouse melanoma model results in declined lactic acid levels, elevated cytotoxic lymphocyte infiltration, and improved tumor responsiveness to immunotherapy. These results provide credence to the hypothesis that RHBDF1 plays a significant role in melanoma regulation and suggest that blocking RHBDF1 may be an efficient technique for reestablishing the tumor immune microenvironment (TIME) in melanoma and halting its progression.

High-resolution melting (HRM)-based detection of polymorphisms in the malic enzyme and glucose-6-phosphate isomerase genes for Leishmania infantum genotyping.

BACKGROUND: Leishmaniasis is a zoonotic disease endemic in the Mediterranean region where Leishmania infantum is the causative agent of human and canine infection. Characterization of this parasite at the subspecies level can be useful in epidemiological studies, to evaluate the clinical course of the disease (e.g. resistant strains, visceral and cutaneous forms of leishmaniasis) as well as to identify infection reservoirs. Multilocus enzyme electrophoresis (MLEE), a method currently recognized as the reference method for characterizing and identifying strains of Leishmania, is cumbersome and time-consuming and requires cultured parasites. These disadvantages have led to the development of other methods, such as multilocus microsatellite typing (MLMT) and multilocus sequence typing (MLST), for typing Leishmania parasites; however, these methods have not yet been applied for routine use. In this study, we first used MLST to identify informative polymorphisms on single-copy genes coding for metabolic enzymes, following which we developed two rapid genotyping assays based on high-resolution melting (HRM) analysis to explore these polymorphisms in L. infantum parasites. METHODS: A customized sequencing panel targeting 14 housekeeping genes was designed and MLST analysis was performed on nine L. infantum canine and human strains/isolates. Two quantitative real-time PCR-HRM assays were designed to analyze two informative polymorphisms on malic enzyme (ME) and glucose-6-phosphate isomerase (GPI) genes (390T/G and 1831A/G, respectively). The two assays were applied to 73 clinical samples/isolates from central/southern Italy and Pantelleria island, and the results were confirmed by DNA sequencing in a subset of samples. RESULTS: The MLST analysis, together with sequences available in the Genbank database, enabled the identification of two informative polymorphisms on the genes coding for ME and GPI. The fast screening of these polymorphisms using two HRM-based assays in 73 clinical samples/isolates resulted in the identification of seven genotypes. Overall, genotype 1 (sequence type 390T/1831G) was the most highly represented (45.2%) in the overall sample and correlated with the most common L. infantum zymodemes (MON-1, MON-72). Interestingly, in Pantelleria island, the most prevalent genotype (70.6%) was genotype 6 (sequence type 390T/1831A). CONCLUSIONS: Applying our HRM assays on clinical samples allowed us to identify seven different genotypes without the need for parasite isolation and cultivation. We have demonstrated that these assays could be used as fast, routine and inexpensive tools for epidemiological surveillance of L. infantum or for the identification of new infection reservoirs.

The novel compound heterozygous variants identified in a Chinese family with glucose phosphate isomerase deficiency and pathogenicity analysis.

BACKGROUND AND AIMS: Glucose phosphate isomerase (GPI) deficiency is an extremely rare autosomal recessive disorder caused by mutations in the GPI gene. In this research, the proband displaying typical manifestations of haemolytic anaemia and his family members were recruited to analyse the pathogenicity of the detected variants. METHODS: Peripheral blood samples were collected from the family members and genomic DNA was extracted and targeted for capture and sequencing. The effect of the candidate pathogenic variants on splicing was further investigated using the minigene splicing system. The computer simulation was also used for further analysis of the detected data. RESULTS: The proband carried the compound heterozygous variants c.633 + 3 A > G and c.295G > T in the GPI gene, which have never been reported before. In the genealogy, co-segregation of the mutant genotype with the phenotype was established. The minigene study showed that intronic mutations resulted in abnormal pre-mRNA splicing. Specifically, the two aberrant transcripts: r.546_633del and r.633 + 1_633 + 2insGT were transcribed by the minigene plasmid expressing the c.633 + 3 A > G variant. The missense mutation c.295G > T in exon 3 resulted in altering glycine at codon 87 to cysteine which was predicted to be pathogenic in an in silico analysis. Deeper analyses revealed that the Gly87Cys missense mutation led to steric hindrance. Compared to the wild-type, the mutation G87C led to a significant increase in intermolecular forces. CONCLUSION: Overall, the novel compound heterozygous variants in the GPI gene contributed to the etiology of the disease. Genetic testing can assist in the diagnosis. The novel gene variants identified in the present study has further expanded the mutational spectrum of GPI deficiency, which can better guide family counselling.

Effects of phthalate and bisphenol plasticizers on the activity of glycolytic enzymes of the moth Spodoptera littoralis.

Environmental plastic pollution has significantly increased in the recent decades, and severely impacts economies, human and biodiversity health. Plastics are made of several chemical additives, including bisphenol and phthalate plasticizers such as bisphenol A (BPA) and Di(2-ethylhexyl)phthalate (DEHP). In some animal species, both BPA and DEHP are known as endocrine disruptor compounds, and can alter physiological and metabolic homeostasis, reproduction, development and/or behavior. To date, the impacts of BPA and DEHP have mainly focused on vertebrates, and to a lesser extent, on aquatic invertebrates. Yet, the few studies which examined the effects of DEHP on terrestrial insects also revealed the impacts this pollutant can have on development, hormone titrations, and metabolic profiles. In particular, it has been hypothesized in the Egyptian cotton leafworm Spodoptera littoralis that the observed metabolic alterations could result from the energetic costs necessary for DEHP detoxification or to the dysregulation of hormonally-controlled enzymatic activities. To get additional insights into the physiological effects of bisphenol and phthalate plasticizers on the moth S. littoralis, larvae were fed with food contaminated by BPA, DEHP, or the mixture of both compounds. Then, activities of four glycolytic enzymes, hexokinase, phosphoglucose isomerase, phosphofructokinase, and pyruvate kinase were measured. BPA and/or DEHP had no effects on the activities of phosphofructokinase and pyruvate kinase. Conversely, BPA-contaminated larvae were characterized by a 1.9-fold increase in phosphoglucose isomerase activity, and BPA + DEHP-fed larvae had highly variable hexokinase activity. Overall, since no disruption of glycolytic enzyme was observed in DEHP-contaminated larvae, our work tended to demonstrate that exposure to bisphenol and DEHP increased the amount of oxidative stress experienced.

[Value of autocrine motility factors in the prediction of the disease progression of PBC- associated hepatocellular carcinoma].

Objective: To clarify the value of autocrine motility factor (ATX) in predicting the disease progression of primary biliary cholangitis (PBC)-associated hepatocellular carcinoma (HCC). Methods: A prospective cohort of 179 newly diagnosed autoimmune liver disease (PBC) patients admitted to the Department of Hepatology at the Fifth Medical Center of the People's Liberation Army General Hospital from January 2016 to January 2018 was selected. All PBC patients received ursodeoxycholic acid (UDCA) treatment and were followed up.The endpoint of the follow-up was the occurrence of primary liver cancer. The relationship between ATX and the clinical characteristics of patients and its significance in predicting disease progression and HCC were analyzed. Results: The peripheral blood ATX level was significantly higher in PBC patients than that of alcoholic cirrhosis (t = 3.278, P = 0.001) and healthy controls (t = 6.594, P < 0.001), but there was no significant difference in ATX levels compared with patients with non-PBC- associated HCC (t = -0.240, P = 0.811). The expression of ATX in liver tissue of PBC patients was significantly higher than that of healthy individuals (Z = -3.633, P < 0.001) and patients with alcoholic cirrhosis (Z = -3.283, P < 0.001), while the expression of ATX in the advanced stage was significantly higher than that in early-stage PBC patients (Z = -2.018, P = 0.034). There was a significant difference in baseline ATX levels between PBC patients without HCC and PBC patients with HCC (228.451 ± 124.093 ng/ml vs. 301.583 ± 100.512 ng/ml, t = 2.339, P = 0.021). Multivariate logistic regression analysis showed that ATX was an independent predictor of PBC progression to HCC (OR = 1.245, 95%CI 1.097-1.413). The baseline peripheral blood ATX level in predicting AUROC of PBC-associated HCC was 0.714, 95%CI 0.597-0.857 and the sensitivity and specificity were 84.6%, and 59.0%, respectively. The optimal cutoff value for predicting serum ATX levels in the occurrence of HCC was 235.254 ng/ml. Conclusion: Patients with PBC have significantly higher levels of ATX expression in their peripheral blood and liver tissue, which can be utilized to assess treatment effectiveness and predict disease progression.

Exploring the chondroprotective effect of Chaenomeles speciosa on Glucose-6-Phosphate Isomerase model mice using an integrated approach of network pharmacology and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) has been used in China for a long time and is gradually gaining more and more recognition worldwide. Chaenomeles speciosa (CSP) (Chinese Pinyin: mugua) is a medicinal and food herb that has long been used as a folk medicine for rheumatic diseases, yet its bioactive components and therapeutic mechanisms are not clear. AIM OF THE STUDY: Exploring anti-inflammatory and chondroprotective effects of CSP on rheumatoid arthritis (RA) and its possible targets of action. MATERIALS AND METHODS: In this study, we performed an integrated approach of network pharmacology, molecular docking and experimental studies to explore the potential mechanism of action of CSP in the treatment of cartilage damage in RA. RESULTS: Studies have shown that Quercetin, ent-Epicatechin and Mairin may be the main active compounds of CSP in the treatment of RA, while AKT1, VEGFA, IL-1ß, IL-6, MMP9 etc. are considered as core target proteins to which the main active compounds in CSP bind, as further confirmed by molecular docking. In addition, the potential molecular mechanism of CSP for the treatment of cartilage damage in RA predicted by network pharmacology analysis was validated by in vivo experiments. CSP was found to downregulate the expression of AKT1, VEGFA, IL-1ß, IL-6, MMP9, ICAM1, VCAM1, MMP3, MMP13 and TNF-α and increase the expression of COL-2 in the joint tissue of Glucose-6-Phosphate Isomerase (G6PI) model mice. Thus CSP contributes to the treatment of rheumatoid arthritis cartilage destruction. CONCLUSION: This study showed that CSP has multi-component, multi-target and multi-pathway characteristics in treating cartilage damage in RA, which can achieve the effect of treating RA by inhibiting the expression of inflammatory factors, reducing neovascularization and alleviating the damage to cartilage caused by the diffusion of synovial vascular opacities, and reducing the degradation of cartilage by MMPs to play a protective role in RA cartilage damage. In conclusion, this study indicates that CSP is a candidate Chinese medicine for further research in treating cartilage damage in RA.

A Novel Glucose-6-Phosphate Isomerase Exists in Chicken Breast Meat: A Selenium-Containing Enzyme that Should Be Re-recognized Through New Eyes.

Glucose-6-phosphate isomerase (GPI) is a highly conserved glycolytic enzyme in nature, and less information was available for GPI from hens. In this study a newly discovered selenocysteine (Sec)-containing GPI in common chicken breast meat was first isolated, purified and identified. Data about LC-MS/MS, FTIR and Se species analyses show that the molecular weight of the enzyme is 62,091 Da and only one Sec is inserted at the 403rd position in the highly conserved primary domain SIS_PGI with sugar conversion function. The enzyme shows excellent activity against hydroxyl radicals as vitamin C (Vc) in vitro. It is deduced that the Sec-containing GPI in the chicken meat may depend on Sec in its molecular structure to resist reactive oxygen species (ROS) stress produced by the accompanying biochemical reactions in cells, to protect its stability and maintain its efficient function that catalyzes the conversion of glucose-6-phosphate to fructose-6-phosphate in the critical glycolytic pathway.

Pathogenic antibody response to glucose-6-phosphate isomerase targets a modified epitope uniquely exposed on joint cartilage.

OBJECTIVES: To identify the arthritogenic B cell epitopes of glucose-6-phosphate isomerase (GPI) and their association with rheumatoid arthritis (RA). METHODS: IgG response towards a library of GPI peptides in patients with early RA, pre-symptomatic individuals and population controls, as well as in mice, were tested by bead-based multiplex immunoassays and ELISA. Monoclonal IgG were generated, and the binding specificity and affinity were determined by ELISA, gel size exclusion chromatography, surface plasma resonance and X-ray crystallography. Arthritogenicity was investigated by passive transfer experiments. Antigen-specific B cells were identified by peptide tetramer staining. RESULTS: Peptide GPI293-307 was the dominant B cell epitope in K/BxN and GPI-immunised mice. We could detect B cells and low levels of IgM antibodies binding the GPI293-307 epitopes, and high affinity anti-GPI293-307 IgG antibodies already 7 days after GPI immunisation, immediately before arthritis onset. Transfer of anti-GPI293-307 IgG antibodies induced arthritis in mice. Moreover, anti-GPI293-307 IgG antibodies were more frequent in individuals prior to RA onset (19%) than in controls (7.5%). GPI293-307-specific antibodies were associated with radiographic joint damage. Crystal structures of the Fab-peptide complex revealed that this epitope is not exposed in native GPI but requires conformational change of the protein in inflamed joint for effective recognition by anti-GPI293-307 antibodies. CONCLUSIONS: We have identified the major pathogenic B cell epitope of the RA-associated autoantigen GPI, at position 293-307, exposed only on structurally modified GPI on the cartilage surface. B cells to this neo-epitope escape tolerance and could potentially play a role in the pathogenesis of RA.

Preparation of mesoporous silica nanocarriers targeting glucose-6-phosphate isomerase inhibition and application in the treatment of rheumatoid arthritis.

Glucose 6-phosphate isomerase (G6PI) is an indicator to assist in diagnosis of rheumatoid arthritis (RA) and monitor the disease. It also plays a key role in proliferating RA synovial tissues, pannus formation, and invasion and destruction of articular cartilage. In this study, we synthesized nanoparticles targeting G6PI (siG6PI-MSN) using mesoporous silica nanocarriers (MSN) and small interfering RNA (siRNA), followed by identifying the characteristics and functions, and preliminarily exploring their application in the treatment of RA in vivo with a type II collagen-induced arthritis (CIA) rat model. It showed that the synthetic functionalized carrier had a regular pore structure and a specific volume and surface area. No obvious hemolysis or toxicity of the carrier was found when its concentration was below 100 µg/ml. Cytological results in vitro suggested that siG6PI-MSN significantly inhibited G6PI expression and reduced the ability of proliferation, migration, and invasion of FLSs, compared with the siNC-MSN group. In vivo results in the CIA rat model showed that the arthritis index and degree of joint swelling among rats in the siG6PI-MSN-treatment group were significantly lower than those in the control group. Moreover, the number of FLSs in Synovium and the levels of TNF α and IL-1 ß were also significantly decreased in the siG6PI-MSN group. Histopathology of the synovial tissue and cartilage revealed siG6PI-MSN treatment significantly reduced the pathological manifestations of arthritis. In conclusion, siG6PI-MSN effectively suppresses the proliferation and invasive growth of synovial tissue and improve joint swelling and inflammatory infiltration, thereby preventing joint damage in RA. This carrier may be a new therapeutic measure for RA, with potential social and economic benefits.

Cytosolic phosphoglucose isomerase is essential for microsporogenesis and embryogenesis in Arabidopsis.

Phosphoglucose isomerase (PGI) catalyzes the interconversion of fructose-6-phosphate and glucose-6-phosphate, which impacts cell carbon metabolic flow. Arabidopsis (Arabidopsis thaliana) contains two nuclear PGI genes respectively encoding plastidial PGI1 and cytosolic PGI (cPGI). The loss of PGI1 impairs the conversion of F6P of the Calvin-Benson cycle to G6P for the synthesis of transitory starch in leaf chloroplasts. Since cpgi knockout mutants have not yet been obtained, they are thought to be lethal. The cpgi lethality can be rescued by expressing CaMV 35S promoter (p35S)-driven cPGI; however, the complemented line is completely sterile due to pollen degeneration. Here, we generated a cpgi mutant expressing p35S::cPGI-YFP in which YFP fluorescence in developing anthers was undetectable specifically in the tapetum and in pollen, which could be associated with male sterility. We also generated RNAi-cPGI knockdown lines with strong cPGI repression in floral buds that exhibited reduced male fertility due to the degeneration of most pollen. Histological analyses indicated that the synthesis of intersporal callose walls was impaired, causing microsporocytes to fail to separate haploid daughter nuclei to form tetrads, which might be responsible for subsequent pollen degeneration. We successfully isolated cpgi knockout mutants in the progeny of a heterozygous cpgi mutant floral-dipped with sugar solutions. The rescued cpgi mutants exhibited diminished young vegetative growth, reduced female fertility, and impaired intersporal callose wall formation in a meiocyte, and, thus, male sterility. Collectively, our data suggest that cPGI plays a vital role in carbohydrate partitioning, which is indispensable for microsporogenesis and early embryogenesis.

Glucose 6 Phosphate Isomerase Deficiency, a Rare Hemolytic Anemia Misdiagnosed as Hereditary Spherocytosis.

Hereditary hemolytic anemias are a heterogenous group of disorders that include membranopathies, enzymopathies, and hemoglobinopathies. Genetic testing is helpful in the diagnostic workup when the clinical and laboratory workup is not conclusive. Here, we present a case of a 21-month-old female who was initially diagnosed with hereditary spherocytosis based on the presence of a variant of unknown significance in the SPTB gene. Further genetic workup revealed a homozygous glucose 6 phosphate isomerase mutation and the patient was ultimately diagnosed with glucose 6 phosphate isomerase deficiency.

Three amino acid substitutions contributing to thermostability of phosphoglucose isomerase in the Glanville fritillary butterfly.

Temperature is one of the most important environmental factors that affect organisms, especially ectotherms, due to its effects on protein stability. Understanding the general rules that govern thermostability changes in proteins to adapt high-temperature environments is crucial. Here, we report the amino acid substitutions of phosphoglucose isomerase (PGI) related to thermostability in the Glanville fritillary butterfly (Melitaea cinxia, Lepidoptera: Nymphalidae). The PGI encoded by the most common allele in M. cinxia in the Chinese population (G3-PGI), which is more thermal tolerant, is more stable under heat stress than that in the Finnish population (D1-PGI). There are 5 amino acid substitutions between G3-PGI and D1-PGI. Site-directed mutagenesis revealed that the combination of amino acid substitutions of H35Q, M49T, and I64V may increase PGI thermostability. These substitutions alter the 3D structure to increase the interaction between 2 monomers of PGI. Through molecular dynamics simulations, it was found that the amino acid at site 421 is more stable in G3-PGI, confining the motion of the α-helix 420-441 and stabilizing the interaction between 2 PGI monomers. The strategy for high-temperature adaptation through these 3 amino acid substitutions is also adopted by other butterfly species (Boloria eunomia, Aglais urticae, Colias erate, and Polycaena lua) concurrent with M. cinxia in the Tianshan Mountains of China, i.e., convergent evolution in butterflies.

Using phosphoglucose isomerase-deficient (pgi1Δ) Saccharomyces cerevisiae to map the impact of sugar phosphate levels on D-glucose and D-xylose sensing.

BACKGROUND: Despite decades of engineering efforts, recombinant Saccharomyces cerevisiae are still less efficient at converting D-xylose sugar to ethanol compared to the preferred sugar D-glucose. Using GFP-based biosensors reporting for the three main sugar sensing routes, we recently demonstrated that the sensing response to high concentrations of D-xylose is similar to the response seen on low concentrations of D-glucose. The formation of glycolytic intermediates was hypothesized to be a potential cause of this sensing response. In order to investigate this, glycolysis was disrupted via the deletion of the phosphoglucose isomerase gene (PGI1) while intracellular sugar phosphate levels were monitored using a targeted metabolomic approach. Furthermore, the sugar sensing of the PGI1 deletants was compared to the PGI1-wildtype strains in the presence of various types and combinations of sugars. RESULTS: Metabolomic analysis revealed systemic changes in intracellular sugar phosphate levels after deletion of PGI1, with the expected accumulation of intermediates upstream of the Pgi1p reaction on D-glucose and downstream intermediates on D-xylose. Moreover, the analysis revealed a preferential formation of D-fructose-6-phosphate from D-xylose, as opposed to the accumulation of D-fructose-1,6-bisphosphate that is normally observed when PGI1 deletants are incubated on D-fructose. This may indicate a role of PFK27 in D-xylose sensing and utilization. Overall, the sensing response was different for the PGI1 deletants, and responses to sugars that enter the glycolysis upstream of Pgi1p (D-glucose and D-galactose) were more affected than the response to those entering downstream of the reaction (D-fructose and D-xylose). Furthermore, the simultaneous exposure to sugars that entered upstream and downstream of Pgi1p (D-glucose with D-fructose, or D-glucose with D-xylose) resulted in apparent synergetic activation and deactivation of the Snf3p/Rgt2p and cAMP/PKA pathways, respectively. CONCLUSIONS: Overall, the sensing assays indicated that the previously observed D-xylose response stems from the formation of downstream metabolic intermediates. Furthermore, our results indicate that the metabolic node around Pgi1p and the level of D-fructose-6-phosphate could represent attractive engineering targets for improved D-xylose utilization.

GPI: An indicator for immune infiltrates and prognosis of human breast cancer from a comprehensive analysis.

Glucose-6-phosphate isomerase (GPI) plays an important part in gluconeogenesis and glycolysis through the interconversion of d-glucose-6-phosphate and d-fructose-6-phosphate, and its clinical significance still remains unclear in breast cancer (BRCA). We analyzed the expressions of GPI in BRCA patients to determine prognostic values. Our results showed that the expression levels of GPI were upregulated in BRCA patients, and a high GPI expression is correlated with poor overall survival (OS) in BRCA. At the same time, a high GPI expression is correlated with poor clinicopathological characteristics, such as stage III, over 60 years old, N3, HER2 negative, and estrogen receptor (ER) positive. Further analysis of the influence of GPI on the prognosis of BRCA suggested that 50 genes and 10 proteins were positively correlated with GPI, and these genes and proteins were mainly involved in cell cycle signaling pathways. In addition, in this study, we observed that GPI was closely related to N 6-methyladenosine (m6A) RNA methylation modification and immune cell infiltration and ferroptosis-related gene expression in BRCA, and there was a difference in m6A RNA methylation alterations, immune cell infiltration, and ferroptosis-related gene expression between the high GPI expression group and the low GPI expression group. Finally, we found that GPI in BRCA had 2.6% gene alterations, and BRCA patients with gene alteration of GPI had a poor prognosis in disease-free survival (DFS). Altogether, our work strongly suggested that GPI may serve as a new prognostic biomarker for BRCA patients.

A structural basis for the functional differences between the cytosolic and plastid phosphoglucose isomerase isozymes.

Phosphoglucose isomerase (PGI) catalyzes the interconversion between glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P), thereby regulating sucrose synthesis in plant cells. In general, plants contain a pair of PGI isozymes located in two distinct compartments of the cell (cytosol and plastid) with differences in both the primary structure and the higher-order structure. Previously, we showed that the activity of cytosolic PGI (PGIc) is more robust (activity, thermal stability, substrate turnover rate, etc.) than that of the plastid counterpart (PGIp) in multiple organisms, including wheat, rice, and Arabidopsis. The crystal structures of apoTaPGIc (an isotype cytosol PGIc in Triticum aestivum), TaPGIc-G6P complex, and apoTaPGIp (an isotype plastid PGIp in Triticum aestivum) were first solved in higher plants, especially in crops. In this study, we detailed the structural characteristics related to the biochemical properties and functions of TaPGIs in different plant organelles. We found that the C-terminal domains (CTDs) of TaPGIc and TaPGIp are very different, which affects the stability of the dimerized enzyme, and that Lys213TaPGIc/Lys193TaPGIp and its surrounding residues at the binding pocket gateway may participate in the entrance and exit of substrates. Our findings provide a good example illuminating the evolution of proteins from primary to higher structures as a result of physical barriers and adaptation to the biochemical environment.

Central Sugar Metabolism and the Cell Wall.

The human opportunistic pathogen Aspergillus fumigatus is recognized for its versatile cell wall when it comes to remodeling its components in adaptation to external threats, and this remodeling renders it refractory to antifungals targeting cell wall biosynthesis. A specific role for general sugar metabolism in the regulation of the synthesis of cell wall polymers has been previously demonstrated. Delving deeper into central sugar metabolism may reveal unexpected fundamental aspects in cell wall construction, as shown by the work of Zhou and coworkers (Y. Zhou, K. Yan, Q. Qin, O.G. Raimi, et al., mBio 13:e01426-22, 2022, https://doi.org/10.1128/mbio.01426-22) on the roles of the phosphoglucose isomerase of A. fumigatus in cell wall biosynthesis.