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ABSTRACT: Chronic heat stress has detrimental effects on the growth performance of broilers, and the potential mechanism is under exploration. In this study, the protein carbonyl modification was introduced to glycolytic enzymes to evaluate its relationship with the growth performance of heat-stressed (HS) broilers. A total of 144 male 28-day-old broilers were assigned to 3 treatments: the normal control group (NC, raised at 22°C with free access to feed and water), the HS group (raised at 32°C with free access to feed and water), and the pair-fed group (PF, raised at 22°C with an amount of feed equal to that consumed by the HS group on a previous day). Results showed that heat stress decreased the average daily growth, increased the feed-to-gain ratio (F/G), decreased breast muscle rate, and increased abdominal fat rate compared with the NC and PF groups (P < 0.05). Higher cloacal temperature and serum creatine kinase activity were found in the HS group than those of the NC and PF groups (P < 0.05). Heat stress increased the contents of carbonyl, advanced glycation end-products, malonaldehyde, and the activities of catalase, glutathione peroxidase, and total antioxidant capacity compared with the NC and PF groups (P < 0.05). Heat stress increased the contents of glucose and lactate, declined the glycogen content, and lowered the relative protein expressions of pyruvate kinase muscle type, lactate dehydrogenase A type (LDHA), and citrate synthase compared to those of the NC group (P < 0.05). In contrast to the NC and PF groups, heat stress intensified the carbonylation levels of phosphoglucomutase 1, triosephosphate isomerase 1, β-enolase, and LDHA, which were positively correlated with the F/G (P < 0.05). These findings demonstrate that heat stress depresses growth performance on account of oxidative stress and glycolysis disorders. It further increases the carbonylation of glycolytic enzymes, which potentially correlates with the F/G by disturbing the mode of energy supply of broilers.

RGG-motif proteins play a crucial role in determining mRNA fate. Suppressor of clathrin deficiency 6 (Scd6) is a conserved RGG-motif containing RNP condensate-resident, translation repressor, and decapping activator protein in Saccharomyces cerevisiae. Identifying protein factors that can modulate Scd6 function is critical to understanding the regulation of mRNA fate by Scd6. In this study, using an approach that combined mRNA tethering assay with flow cytometry, we screened 50 genes for their role in modulating the translation repression activity of Scd6. We identified eight conserved modulators with human homologs. Of these, we further characterised in detail guanine nucleotide exchange factor (GEF) Rom2 (Rho1 multicopy suppressor) and glycolytic enzyme Tdh3 (Triose phosphate dehydrogenase 3), which, respectively, impede and promote translation repression activity of Scd6. Our study reveals that Rom2 negatively regulates the arginine methylation of Scd6 and antagonises its localisation to P-bodies. Tdh3, on the other hand, promotes Scd6 interaction with Hmt1, thereby promoting the arginine methylation of Scd6 and enhanced eIF4G1 interaction, which is known to promote its repression activity. Identifying these novel modulators provides exciting new insights into the role of a metabolic enzyme of the glycolytic pathway and guanine nucleotide exchange factor implicated in the cell wall integrity pathway in regulating Scd6 function and, thereby, cytoplasmic mRNA fate., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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Melanoma cells exhibit increased aerobic glycolysis, which represents a major biochemical alteration associated with malignant transformation; thus, glycolytic enzymes could be exploited to selectively target cancer cells in cancer therapy. Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) switches glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate by coupling with the reduction of NAD+ to NADH. Here, we demonstrated that GAPDHS displays significantly higher expression in uveal melanoma (UM) than in normal controls. Functionally, the knockdown of GAPDHS in UM cell lines hindered glycolysis by decreasing glucose uptake, lactate production, adenosine triphosphate (ATP) generation, cell growth and proliferation; conversely, overexpression of GAPDHS promoted glycolysis, cell growth and proliferation. Furthermore, we identified that SOX10 knockdown reduced the activation of GAPDHS, leading to an attenuated malignant phenotype, and that SOX10 overexpression promoted the activation of GAPDHS, leading to an enhanced malignant phenotype. Mechanistically, SOX10 exerted its function by binding to the promoter of GAPDHS to regulate its expression. Importantly, SOX10 abrogation suppressed in vivo tumor growth and proliferation. Collectively, the results reveal that GAPDHS, which is regulated by SOX10, controls glycolysis and contributes to UM tumorigenesis, highlighting its potential as a therapeutic target.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyzing the sixth step of glycolysis has been investigated for allosteric features that might be used as potential target for specific inhibition of Staphylococcus aureus (S.aureus). X-ray structure of bacterial enzyme for which a tunnel-like opening passing through the center previously proposed as an allosteric site has been subjected to six independent 500 ns long Molecular Dynamics simulations. Harmonic bond restraints were employed at key residues to underline the allosteric feature of this region. A noticeable reduction was observed in the mobility of NAD+ binding domains when restrictions were applied. Also, a substantial decrease in cross-correlations between distant Cα fluctuations was detected throughout the structure. Mutual information (MI) analysis revealed a similar decrease in the degree of correspondence in positional fluctuations in all directions everywhere in the receptor. MI between backbone and side-chain torsional variations changed its distribution profile and decreased considerably around the catalytic sites when restraints were employed. Principal component analysis clearly showed that the restrained state sampled a narrower range of conformations than apo state, especially in the first principal mode due to restriction in the conformational flexibility of NAD+ binding domain. Clustering the trajectory based on catalytic site residues displayed a smaller repertoire of conformations for restrained state compared to apo. Representative snapshots subjected to k-shortest pathway analysis revealed the impact of bond restraints on the allosteric communication which displayed distinct optimal and suboptimal pathways for two states, where observed frequencies of critical residues Gln51 and Val283 at the proposed site changed considerably. [Display omitted] • Allosteric potential of a proposed site in Sa GAPDH was explored via bond restraints. • Reduced mobility and change in communication pathways upon perturbation. • Significant decrease in mutual correspondence upon perturbation of the allosteric site. • Restricted conformational space when restraints were employed. • Perturbation of the predicted site changed the dynamic feature of the receptor. [ABSTRACT FROM AUTHOR]

Three allosteric glycolytic enzymes, phosphofructokinase, glyceraldehyde-3 phosphate dehydrogenase and pyruvate kinase, associated with bacterial, parasitic and human species, were explored to identify potential allosteric sites that would be used as prime targets for species-specific drug design purposes using a newly developed approach which incorporates solvent mapping, elastic network modeling, sequence and structural alignments. The majority of binding sites detected by solvent mapping overlapped with the interface regions connecting the subunits, thus appeared as promising target sites for allosteric regulation. Each binding site was then evaluated by its ability to alter the global dynamics of the receptor defined by the percentage change in the frequencies of the lowest-frequency modes most significantly and as anticipated, the most effective ones were detected in the vicinity of the well-reported catalytic and allosteric sites. Furthermore, some of our proposed regions intersected with experimentally resolved sites which are known to be critical for activity regulation, which further validated our approach. Despite the high degree of structural conservation encountered between bacterial/parasitic and human glycolytic enzymes, the majority of the newly presented allosteric sites exhibited a low degree of sequence conservation which further increased their likelihood to be used as species-specific target regions for drug design studies.

Objective This study investigated the effects of different dietary energy sources on early postmortem muscle metabolism of finishing pigs. Methods Seventy-two barrow (Duroc×Landrace×Yorkshire, DLY) pigs (65.0±2.0 kg) were allotted to three iso-energetic and iso-nitrogenous diets: A (44.1% starch, 5.9% crude fat, and 12.6% neutral detergent fibre [NDF]), B (37.6% starch, 9.5% crude fat, and 15.4% NDF) or C (30.9% starch, 14.3% crude fat, and 17.8% NDF). After the duration of 28-day feeding experiment, 24 pigs (eight per treatment) were slaughtered and the M. longissimus lumborum (LL) samples at 45 min postmortem were collected. Results Compared with diet A, diet C resulted in greater adenosine triphosphate and decreased phosphocreatine (PCr) concentrations, greater activity of creatine kinase and reduced percentage bound activities of hexokinase (HK), and pyruvate kinase (PK) in LL muscles (p

Chronic heat stress has detrimental effects on the growth performance of broilers, and the potential mechanism is under exploration. In this study, the protein carbonyl modification was introduced to glycolytic enzymes to evaluate its relationship with the growth performance of heat-stressed (HS) broilers. A total of 144 male 28-day-old broilers were assigned to 3 treatments: the normal control group (NC , raised at 22°C with free access to feed and water), the HS group (raised at 32°C with free access to feed and water), and the pair-fed group (PF , raised at 22°C with an amount of feed equal to that consumed by the HS group on a previous day). Results showed that heat stress decreased the average daily growth, increased the feed-to-gain ratio (F/G), decreased breast muscle rate, and increased abdominal fat rate compared with the NC and PF groups (P < 0.05). Higher cloacal temperature and serum creatine kinase activity were found in the HS group than those of the NC and PF groups (P < 0.05). Heat stress increased the contents of carbonyl, advanced glycation end-products, malonaldehyde, and the activities of catalase, glutathione peroxidase, and total antioxidant capacity compared with the NC and PF groups (P < 0.05). Heat stress increased the contents of glucose and lactate, declined the glycogen content, and lowered the relative protein expressions of pyruvate kinase muscle type, lactate dehydrogenase A type (LDHA), and citrate synthase compared to those of the NC group (P < 0.05). In contrast to the NC and PF groups, heat stress intensified the carbonylation levels of phosphoglucomutase 1, triosephosphate isomerase 1, β-enolase, and LDHA, which were positively correlated with the F/G (P < 0.05). These findings demonstrate that heat stress depresses growth performance on account of oxidative stress and glycolysis disorders. It further increases the carbonylation of glycolytic enzymes, which potentially correlates with the F/G by disturbing the mode of energy supply of broilers. [ABSTRACT FROM AUTHOR]

Identification of cues originating from skeletal muscle that govern bone formation is essential for understanding the crosstalk between muscle and bone and for developing therapies for degenerative bone diseases. Here, we identified that skeletal muscle secreted multiple extracellular vesicles (Mu-EVs). These Mu-EVs traveled through the bloodstream to reach bone, where they were phagocytized by bone marrow mesenchymal stem/stromal cells (BMSCs). Mu-EVs promoted osteogenic differentiation of BMSCs and protected against disuse osteoporosis in mice. The quantity and bioactivity of Mu-EVs were tightly correlated with the function of skeletal muscle. Proteomic analysis revealed numerous proteins in Mu-EVs, some potentially regulating bone metabolism, especially glycolysis. Subsequent investigations indicated that Mu-EVs promoted the glycolysis of BMSCs by delivering lactate dehydrogenase A into these cells. In summary, these findings reveal that Mu-EVs play a vital role in BMSC metabolism regulation and bone formation stimulation, offering a promising approach for treating disuse osteoporosis. [Display omitted] • Skeletal muscle secretes Mu-EVs, which can travel via blood vessels to bone • Supplementation with Mu-EVs alleviates disuse osteoporosis after muscle atrophy • Mu-EVs promote osteogenic differentiation of BMSCs by enhancing glycolysis • Glycolic profiles transported by Mu-EVs function as non-canonical myokines In their study, Ma et al. demonstrate that skeletal muscle secretes abundant extracellular vesicles (Mu-EVs). These Mu-EVs promote glycolysis in BMSCs by delivering glycolic profiles to enhance bone formation. The quantity and bioactivity of Mu-EVs are found to correlate with skeletal muscle function. Importantly, supplementation with Mu-EVs is shown to alleviate disuse osteoporosis following muscle atrophy, suggesting a potential therapeutic strategy. [ABSTRACT FROM AUTHOR]

Treponema pallidum (T. pallidum), an obligate extracellular bacterium, is the causative agent of sexually transmitted bacterial diseases. In this study, the glycolytic enzyme enolase (Tp Eno) of T. pallidum were injected intramuscularly into C57BL/6 mice, resulting in higher levels of specific anti -Tp Eno antibodies and Tp Eno-specific splenocyte proliferation than those in the mice immunized with recombinant protein Tp Eno. Cytokine (IL-4, IL-6, IL-10, IFN-γ, and TNF-α) analysis of splenocytes showed that the Tp Eno could slightly trigger the Th1-biased immune response. Furthermore, immunization of mice with Tp Eno elicited a significant production of IFN-γ by CD4+ T-cells in the spleen. Subsequently, mice were inoculated intradermally (between the scapulae), intraperitoneally, intrarectally and via the corpora cavernosa with 2.5 × 106 organisms per site (1 × 107 total organisms). The bacterial organ burden detected in the blood, spleen, liver, testes or brain of immunized mice suggested that Tp Eno enhances protective immunity to inhibit T. pallidum colonization in distal tissues. Therefore, Tp Eno vaccination enhances Tp Eno-specific immunogenicity and provides protection against T. pallidum dissemination. • The C57BL/6 mice was attempted to evaluate immunogenicity and protection of Tp Eno against T. pallidum infection. • Tp Eno represents a promising new vaccine candidate that can stop the spread of T. pallidum. • Spirochetes are present in local sites of inoculation and disseminate to other tissues after 30 days post-inoculation in mice. [ABSTRACT FROM AUTHOR]

The use of mixed sugars containing glucose and xylose in lignocellulosic biomass is desirable for the microbial production of chemicals and fuels. We investigated the effect of individual or simultaneous overexpression of glycolytic genes on d -lactate production from a mixture of glucose and xylose by a recombinant xylose-assimilating Corynebacterium glutamicum strain. The individual overexpression of genes encoding phosphofructokinase (PFK) and triosephosphate isomerase (TPI) increased d -lactate production rate by 71% and 34%, respectively, with corresponding increases (2.4- and 1.8-fold) in the glucose consumption; however, the amount of xylose consumed not altered. d -Lactate yield was also increased by 5.5%, but only in the strain overexpressing the gene encoding PFK. In the parent strain and the strains overexpressing the genes encoding PFK or TPI, a reduction in d -lactate production occurred at approximately 900 mM after 32 h. However, the strain that simultaneously overexpressed the genes encoding PFK and TPI continued to produce d -lactate after 32 h, with the eventual production of 1326 mM after production for 80 h in mineral salts medium. Our findings contribute to the cost-effective, large-scale production of d -lactate from mixed sugars. Highlights • d -Lactate was produced from mixed sugars by metabolically engineered Corynebacterium glutamicum. • Overexpressing phosphofructokinase and triosephosphate isomerase genes was critical for high d -lactate production from mixed sugars. • The highest titer of d -lactate in this study reached 1326 mM (119.3 g/L) from a mixture of glucose and xylose. [ABSTRACT FROM AUTHOR]

Abstract Female Aedes aegypti mosquitoes are vectors of arboviruses that cause diseases of public health significance. The discovery of new metabolic targets is crucial for improving mosquito control strategies. We recently demonstrated that glucose oxidation supports ammonia detoxification in A. aegypti. Pyruvate kinase (PK, EC 2.7.1.40) catalyzes the last step of the glycolytic pathway. In most organisms, one or more allosteric effectors control PK activity. However, the kinetic properties and structure of PK in mosquitoes have not been previously reported. In this study, two alternatively spliced mRNA variants (AaPK1 and AaPK2) that code for PKs were identified in the A. aegypti genome. The AaPK1 mRNA variant, which encodes a 529 amino acid protein with an estimated molecular weight of ∼57 kDa, was cloned. The protein was expressed in Escherichia coli and purified. The AaPK1 kinetic properties were identified. The recombinant protein was also crystallized and its 3D structure determined. We found that alanine, glutamine, proline, serine and fructose-1-phosphate displayed a classic allosteric activation on AaPK1. Ribulose-5-phosphate acted as an allosteric inhibitor of AaPK1 but its inhibitory effect was reversed by alanine, glutamine, proline and serine. Additionally, the allosteric activation of AaPK1 by amino acids was weakened by fructose-1,6-bisphosphate, whereas the allosteric activation of AaPK1 by alanine and serine was diminished by glucose-6-phosphate. The AaPK1 structure shows the presence of fructose-1,6-bisphosphate in the allosteric site. Together, our results reveal that specific amino acids and phosphorylated sugars tightly regulate conformational dynamics and catalytic changes of AaPK1. The distinctive AaPK1 allosteric properties support a complex role for this enzyme within mosquito metabolism. Graphical abstract Image 1 Highlights • The three-dimensional structure of recombinant pyruvate kinase 1 from Aedes aegypti (AaPK1) was determined by x-ray crystallography in complex with fructose-1,6-biphosphate. • Fructose-1-phosphate and specific amino acids are allosteric activators of AaPK1. • The allosteric inhibitory effect of ribulose-5-phosphate on AaPK1 was modulated by several amino acids. • Fructose-1,6-bisphosphate binding alone or in combination with amino acids increases the melting temperature of AaPK1. • The binding of fructose-1,6-bisphosphate alone and in combination with specific amino acids increases the melting temperature of AaPK1. [ABSTRACT FROM AUTHOR]

Protein phosphorylation is a key modulator in glycolysis metabolism and regulates the activity of glycometabolic enzymes. The phosphorylation levels of sarcoplasmic proteins were investigated in relationship to the glycolysis rate in postmortem ovine muscle in the present study. The global phosphorylation levels of sarcoplasmic proteins increased early postmortem and then decreased afterwards in postmortem ovine muscle. Protein bands of different phosphorylation levels were identified as glycometabolism‐related enzymes. The phosphorylation levels of Glycogen phosphorylase and Pyruvate kinase maybe one possible reason for the different glycolytic rates at 0.5 h postmortem. The glycolytic rate attributes were negatively correlated with four bands which were probably phosphofructokinase, enolase and adenylate kinase isoenzyme. In summary, the phosphorylation of sarcoplasmic proteins is related to muscle pH decline early postmortem, influencing the meat rigour mortis and quality development. Quantification of global phosphorylation levels of sarcoplasmic proteins. [ABSTRACT FROM AUTHOR]

Trichomonas vaginalis is the protist parasite that causes the most common, non-viral sexually transmitted infection called trichomonosis. Enolase is a moonlighting protein that apart from its canonical function as a glycolytic enzyme, serves as a plasminogen receptor on the cell surface of T. vaginalis and, in consequence, it has been stablished as a virulence factor in this parasite. In the Trichomonas vaginalis sequence database there are nine genes annotated as enolase. In this work, we analyzed these genes as well as their products. We found that seven out of nine genes might indeed perform enolase activity, whereas two genes might have been equivocally identified, or they might be pseudogenes. Furthermore, a combination of qRT-PCR and proteomic approaches was used to assess, for the first time, the expression of these genes in the highly virulent mexican isolate of T. vaginalis CNCD-147 at different iron concentrations. We could find peptides corresponding to enolases encoded by genes TVAG_464170, TVAG_043500 and TVAG_329460. Moreover, we identified two distinctive characteristics within enolases from Trichomonas vaginalis. One of them corresponds to three key substitutions within one of the loops of the active site, compared to host enolase. The other, is a unique N-terminal motif, composed of 15 to 18 residues, on all the potentially active enolases, whose function still has to be stablished. Both differential features merit further studies as potential drug and vaccine targets as well as diagnosis markers. These findings offer new possibilities to fight trichomonosis. [ABSTRACT FROM AUTHOR]

A quantitative analysis of protein phosphorylation in ovine LTL muscle with different color stability was performed in the present study using TMT labeling in combination with TiO 2 phosphopeptide enrichment. A total of 3412 phosphopeptides assigned to 1070 phosphoproteins were identified by mass spectrometry, of which 243 proteins were detected to be differentially phosphorylated between muscles of different color stability. Among these differentially phosphorylated proteins, 27 phosphoproteins were identified to be key color-related proteins by informatics analysis. Proteins involved in carbohydrate metabolism, especially glycolytic enzymes, were the largest cluster of protein determined to be color-related. In addition, the phosphorylation of myoglobin at Ser133 plays a negative role in the regulation of meat color stability. In summary, this study revealed that the phosphorylation of some glycolytic enzymes and myoglobin at specific serine residues may play critical roles in the regulation of meat color stability. [ABSTRACT FROM AUTHOR]

The phosphorylation of sarcoplasmic proteins in postmortem muscles was investigated in relationship to color stability in the present study. Although no difference was observed in the global phosphorylation level of sarcoplasmic proteins, difference was determined in the phosphorylation levels of individual protein bands from muscles with different color stability. Correlation analysis and liquid chromatography – tandem mass spectrometry (LC-MS/MS) identification of phosphoproteins showed that most of the color stability-related proteins were glycolytic enzymes. Interestingly, the phosphorylation level of myoglobin was inversely related to meat color stability. As the phosphorylation of myoglobin increased, color stability based on a ∗ value decreased and metMb content increased. In summary, the study revealed that protein phosphorylation might play a role in the regulation of meat color stability probably by regulating glycolysis and the redox stability of myoglobin, which might be affected by the phosphorylation of myoglobin. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the effects of different dietary starch types on early postmortem muscle energy metabolism in finishing pigs. Ninety barrows (68.0 ± 2.0 kg) were randomly allotted to three experimental diets with five replicates of six pigs, containing pure waxy maize starch (WMS), nonwaxy maize starch (NMS), and pea starch (PS) (amylose/amylopectin were 0.07, 0.19 and 0.28 respectively). Compared with the WMS diet, pigs fed the PS diet exhibited greater creatine kinase activity, higher adenosine triphosphate and adenosine diphosphate contents, lower phosphocreatine (PCr), adenosine monophosphate and glycogen contents, and lower glycolytic potential ( P < 0.05). Moreover, the PS diet led to reduced percentage of bound hexokinase activity, decreased level of phosphorylated AKT ( P < 0.05) and increased level of hypoxia-inducible factor-1α ( P < 0.05). In conclusion, diet with high amylose content might promote PCr degradation and inhibit the rate of glycolysis, followed by attenuation of early postmortem glycolysis in finishing pigs. [ABSTRACT FROM AUTHOR]

The non-starch polysaccharides (NSPs) of two jelly-fig cultivars were extracted to compare their effects on the delay of glucose release and in vitro starch digestion with those of commercial agar. The ability of jelly-fig NSPs to retard glucose diffusion was investigated. The NSPs of both jelly-fig cultivars regulated glucose absorption. The Hongjiu cultivar had the highest rate of α-amylase inhibition (63.17 ± 0.04%). Jelly-fig NSPs reduced amylose leaching by 19–32%. The Hongjiu cultivar showed stronger cholesterol absorption ability compared with other NSPs. In vitro starch digestion indicated that jelly-fig NSPs were beneficial to the retardation of glucose release. The viscosity of jelly-fig NSPs was similar to those in in vitro starch digestion, indicating that the effects of jelly-fig on glucose release were strongly associated with viscosity. Scanning electron microscopy (SEM) indicated that jelly-fig NSP-treated starch granules were densely packed with smooth surfaces. The Hongjiu cultivar starch granules (34.93 μm) were substantially smaller than those of the control group (115 μm), indicating that starch granule gelatinization and swelling were attenuated. The NSPs of Hongjiu cultivar demonstrate potential in retarding glucose release in combination with starch sources to lower their glycemic index. This provides a novel natural source for blood glucose-controlling component in food products. • The jelly fig NSPs possess the potential for retarding glucose release. • The jelly fig NSPs exhibited regulation of starch degradation. • The jelly fig NSPs showed stronger cholesterol absorption ability. • Viscosity was a major contributing factor in retarding glucose release. [ABSTRACT FROM AUTHOR]

Triose phosphate isomerase (TPI) deficiency is a rare, but highly debilitating, inherited metabolic disease. Almost all patients suffer severe neurological effects and the most severely affected are unlikely to live beyond early childhood. Here, we describe an in silico study into well-characterised variants which are associated with the disease alongside an investigation into 79 currently uncharacterised TPI variants which are known to occur in the human population. The majority of the disease-associated mutations affected amino acid residues close to the dimer interface or the active site. However, the location of the altered amino acid residue did not predict the severity of the resulting disease. Prediction of the effect on protein stability using a range of different programs suggested a relationship between the degree of instability caused by the sequence variation and the severity of the resulting disease. Disease-associated variations tended to affect well-conserved residues in the protein's sequence. However, the degree of conservation of the residue was not predictive of disease severity. The majority of the 79 uncharacterised variants are potentially associated with disease since they were predicted to destabilise the protein and often occur in well-conserved residues. We predict that individuals homozygous for the corresponding mutations would be likely to suffer from TPI deficiency. [ABSTRACT FROM AUTHOR]

The characterisation of the gene encoding Trypanosoma cruzi CL Brener phosphofructokinase (PFK) and the biochemical properties of the expressed enzyme are reported here. In contradiction with previous reports, the PFK genes of CL Brener and YBM strain T. cruzi were found to be similar to their Leishmania mexicana and Trypanosoma brucei homologs in terms of both kinetic properties and size, with open reading frames encoding polypeptides with a deduced molecular mass of 53,483. The predicted amino acid sequence contains the C-terminal glycosome-targeting tripeptide SKL; this localisation was confirmed by immunofluorescence assays. In sequence comparisons with the genes of other eukaryotes, it was found that, despite being an adenosine triphosphate-dependent enzyme, T. cruzi PFK shows significant sequence similarity with inorganic pyrophosphate-dependent PFKs.

Glycolytic enzymes, such as enolase, have been described as multifunctional complex proteins that also display non-glycolytic activities, termed moonlighting functions. Although enolase multifunctionality has been described for several organisms, the conservation of enolase alternative functions through different phyla has not been explored with more details. A useful strategy to investigate moonlighting functions is the use of systems biology tools, which allow the prediction of protein functions/interactions by graph design and analysis. In this work, available information from protein–protein interaction (PPI) databases were used to design enolase PPI networks for four eukaryotic organisms, namely Homo sapiens, Drosophila melanogaster , Caenorhabditis elegans , and Saccharomyces cerevisiae , covering a wide spectrum of this domain of life. PPI networks with number of nodes ranging from 140 to 411 and up to 15,855 connections were generated, and modularity and centrality analyses, and functional enrichment were performed for all of them. The performed analyses showed that enolase is a central node within the networks, and that, in addition to its canonical interactions with proteins related to glycolysis and energetic metabolism, it is also part of protein clusters related to different biological processes, like transcription, development, and apoptosis, among others. Some of these non-glycolytic clusters, are partially conserved between networks, in terms of overall sharing of orthologs, overall cluster structure, and/or at the levels of key regulatory proteins within clusters. Overall, our results provided evidences of enolase multifunctionality and evolutionary conservation of enolase PPIs at all these levels. [ABSTRACT FROM AUTHOR]

We previously reported on the impacts of the overexpression of individual genes of the glycolytic pathway encoding glucokinase (GLK), glyceraldehyde phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), triosephosphate isomerase (TPI), and bisphosphate aldolase (FBA) on D-lactate productivity in Corynebacterium glutamicum under oxygen-deprived conditions. Searching for synergies, in the current study, we simultaneously overexpressed the five glycolytic genes in a stepwise fashion to evaluate the effect of the cumulative overexpression of glycolytic genes on D-lactate production. Interestingly, the final D-lactate concentration markedly differed depending on whether or not the PFK encoding gene was overexpressed when combined with overexpressing other glycolytic genes. The simultaneous overexpression of the GLK, GAPDH, TPI, and FBA encoding genes led to the highest initial D-lactate concentration at 10 h. However, this particular recombinant strain dramatically slowed producing D-lactate when a concentration of 1300 mM was reached, typically after 32 h. In contrast, the strain overexpressing the PFK encoding gene together with the GLK, GAPDH, TPI, and FBA encoding genes showed 12.7 % lower initial D-lactate concentration at 10 h than that observed with the strain overexpressing the genes coding for GLK, GAPDH, TPI, and FBA. However, this recombinant strain continued to produce D-lactate after 32 h, reaching 2169 mM after a mineral salts medium bioprocess incubation period of 80 h. These results suggest that overexpression of the PFK encoding gene is essential for achieving high production of D-lactate. Our findings provide interesting options to explore for using C. glutamicum for cost-efficient production of D-lactate at the industrial scale. [ABSTRACT FROM AUTHOR]

The sarcoplasmic proteome of beef Longissimus lumborum demonstrating animal-to-animal variation in color stability was examined to correlate proteome profile with color. Longissimus lumborum (36 h post-mortem) muscles were obtained from 73 beef carcasses, aged for 13 days, and fabricated to 2.5-cm steaks. One steak was allotted to retail display, and another was immediately vacuum packaged and frozen at − 80 °C. Aerobically packaged steaks were stored under display, and color was evaluated on days 0 and 11. The steaks were ranked based on redness and color stability on day 11, and ten color-stable and ten color-labile carcasses were identified. Sarcoplasmic proteome of frozen steaks from the selected carcasses was analyzed. Nine proteins were differentially abundant in color-stable and color-labile steaks. Three glycolytic enzymes (phosphoglucomutase-1, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase M2) were over-abundant in color-stable steaks and positively correlated ( P < 0.05) to redness and color stability. These results indicated that animal variations in proteome contribute to differences in beef color. [ABSTRACT FROM AUTHOR]