PKM2 functions as a histidine kinase to phosphorylate PGAM1 and increase glycolysis shunts in cancer.

Phosphoglycerate mutase 1 (PGAM1) is a key node enzyme that diverts the metabolic reactions from glycolysis into its shunts to support macromolecule biosynthesis for rapid and sustainable cell proliferation. It is prevalent that PGAM1 activity is upregulated in various tumors; however, the underlying mechanism remains unclear. Here, we unveil that pyruvate kinase M2 (PKM2) moonlights as a histidine kinase in a phosphoenolpyruvate (PEP)-dependent manner to catalyze PGAM1 H11 phosphorylation, that is essential for PGAM1 activity. Moreover, monomeric and dimeric but not tetrameric PKM2 are efficient to phosphorylate and activate PGAM1. In response to epidermal growth factor signaling, Src-catalyzed PGAM1 Y119 phosphorylation is a prerequisite for PKM2 binding and the subsequent PGAM1 H11 phosphorylation, which constitutes a discrepancy between tumor and normal cells. A PGAM1-derived pY119-containing cell-permeable peptide or Y119 mutation disrupts the interaction of PGAM1 with PKM2 and PGAM1 H11 phosphorylation, dampening the glycolysis shunts and tumor growth. Together, these results identify a function of PKM2 as a histidine kinase, and illustrate the importance of enzyme crosstalk as a regulatory mode during metabolic reprogramming and tumorigenesis.

Exercise-induced ß-hydroxybutyrate promotes Treg cell differentiation to ameliorate colitis in mice.

Increasing attention is being paid to the mechanistic investigation of exercise-associated chronic inflammatory disease improvement. Ulcerative colitis (UC) is one type of chronic inflammatory bowel disease with increasing incidence and prevalence worldwide. It is known that regular moderate aerobic exercise (RMAE) reduces the incidence or risk of UC, and attenuates disease progression in UC patients. However, the mechanisms of this RMAE's benefit are still under investigation. Here, we revealed that ß-hydroxybutyrate (ß-HB), a metabolite upon prolonged aerobic exercise, could contribute to RMAE preconditioning in retarding dextran sulfate sodium (DSS)-induced mouse colitis. When blocking ß-HB production, RMAE preconditioning-induced colitis amelioration was compromised, whereas supplementation of ß-HB significantly rescued impaired ß-HB production-associated defects. Meanwhile, we found that RMAE preconditioning significantly caused decreased colonic Th17/Treg ratio, which is considered to be important for colitis mitigation; and the downregulated Th17/Treg ratio was associated with ß-HB. We further demonstrated that ß-HB can directly promote the differentiation of Treg cell rather than inhibit Th17 cell generation. Furthermore, ß-HB increased forkhead box protein P3 (Foxp3) expression, the core transcriptional factor for Treg cell, by enhancing histone H3 acetylation in the promoter and conserved noncoding sequences of the Foxp3 locus. In addition, fatty acid oxidation, the key metabolic pathway required for Treg cell differentiation, was enhanced by ß-HB treatment. Lastly, administration of ß-HB without exercise significantly boosted colonic Treg cell and alleviated colitis in mice. Together, we unveiled a previously unappreciated role for exercise metabolite ß-HB in the promotion of Treg cell generation and RMAE preconditioning-associated colitis attenuation.

Cysteine and homocysteine can be exploited by GPX4 in ferroptosis inhibition independent of GSH synthesis.

Ferroptosis is inhibited by glutathione peroxidase 4 (GPX4), an antioxidant enzyme that uses reduced glutathione (GSH) as a cofactor to detoxify lipid hydroperoxides. As a selenoprotein, the core function of GPX4 is the thiol-dependent redox reaction. In addition to GSH, other small molecules such as cysteine and homocysteine also contain thiols; yet, whether GPX4 can exploit cysteine and homocysteine to directly detoxify lipid hydroperoxides and inhibit ferroptosis has not been addressed. In this study, we found that cysteine and homocysteine inhibit ferroptosis in a GPX4-dependent manner. However, cysteine inhibits ferroptosis independent of GSH synthesis, and homocysteine inhibits ferroptosis through non-cysteine and non-GSH pathway. Furthermore, we used molecular docking and GPX4 activity analysis to study the binding patterns and affinity between GPX4 and GSH, cysteine, and homocysteine. We found that besides GSH, cysteine and homocysteine are also able to serve as substrates for GPX4 though the affinities of GPX4 with cysteine and homocysteine are lower than that with GSH. Importantly, GPX family and the GSH synthetase pathway might be asynchronously evolved. When GSH synthetase is absent, for example in Flexibacter, the fGPX exhibits higher affinity with cysteine and homocysteine than GSH. Taken together, the present study provided the understanding of the role of thiol-dependent redox systems in protecting cells from ferroptosis and propose that GSH might be a substitute for cysteine or homocysteine to be used as a cofactor for GPX4 during the evolution of aerobic metabolism.

DNA repair byproduct 8-oxoguanine base promotes myoblast differentiation.

Muscle contraction increases the level of reactive oxygen species (ROS), which has been acknowledged as key signaling entities in muscle remodeling and to underlie the healthy adaptation of skeletal muscle. ROS inevitably endows damage to various cellular molecules including DNA. DNA damage ought to be repaired to ensure genome integrity; yet, how DNA repair byproducts affect muscle adaptation remains elusive. Here, we showed that exercise elicited the generation of 8-oxo-7,8-dihydroguanine (8-oxoG), that was primarily found in mitochondrial genome of myofibers. Upon exercise, TA muscle's 8-oxoG excision capacity markedly enhanced, and in the interstitial fluid of TA muscle from the post-exercise mice, the level of free 8-oxoG base was significantly increased. Addition of 8-oxoG to myoblasts triggered myogenic differentiation via activating Ras-MEK-MyoD signal axis. 8-Oxoguanine DNA glycosylase1 (OGG1) silencing from cells or Ogg1 KO from mice decreased Ras activation, ERK phosphorylation, MyoD transcriptional activation, myogenic regulatory factors gene (MRFs) expression. In reconstruction experiments, exogenously added 8-oxoG base enhanced the expression of MRFs and accelerated the recovery of the injured skeletal muscle. Collectively, these data not only suggest that DNA repair metabolite 8-oxoG function as a signal entity for muscle remodeling and contribute to exercise-induced adaptation of skeletal muscle, but also raised the potential for utilizing 8-oxoG in clinical treatment to skeletal muscle damage-related disorders.

Ginseng Pectin WGPA Alleviates Exercise-Induced Fatigue by Enhancing Gluconeogenesis.

With the development of medicine and sport science, growing attention has been paid to the recovery of exercise-induced fatigue. Ginseng pectin has been shown to be important for a variety of biological functions. Although many studies suggest that ginseng pectin plays an important role in the alleviation of exercise-induced fatigue, the underlying mechanism still remains unclear. In this study, C57BL/6J mice were subjected to a wheel apparatus for exhaustive exercise and fed with ginseng pectin WGPA (acidic fraction of water-soluble ginseng polysaccharides) afterwards. Subsequently, a series of physiological and biochemical indexes, such as blood lactic acid, blood glucose, muscle glycogen, insulin, and glucagon, is evaluated. Meanwhile, enzymatic activity and mRNA level of key enzymes involved in hepatic gluconeogenesis are analyzed. Our results demonstrate that the treatment of ginseng pectin WGPA can result in enhanced gluconeogenesis and decreased insulin and in turn facilitate the recovery of exercise-induced fatigue. In response to WGPA treatment, both phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6 phosphatase (G6Pase) activity were upregulated, indicating that these two enzymes play a critical role in WGPA-induced upregulation in gluconeogenesis. Moreover, mRNA level of G6Pase, but not PEPCK, was increased upon WGPA treatment, suggesting that G6Pase expression is regulated by WGPA. Importantly, the presence of WGPA downregulated insulin both in vivo and in vitro, suggesting the upregulation in gluconeogenesis may be due to alterations in insulin. Together, we provide evidence that ginseng pectin WGPA is able to alleviate exercise-induced fatigue by reducing insulin and enhancing gluconeogenesis.

Path Planning and Trajectory Tracking for Automatic Guided Vehicles.

Automated guided vehicle technology has become a hot area of scientific research due to its increasing use in manufacturing and logistics. Its main features are programming and control, remote computer eye tracking, command receiving and execution, autonomous route planning, and autonomous driving execution of tasks, with the advantages of high intelligence and flexibility. In this work, a simple vehicle model is used to study the route planning and tracking control of automatic guided vehicles. This paper uses wireless communication to find the optimal route planning problem. Using geometric methods, we develop a model of the working environment of the mobile automatic guided vehicle and develop a route finding algorithm. Based on the kinematic model, an advanced routing controller is designed to conduct experimental simulation of two trajectories and verify the effectiveness of the trajectory tracking controller. When the time is after 2 s, the position error is almost completely zero. In the path planning, when the number of iterations is greater than 10, the path length remains constant, verifying the effectiveness of the method in this paper.

ACLY ubiquitination by CUL3-KLHL25 induces the reprogramming of fatty acid metabolism to facilitate iTreg differentiation.

Inducible regulatory T (iTreg) cells play a central role in immune suppression. As iTreg cells are differentiated from activated T (Th0) cells, cell metabolism undergoes dramatic changes, including a shift from fatty acid synthesis (FAS) to fatty acid oxidation (FAO). Although the reprogramming in fatty acid metabolism is critical, the mechanism regulating this process during iTreg differentiation is still unclear. Here we have revealed that the enzymatic activity of ATP-citrate lyase (ACLY) declined significantly during iTreg differentiation upon transforming growth factor ß1 (TGFß1) stimulation. This reduction was due to CUL3-KLHL25-mediated ACLY ubiquitination and degradation. As a consequence, malonyl-CoA, a metabolic intermediate in FAS that is capable of inhibiting the rate-limiting enzyme in FAO, carnitine palmitoyltransferase 1 (CPT1), was decreased. Therefore, ACLY ubiquitination and degradation facilitate FAO and thereby iTreg differentiation. Together, we suggest TGFß1-CUL3-KLHL25-ACLY axis as an important means regulating iTreg differentiation and bring insights into the maintenance of immune homeostasis for the prevention of immune diseases.

Butyrate enhances CPT1A activity to promote fatty acid oxidation and iTreg differentiation.

Inducible regulatory T (iTreg) cells play a crucial role in immune suppression and are important for the maintenance of immune homeostasis. Mounting evidence has demonstrated connections between iTreg differentiation and metabolic reprogramming, especially rewiring in fatty acid oxidation (FAO). Previous work showed that butyrate, a specific type of short-chain fatty acid (SCFA) readily produced from fiber-rich diets through microbial fermentation, was critical for the maintenance of intestinal homeostasis and capable of promoting iTreg generation by up-regulating histone acetylation for gene expression as an HDAC inhibitor. Here, we revealed that butyrate could also accelerate FAO to facilitate iTreg differentiation. Moreover, butyrate was converted, by acyl-CoA synthetase short-chain family member 2 (ACSS2), into butyryl-CoA (BCoA), which up-regulated CPT1A activity through antagonizing the association of malonyl-CoA (MCoA), the best known metabolic intermediate inhibiting CPT1A, to promote FAO and thereby iTreg differentiation. Mutation of CPT1A at Arg243, a reported amino acid required for MCoA association, impaired both MCoA and BCoA binding, indicating that Arg243 is probably the responsible site for MCoA and BCoA association. Furthermore, blocking BCoA formation by ACSS2 inhibitor compromised butyrate-mediated iTreg generation and mitigation of mouse colitis. Together, we unveil a previously unappreciated role for butyrate in iTreg differentiation and illustrate butyrate-BCoA-CPT1A axis for the regulation of immune homeostasis.

Pyruvate Kinase M2 Promotes the Activation of Dendritic Cells by Enhancing IL-12p35 Expression.

Dendritic cells (DCs) play a central role in both innate and adaptive immunity. Emerging evidence has demonstrated metabolic reprogramming during DC activation. However, how DC activation is linked with metabolic reprogramming remains unclear. Here we show that pyruvate kinase M2 (PKM2), the rate-limiting enzyme in the last step of glycolysis, is critical for LPS-induced DC activation. Upon DC activation, JNK signaling stimulated p300 association with PKM2 for the acetylation of lysine 433, a classic posttranslational modification critical for PKM2 destabilization and nuclear re-localization. Subsequently, nuclear PKM2 partnered with c-Rel to enhance Il12p35 expression, which is important for Th1 cell differentiation. Meanwhile, decreased enzymatic activity of PKM2 due to detetramerization facilitated glycolysis and fatty acid synthesis, helping DCs meet their need for biomacromolecules. Together, we provide evidence for metabolic control of DC activation and offer insights into aberrant immune responses due to dysregulated Th1 functions.

Preventative delivery of IL-35 by Lactococcus lactis ameliorates DSS-induced colitis in mice.

Ulcerative colitis (UC) is one of the two major forms of inflammatory bowel disease (IBD) characterized by superficial mucosal inflammation, rectal bleeding, diarrhea, and abdominal pain. Anti-inflammatory and immunosuppressive drugs have been used in the therapy of human UC. Interleukin (IL)-35, which functions as an anti-inflammatory cytokine, has been shown to play a potential therapeutic role in a UC-like mouse colitis induced by dextran sodium sulfate (DSS). However, the contribution of IL-35 via oral administration to colitis prevention has not been determined. In order to explore its preventative potentiality, a dairy Lactococcus lactis NZ9000 strain was engineered to express murine IL-35 (NZ9000/IL-35), and this recombinant bacteria was applied to prevent and limit the development of DSS-induced mouse colitis. We found that oral administration of NZ9000/IL-35 induced the accumulation of IL-35 in the gut lumen of normal mice. When administrated preventatively, NZ9000/IL-35-gavaged mice exhibited decreased weight loss, DAI score, colon shortening as well as colitis-associated histopathological changes in colon, indicating that the oral administration of NZ9000/35 contributed to the suppression of DSS-induced colitis progression. Moreover, much less Th17 cells and higher level of Treg cells in lamina propria, as well as increased colon and serum levels of IL-10 with a concomitant reduced pro-inflammatory cytokines, IL-6, IL-17A, IFN-γ, and TNF-α were apparently regulated by NZ9000/IL-35 in colitis mice. Together, we put forward direct evidence pinpointing the effectiveness of NZ9000/IL-35 in preventing UC-like mouse colitis, implying a potential candidate of this recombinant Lactococcus lactis that prevent the progression of IBD.

Abrogation of Lupus Nephritis in Somatic Hypermutation-Deficient MRL/lpr Mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease posing threats to multiple organs in the human body. As a typical manifestation of SLE, lupus nephritis is characterized by a series of pathological changes in glomerulus as well as accumulation of pathogenic autoreactive IgG with complement in the kidney that dramatically disrupts renal functions. Activation-induced deaminase (AID), which governs both somatic hypermutation (SHM) and class-switch recombination (CSR), has been shown to be essential for the regulation of SLE. However, the relative contributions of SHM and CSR to SLE pathology have not been determined. Based on the available AIDG23S mice, we successfully established an AIDG23S MRL/lpr mouse model, in which SHM is specifically abolished, although CSR is largely unaffected. We found that the abrogation of SHM effectively alleviated SLE-associated histopathological alterations, such as expansion of the mesangial matrix and thickening of the basement membrane of Bowman's capsule as well as infiltration of inflammatory cells. Compared with SLE mice, AIDG23S MRL/lpr mice exhibited decreased proteinuria, blood urea nitrogen, and creatinine, indicating that the loss of SHM contributed to the recovery of renal functions. As a consequence, the life span of those SHM-deficient MRL/lpr mice was extended. Together, we provide direct evidence pinpointing a vital role of SHM in the control of SLE development.

O-GlcNAcylation destabilizes the active tetrameric PKM2 to promote the Warburg effect.

The Warburg effect, characterized by increased glucose uptake and lactate production, is a well-known universal across cancer cells and other proliferating cells. PKM2, a splice isoform of the pyruvate kinase (PK) specifically expressed in these cells, serves as a major regulator of this metabolic reprogramming with an adjustable activity subjected to numerous allosteric effectors and posttranslational modifications. Here, we have identified a posttranslational modification on PKM2, O-GlcNAcylation, which specifically targets Thr405 and Ser406, residues of the region encoded by the alternatively spliced exon 10 in cancer cells. We show that PKM2 O-GlcNAcylation is up-regulated in various types of human tumor cells and patient tumor tissues. The modification destabilized the active tetrameric PKM2, reduced PK activity, and led to nuclear translocation of PKM2. We also observed that the modification was associated with an increased glucose consumption and lactate production and enhanced level of lipid and DNA synthesis, indicating that O-GlcNAcylation promotes the Warburg effect. In vivo experiments showed that blocking PKM2 O-GlcNAcylation attenuated tumor growth. Thus, we demonstrate that O-GlcNAcylation is a regulatory mechanism for PKM2 in cancer cells and serves as a bridge between PKM2 and metabolic reprogramming typical of the Warburg effect.

Studies on construction of a recombinant Eimeria tenella SO7 gene expressing Escherichia coli and its protective efficacy against homologous infection.

Eimeria spp. are the causative agents of coccidiosis, a major disease affecting the poultry industry. A recombinant non-antibiotic Escherichia coli that expresses the Eimeria tenella SO7 gene was constructed and its protective efficacy against homologous infection in chickens was determined. The three-day-old chickens were orally immunized with the recombinant non-antibiotic SO7 gene expressing E. coli and boosted two weeks later. Four weeks after the second immunization, the chickens were challenged with 5 × 10(4) homologous sporulated oocysts. The protective effects of the recombinant non-antibiotic E. coli were determined by measuring body weight change, mortality, histopathology, lesion scores, oocyst counts, the specific antibody response and the frequency of CD4(+) and CD8(+) lymphocytes in peripheral blood. The results showed that immunization with SO7 expressing E. coli resulted in significantly improved body weight gain, reduced lesion scores and oocyst shedding in immunized chickens compared to controls. Furthermore, administration of recombinant SO7 expressing E. coli leads to a significant increase in serum antibody, CD4(+) and CD8(+) T cells in peripheral blood of chickens. These results, therefore, suggest that the recombinant non-antibiotic E. coli that expresses the SO7 gene is able to effectively stimulate host protective immunity as evidenced by the induction of development of both humoral and cell-mediated immune responses against homologous challenge in chickens.