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1.
Mil Med Res ; 9(1): 22, 2022 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-35596191

RESUMO

BACKGROUND: Sepsis involves life-threatening organ dysfunction and is caused by a dysregulated host response to infection. No specific therapies against sepsis have been reported. Celastrol (Cel) is a natural anti-inflammatory compound that shows potential against systemic inflammatory diseases. This study aimed to investigate the pharmacological activity and molecular mechanism of Cel in models of endotoxemia and sepsis. METHODS: We evaluated the anti-inflammatory efficacy of Cel against endotoxemia and sepsis in mice and macrophage cultures treated with lipopolysaccharide (LPS). We screened for potential protein targets of Cel using activity-based protein profiling (ABPP). Potential targets were validated using biophysical methods such as cellular thermal shift assays (CETSA) and surface plasmon resonance (SPR). Residues involved in Cel binding to target proteins were identified through point mutagenesis, and the functional effects of such binding were explored through gene knockdown. RESULTS: Cel protected mice from lethal endotoxemia and improved their survival with sepsis, and it significantly decreased the levels of pro-inflammatory cytokines in mice and macrophages treated with LPS (P < 0.05). Cel bound to Cys424 of pyruvate kinase M2 (PKM2), inhibiting the enzyme and thereby suppressing aerobic glycolysis (Warburg effect). Cel also bound to Cys106 in high mobility group box 1 (HMGB1) protein, reducing the secretion of inflammatory cytokine interleukin (IL)-1ß. Cel bound to the Cys residues in lactate dehydrogenase A (LDHA). CONCLUSION: Cel inhibits inflammation and the Warburg effect in sepsis via targeting PKM2 and HMGB1 protein.


Assuntos
Endotoxemia , Proteína HMGB1 , Sepse , Animais , Anti-Inflamatórios/uso terapêutico , Citocinas/uso terapêutico , Endotoxemia/tratamento farmacológico , Proteína HMGB1/metabolismo , Proteína HMGB1/uso terapêutico , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Lipopolissacarídeos/uso terapêutico , Camundongos , Triterpenos Pentacíclicos , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Piruvato Quinase/uso terapêutico , Sepse/tratamento farmacológico
2.
Front Endocrinol (Lausanne) ; 13: 838204, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35418943

RESUMO

Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovaries. In this study, we induced a young-adult PCOS rat model by oral administration of letrozole combined with a high-fat diet and then treated with mogroside V (MV) to evaluate the protective effects of MV on endocrine and follicle development in young-adult PCOS rats. MV (600 mg/kg/day) administration not only significantly reduced the body weight and ovary weight, but also attenuated the disrupted estrous cycle and decreased the level of testosterone. MV restored the follicular development, especially by increasing the number of corpus luteum and the thickness of the granular layer in young-adult POCS rats. Moreover, metabolomics showed that MV markedly increased the levels of D-Glucose 6-phosphate, lactate and GTP, while decreased the level of pyruvate. Bioinformatic analysis revealed that MV recovered multiple metabolism-related processes including gluconeogenesis, glycolysis and glucose metabolic process. Further real-time quantitative PCR analysis showed that MV upregulated the expression of lactate dehydrogenase A (Ldha), hexokinase 2 (Hk2) and pyruvate kinase M2 (Pkm2). Western blotting and immunohistochemistry analysis showed that MV restored the expression of lactate dehydrogenase A (Ldha), hexokinase 2 (Hk2) and pyruvate kinase M2 (Pkm2). Collectively, these findings indicated that MV could effectively improve the ovarian microenvironment by upregulating the expression of LDHA, HK2 and PKM2 in granulosa cells and enhancing lactate and energy production, which may contribute to follicle development and ovulation of young-adult PCOS rats.


Assuntos
Síndrome do Ovário Policístico , Animais , Dieta Hiperlipídica/efeitos adversos , Feminino , Glicólise , Hexoquinase/metabolismo , Hexoquinase/farmacologia , Humanos , Lactato Desidrogenase 5 , Ácido Láctico/efeitos adversos , Letrozol , Ovulação , Síndrome do Ovário Policístico/induzido quimicamente , Síndrome do Ovário Policístico/tratamento farmacológico , Síndrome do Ovário Policístico/metabolismo , Piruvato Quinase/metabolismo , Piruvato Quinase/farmacologia , Ratos , Triterpenos , Microambiente Tumoral
3.
BMC Biol ; 20(1): 87, 2022 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-35418203

RESUMO

BACKGROUND: In all living organisms, DNA replication is exquisitely regulated in a wide range of growth conditions to achieve timely and accurate genome duplication prior to cell division. Failures in this regulation cause DNA damage with potentially disastrous consequences for cell viability and human health, including cancer. To cope with these threats, cells tightly control replication initiation using well-known mechanisms. They also couple DNA synthesis to nutrient richness and growth rate through a poorly understood process thought to involve central carbon metabolism. One such process may involve the cross-species conserved pyruvate kinase (PykA) which catalyzes the last reaction of glycolysis. Here we have investigated the role of PykA in regulating DNA replication in the model system Bacillus subtilis. RESULTS: On analysing mutants of the catalytic (Cat) and C-terminal (PEPut) domains of B. subtilis PykA we found replication phenotypes in conditions where PykA is dispensable for growth. These phenotypes are independent from the effect of mutations on PykA catalytic activity and are not associated with significant changes in the metabolome. PEPut operates as a nutrient-dependent inhibitor of initiation while Cat acts as a stimulator of replication fork speed. Disruption of either PEPut or Cat replication function dramatically impacted the cell cycle and replication timing even in cells fully proficient in known replication control functions. In vitro, PykA modulates activities of enzymes essential for replication initiation and elongation via functional interactions. Additional experiments showed that PEPut regulates PykA activity and that Cat and PEPut determinants important for PykA catalytic activity regulation are also important for PykA-driven replication functions. CONCLUSIONS: We infer from our findings that PykA typifies a new family of cross-species replication control regulators that drive the metabolic control of replication through a mechanism involving regulatory determinants of PykA catalytic activity. As disruption of PykA replication functions causes dramatic replication defects, we suggest that dysfunctions in this new family of universal replication regulators may pave the path to genetic instability and carcinogenesis.


Assuntos
Glicólise , Piruvato Quinase , Bacillus subtilis/genética , Divisão Celular , Replicação do DNA , Piruvato Quinase/genética , Piruvato Quinase/metabolismo
4.
Biomaterials ; 284: 121523, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35462306

RESUMO

Tumor cells intensively engage in metabolic reprogramming for enhancing the availability of glycolytic metabolites and support cell proliferation. As the most important rate-limiting enzyme in aerobic glycolysis, activating the pyruvate kinase muscle isoform 2 (PKM2) from dimers to tetramers has become a key tumor chemotherapy method to control glucose metabolism. Herein, we developed a glycopeptide-based PKM2 nano-activator, which could induce the tetramerization of PKM2 based on serine bonding to Domain C of PKM2. The bound and trapped PKM2 tetramers significantly hindered glycolytic intermediates, prevented the nucleus translocation of dimeric PKM2, and ultimately inhibited the proliferation, chemoresistance and metastasis of tumor. The glycopeptide assembled into nanoparticles under aqueous conditions and in the circulation, which in situ transformed into PKM2 nano-activator with nanofibrillar structure after specifically activated by O-GlcNAcase recognition upregulated in a wide range of human tumors. Moreover, the glycopeptide-based PKM2 nano-activator successfully accumulated at the tumor sites and boosted the chemo-drug sensitivity against prostate and breast cancers. Attributed to these intriguing results, the newly developed glycopeptide-based PKM2 nano-activator can be envisioned a promising candidate for the treatment of tumors by switching catabolic pathways.


Assuntos
Neoplasias da Mama , Piruvato Quinase , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Glicólise , Glicopeptídeos/metabolismo , Humanos , Masculino , Músculos/metabolismo , Isoformas de Proteínas/metabolismo , Piruvato Quinase/metabolismo
5.
Cell Death Dis ; 13(3): 252, 2022 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-35306527

RESUMO

Neuroendocrine differentiation (NED) frequently occurs in androgen-deprivation therapy (ADT)-resistant prostate cancer (PCa) and is typically associated with metabolic pathway alterations, acquisition of lineage plasticity, and malignancy. There is no conventional therapeutic approach for PCa patients with NED pathologic features because the molecular targets are unknown. Here, we evaluated the regulatory mechanism of NED-associated metabolic reprogramming induced by ADT. We detected that the loss of the androgen-responsive transcription factor, zinc finger, and BTB domain containing 10 (ZBTB10), can activate pyruvate kinase L/R (PKLR) to enhance a NED response that is associated with glucose uptake by PCa cells. PKLR exhibits a tumor-promoting effect in PCa after ADT, but ZBTB10 can compensate for the glucose metabolism and NED capacity of PKLR through the direct transcriptional downregulation of PKLR. Targeting PKLR by drug repurposing with FDA-approved compounds can reduce the aggressiveness and NED of ADT-resistant PCa. We demonstrated that PKLR acts as a modulator to activate NED in PCa enhancement by loss of ZBTB10, thereby enabling PCa cells to mount a glycolysis response essential for therapeutic resistance. Our findings highlight the broad relation between NED and metabolic dysfunction to provide gene expression-based biomarkers for NEPC treatment.


Assuntos
Neoplasias de Próstata Resistentes à Castração , Neoplasias da Próstata , Piruvato Quinase/metabolismo , Antagonistas de Androgênios/farmacologia , Androgênios/farmacologia , Regulação para Baixo , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias de Próstata Resistentes à Castração/genética , Piruvato Quinase/genética , Proteínas Repressoras/metabolismo
6.
Hypertension ; 79(5): 932-945, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35232222

RESUMO

BACKGROUND: Metabolic reprogramming is a hallmark of pulmonary arterial hypertension. Platelet activation has been implicated in pulmonary arterial hypertension (PAH), whereas the role of platelet in the pathogenesis of PAH remains unclear. METHODS: First, we explored the platelet function of semaxanib' a inhibitor of VEGF receptor (SU5416)/hypoxia mice and monocrotaline-injected rats PAH model. Then we investigated pulmonary arterial smooth muscle cell aerobic glycolysis after being treated with platelet supernatant. TGF (transforming growth factor)-ßRI, pyruvate kinase muscle 2, and other antagonists were applied to identify the underlying mechanism. In addition, platelet-specific deletion TGF-ß1 mice were exposed to chronic hypoxia and SU5416. Cardiopulmonary hemodynamics, vascular remodeling, and aerobic glycolysis of pulmonary arterial smooth muscle cell were determined. RESULTS: Here, we demonstrate that platelet-released TGF-ß1 enhances the aerobic glycolysis of pulmonary arterial smooth muscle cells after platelet activation via increasing pyruvate kinase muscle 2 expression. Mechanistically, platelet-derived TGF-ß1 regulate spyruvate kinase muscle 2 expression through mTOR (mammalian target of rapamycin)/c-Myc/PTBP-1(polypyrimidine tract binding protein 1)/hnRNPA-1(heterogeneous nuclear ribonucleoprotein A1) pathway. Platelet TGF-ß1 deficiency mice are significantly protected from SU5416 plus chronic hypoxia-induced PAH, including attenuated increases in right ventricular systolic pressure and less pulmonary vascular remodeling. Also, in Pf4cre+ Tgfb1fl/fl mice, pulmonary arterial smooth muscle cells showed lower glycolysis capacity and their pyruvate kinase muscle 2 expression decreased. CONCLUSIONS: Our data demonstrate that TGF-ß1 released by platelet contributes to the pathogenesis of PAH and further highlights the role of platelet in PAH.


Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Animais , Proliferação de Células , Glicólise , Hipertensão Pulmonar/metabolismo , Hipóxia/metabolismo , Mamíferos/metabolismo , Camundongos , Músculos , Miócitos de Músculo Liso/metabolismo , Isoformas de Proteínas/metabolismo , Artéria Pulmonar/metabolismo , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Ratos , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/metabolismo , Regulação para Cima , Remodelação Vascular/fisiologia
7.
Eur J Med Chem ; 234: 114270, 2022 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-35290845

RESUMO

Liver pyruvate kinase (PKL) is a major regulator of metabolic flux and ATP production during liver cell glycolysis and is considered a potential drug target for the treatment of non-alcoholic fatty liver disease (NAFLD). In this study, we report the first ADP-competitive PKL inhibitors and identify several starting points for the further optimization of these inhibitors. Modeling and structural biology guided the optimization of a PKL-specific anthraquinone-based compound. A structure-activity relationship study of 47 novel synthetic derivatives revealed PKL inhibitors with half-maximal inhibitory concentration (IC50) values in the 200 nM range. Despite the difficulty involved in studying a binding site as exposed as the ADP site, these derivatives feature expanded structural diversity and chemical spaces that may be used to improve their inhibitory activities against PKL. The obtained results expand the knowledge of the structural requirements for interactions with the ADP-binding site of PKL.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Piruvato Quinase , Difosfato de Adenosina/farmacologia , Antraquinonas/farmacologia , Humanos , Fígado/metabolismo , Piruvato Quinase/metabolismo
8.
Sci Rep ; 12(1): 4024, 2022 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-35256696

RESUMO

Extracellular PKM2 (exPKM2) levels have been reported to be increased in several cancers and inflammatory diseases, including rheumatoid arthritis (RA). This study aimed to investigate the association of circulating exPKM2 levels with radiographic progression in RA patients and the effect of exPKM2 on osteoclastogenesis. Plasma and synovial fluid exPKM2 levels were significantly elevated in RA patients. Plasma exPKM2 levels were correlated with RA disease activity and were an independent predictor for radiographic progression in RA patients with a disease duration of ≤ 12 months. CD14+ monocytes but not RA fibroblast-like synoviocytes secreted PKM2 upon stimulation with inflammatory mediators. Recombinant PKM2 (rPKM2) increased the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells and resorption pit in osteoclast precursors, dose-dependently, even in the absence of receptor activator of nuclear factor-kappa B ligand (RANKL). rPKM2 treatment upregulated the expression of dendrocyte-expressed seven transmembrane protein (DC-STAMP) and MMP-9 via the ERK pathway. Although rPKM2 did not directly bind to RAW264.7 cells, extracellular application of pyruvate, the end-product of PKM2, showed effects similar to those seen in rPKM2-induced osteoclastogenesis. These results suggest that exPKM2 is a potential regulator of RA-related joint damage and a novel biomarker for subsequent radiographic progression in patients with early-stage RA.


Assuntos
Artrite Reumatoide , Osteogênese , Piruvato Quinase , Artrite Reumatoide/diagnóstico por imagem , Artrite Reumatoide/enzimologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Diferenciação Celular , Humanos , Osteoclastos/metabolismo , Osteoclastos/patologia , Piruvato Quinase/metabolismo , Ligante RANK/metabolismo
9.
J Exp Med ; 219(4)2022 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-35266960

RESUMO

The spleen is an important site of hematopoietic stem/progenitor cell (HSPC) preconditioning and tumor-promoting myeloid cell generation in cancer, but the regulatory mechanism remains unclear. Here, we found that PKR-like endoplasmic reticulum kinase (PERK) mediated HSPC reprogramming into committed MDSC precursors in the spleen via PERK-ATF4-C/EBPß signaling. Pharmacological and genetic inhibition of this pathway in murine and human HSPCs prevented their myeloid descendant cells from becoming MDSCs even with subsequent exposure to tumor microenvironment (TME) factors. In mice, the selective delivery of PERK antagonists to the spleen was not only sufficient but more effective than the tumor-targeted strategy in preventing MDSC activation in the tumor, leading to profound TME reshaping and tumor regression. Clinically, HSPCs in the spleen of cancer patients exhibit increased PERK signaling correlated with enhanced myelopoiesis. Our findings indicate that PERK-mediated HSPC preconditioning plays a crucial role in MDSC generation, suggesting novel spleen-targeting therapeutic opportunities for restraining the tumor-promoting myeloid response at its source.


Assuntos
Mielopoese , Neoplasias , Animais , Retículo Endoplasmático , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Neoplasias/metabolismo , Piruvato Quinase/metabolismo , Baço , Microambiente Tumoral , eIF-2 Quinase
10.
JCI Insight ; 7(5)2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35133981

RESUMO

Diabetic nephropathy (DN) arises from systemic and local changes in glucose metabolism and hemodynamics. We have reported that many glycolytic and mitochondrial enzymes, such as pyruvate kinase M2 (PKM2), were elevated in renal glomeruli of DN-protected patients with type 1 and type 2 diabetes. Here, mice with PKM2 overexpression specifically in podocytes (PPKM2Tg) were generated to uncover the renal protective function of PPKM2Tg as a potential therapeutic target that prevented elevated albumin/creatinine ratio (ACR), mesangial expansion, basement membrane thickness, and podocyte foot process effacement after 7 months of streptozotocin-induced (STZ-induced) diabetes. Furthermore, diabetes-induced impairments of glycolytic rate and mitochondrial function were normalized in diabetic PPKM2Tg glomeruli, in concordance with elevated Ppargc1a and Vegf expressions. Restored VEGF expression improved glomerular maximal mitochondrial function in diabetic PPKM2Tg and WT mice. Elevated VEGF levels were observed in the glomeruli of DN-protected patients with chronic type 1 diabetes and clinically correlated with estimated glomerular filtration (GFR) - but not glycemic control. Mechanistically, the preservations of mitochondrial function and VEGF expression were dependent on tetrameric structure and enzymatic activities of PKM2 in podocytes. These findings demonstrate that PKM2 structure and enzymatic activation in podocytes can preserve the entire glomerular mitochondrial function against toxicity of hyperglycemia via paracrine factors such as VEGF and prevent DN progression.


Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Nefropatias Diabéticas , Podócitos , Piruvato Quinase , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Nefropatias Diabéticas/metabolismo , Humanos , Camundongos , Podócitos/metabolismo , Piruvato Quinase/metabolismo , Regeneração , Fator A de Crescimento do Endotélio Vascular/metabolismo
11.
Org Biomol Chem ; 20(9): 1869-1873, 2022 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-35156979

RESUMO

A biocatalytic cascade based on concerted operation of pyruvate kinase and luciferase with a bioluminescent output was switched reversibly between low and high activity by applying an external magnetic field at different positions or removing it. The enzymes participating in the reaction cascade were bound to magnetic nanoparticles to allow their translocation or aggregation/dispersion to be controlled by the magnetic field. The reaction intensity, measured as the bioluminescent output, was dependent on the effective distances between the enzymes transported on the magnetic nanoparticles controlled by the magnets.


Assuntos
Fluorescência , Luciferases/metabolismo , Nanopartículas de Magnetita/química , Piruvato Quinase/metabolismo , Aliivibrio fischeri/enzimologia , Animais , Biocatálise , Luciferases/química , Medições Luminescentes , Campos Magnéticos , Piruvato Quinase/química , Coelhos
12.
Redox Biol ; 50: 102257, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35149342

RESUMO

T lymphocyte and macrophage infiltration in the aortic wall is critical for abdominal aortic aneurysm (AAA). However, how T lymphocytes interact with macrophages in the pathogenesis of AAA remains largely uncharacterized. In an elastase-induced murine AAA model, we first found that the expression of pyruvate kinase muscle isozyme 2 (PKM2), the last rate-limiting enzyme in glycolysis, was increased in infiltrated T lymphocytes of vascular lesions. T lymphocyte-specific PKM2 deficiency in mice (LckCrePKM2fl/fl) or intraperitoneal administration of the sphingomyelinase inhibitor GW4869 caused a significant attenuation of the elastase-increased aortic diameter, AAA incidence, elastic fiber disruption, matrix metalloproteinases (MMPs) expression, and macrophage infiltration in the vascular adventitia compared with those in PKM2fl/fl mice. Mechanistically, extracellular vesicles (EVs) derived from PKM2-activated T lymphocytes elevated macrophage iron accumulation, lipid peroxidation, and migration in vitro, while macrophages treated with EVs from PKM2-null T lymphocytes or pretreated with the lipid peroxidation inhibitors ferrostatin-1 (Fer-1), liproxstatin-1 (Lip-1), or the iron chelating agent deferoxamine mesylate (DFOM) reversed these effects. In vascular lesions of elastase-induced LckCrePKM2fl/fl mice with AAA, the oxidant system weakened, with downregulated 4-hydroxynonenal (4-HNE) levels and strengthened antioxidant defense systems with upregulated glutathione peroxidase 4 (GPX4) and cystine/glutamate antiporter solute carrier family 7 member 11 (Slc7a11) expressions in macrophages. High-throughput metabolomics showed that EVs derived from PKM2-activated T lymphocytes contained increased levels of polyunsaturated fatty acid (PUFA)-containing phospholipids, which may provide abundant substrates for lipid peroxidation in target macrophages. More importantly, upregulated T lymphocyte PKM2 expression was also found in clinical AAA subjects, and EVs isolated from AAA patient plasma enhanced macrophage iron accumulation, lipid peroxidation, and migration ex vivo. Therefore, from cell-cell crosstalk and metabolic perspectives, the present study shows that PKM2-activated T lymphocyte-derived EVs may drive AAA progression by promoting macrophage redox imbalance and migration, and targeting the T lymphocyte-EV-macrophage axis may be a potential strategy for early warning and treating AAA.


Assuntos
Aneurisma da Aorta Abdominal , Vesículas Extracelulares , Piruvato Quinase , Animais , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/metabolismo , Proteínas de Transporte , Modelos Animais de Doenças , Humanos , Peroxidação de Lipídeos , Macrófagos/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Piruvato Quinase/metabolismo , Linfócitos T/metabolismo , Hormônios Tireóideos
13.
Int J Mol Sci ; 23(3)2022 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-35163274

RESUMO

Eukarya pyruvate kinases possess glutamate at position 117 (numbering of rabbit muscle enzyme), whereas bacteria have either glutamate or lysine. Those with E117 are K+-dependent, whereas those with K117 are K+-independent. In a phylogenetic tree, 80% of the sequences with E117 are occupied by T113/K114/T120 and 77% of those with K117 possess L113/Q114/(L,I,V)120. This work aims to understand these residues' contribution to the K+-independent pyruvate kinases using the K+-dependent rabbit muscle enzyme. Residues 117 and 120 are crucial in the differences between the K+-dependent and -independent mutants. K+-independent activity increased with L113 and Q114 to K117, but L120 induced structural differences that inactivated the enzyme. T120 appears to be key in folding the protein and closure of the lid of the active site to acquire its active conformation in the K+-dependent enzymes. E117K mutant was K+-independent and the enzyme acquired the active conformation by a different mechanism. In the K+-independent apoenzyme of Mycobacterium tuberculosis, K72 (K117) flips out of the active site; in the holoenzyme, K72 faces toward the active site bridging the substrates through water molecules. The results provide evidence that two different mechanisms have evolved for the catalysis of this reaction.


Assuntos
Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Piruvato Quinase/ultraestrutura , Sequência de Aminoácidos/genética , Animais , Apoenzimas/metabolismo , Sítios de Ligação , Catálise , Domínio Catalítico , Ácido Glutâmico/metabolismo , Lisina/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Filogenia , Potássio/metabolismo , Conformação Proteica , Coelhos
14.
Adv Sci (Weinh) ; 9(8): e2104055, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35048565

RESUMO

DNA repair confers the resistance of tumor cells to DNA-damaging anticancer therapies, while how reprogrammed metabolism in tumor cells contributes to such process remains poorly understood. Pyruvate kinase M2 isoform (PKM2) catalyzes the conversion of phosphoenolpyruvate to pyruvate and regulates the last rate-limiting step of glycolysis. Here it is shown that the glycolytic metabolite pyruvate enhances DNA damage repair by facilitating chromatin loading of γH2AX, thereby promoting the radiation resistance of glioma cells. Mechanistically, PKM2 is phosphorylated at serine (S) 222 upon DNA damage and interacts with FACT complex, a histone chaperone comprising SPT16 and SSRP1 subunit. The pyruvate produced by PKM2 directly binds to SSRP1, which increases the association of FACT complex with γH2AX and subsequently facilitates FACT-mediated chromatin loading of γH2AX, ultimately promoting DNA repair and tumor cell survival. Intriguingly, the supplementation of exogenous pyruvate can also sufficiently enhance FACT-mediated chromatin loading of γH2AX and promotes tumor cell survival upon DNA damage. The levels of PKM2 S222 phosphorylation correlate with the malignancy and prognosis of human glioblastoma. The finding demonstrates a novel mechanism by which PKM2-produced pyruvate promotes DNA repair by regulating γH2AX loading to chromatin and establishes a critical role of this mechanism in glioblastoma radiation resistance.


Assuntos
Glioblastoma , Linhagem Celular Tumoral , Cromatina , Proteínas de Ligação a DNA/genética , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Proteínas de Grupo de Alta Mobilidade/genética , Histonas , Humanos , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Ácido Pirúvico/metabolismo , Fatores de Elongação da Transcrição/genética
15.
J Pharmacol Exp Ther ; 380(3): 210-219, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35031585

RESUMO

Etavopivat is an investigational, oral, small molecule activator of erythrocyte pyruvate kinase (PKR) in development for the treatment of sickle cell disease (SCD) and other hemoglobinopathies. PKR activation is proposed to ameliorate the sickling of SCD red blood cells (RBCs) through multiple mechanisms, including reduction of 2,3-diphosphoglycerate (2,3-DPG), which consequently increases hemoglobin (Hb)-oxygen affinity; increased binding of oxygen reduces sickle hemoglobin polymerization and sickling. In addition, PKR activation increases adenosine triphosphate (ATP) produced via glycolytic flux, which helps preserve membrane integrity and RBC deformability. We evaluated the pharmacodynamic response to etavopivat in nonhuman primates (NHPs) and in healthy human subjects and evaluated the effects in RBCs from patients with SCD after ex vivo treatment with etavopivat. A single dose of etavopivat decreased 2,3-DPG in NHPs and healthy subjects. Hb-oxygen affinity was significantly increased in healthy subjects after 24 hours. After daily dosing of etavopivat over 5 consecutive days in NHPs, ATP was increased by 38% from baseline. Etavopivat increased Hb-oxygen affinity and reduced sickling in RBCs collected from patients with SCD with either homozygous hemoglobin S or hemoglobin S and C disease. Collectively, these results demonstrate the ability of etavopivat to decrease 2,3-DPG and increase ATP, resulting in increased Hb-oxygen affinity and improved sickle RBC function. Etavopivat is currently being evaluated in clinical trials for the treatment of SCD. SIGNIFICANCE STATEMENT: Etavopivat, a small molecule activator of the glycolytic enzyme erythrocyte pyruvate kinase, decreased 2,3-diphosphoglycerate in red blood cells (RBCs) from nonhuman primates and healthy subjects and significantly increased hemoglobin (Hb)-oxygen affinity in healthy subjects. Using ex vivo RBCs from donors with sickle cell disease (SCD) (homozygous hemoglobin S or hemoglobin S and C genotype), etavopivat increased Hb-oxygen affinity and reduced sickling under deoxygenation. Etavopivat shows promise as a treatment for SCD that could potentially reduce vaso-occlusion and improve anemia.


Assuntos
Anemia Falciforme , Hemoglobina Falciforme , 2,3-Difosfoglicerato/metabolismo , 2,3-Difosfoglicerato/farmacologia , Trifosfato de Adenosina/metabolismo , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/metabolismo , Animais , Eritrócitos/metabolismo , Hemoglobina Falciforme/metabolismo , Hemoglobina Falciforme/farmacologia , Hemoglobina Falciforme/uso terapêutico , Hemoglobinas/metabolismo , Humanos , Oxigênio/metabolismo , Piruvato Quinase/metabolismo , Piruvato Quinase/farmacologia , Piruvato Quinase/uso terapêutico , Ácido Pirúvico/farmacologia
16.
Clin Cancer Res ; 28(7): 1446-1459, 2022 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-35078861

RESUMO

PURPOSE: DNA-dependent protein kinase catalytic subunit (DNA-PKcs, herein referred as DNA-PK) is a multifunctional kinase of high cancer relevance. DNA-PK is deregulated in multiple tumor types, including prostate cancer, and is associated with poor outcomes. DNA-PK was previously nominated as a therapeutic target and DNA-PK inhibitors are currently undergoing clinical investigation. Although DNA-PK is well studied in DNA repair and transcriptional regulation, much remains to be understood about the way by which DNA-PK drives aggressive disease phenotypes. EXPERIMENTAL DESIGN: Here, unbiased proteomic and metabolomic approaches in clinically relevant tumor models uncovered a novel role of DNA-PK in metabolic regulation of cancer progression. DNA-PK regulation of metabolism was interrogated using pharmacologic and genetic perturbation using in vitro cell models, in vivo xenografts, and ex vivo in patient-derived explants (PDE). RESULTS: Key findings reveal: (i) the first-in-field DNA-PK protein interactome; (ii) numerous DNA-PK novel partners involved in glycolysis; (iii) DNA-PK interacts with, phosphorylates (in vitro), and increases the enzymatic activity of glycolytic enzymes ALDOA and PKM2; (iv) DNA-PK drives synthesis of glucose-derived pyruvate and lactate; (v) DNA-PK regulates glycolysis in vitro, in vivo, and ex vivo; and (vi) combination of DNA-PK inhibitor with glycolytic inhibitor 2-deoxyglucose leads to additive anti-proliferative effects in aggressive disease. CONCLUSIONS: Findings herein unveil novel DNA-PK partners, substrates, and function in prostate cancer. DNA-PK impacts glycolysis through direct interaction with glycolytic enzymes and modulation of enzymatic activity. These events support energy production that may contribute to generation and/or maintenance of DNA-PK-mediated aggressive disease phenotypes.


Assuntos
Proteína Quinase Ativada por DNA , Neoplasias de Próstata Resistentes à Castração , DNA , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Glicólise , Humanos , Masculino , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Proteômica , Piruvato Quinase/metabolismo
17.
Biochim Biophys Acta Mol Cell Res ; 1869(4): 119206, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35026348

RESUMO

Pyruvate kinase isoform M2 (PKM2) is a rate-limiting glycolytic enzyme that is widely expressed in embryonic tissues. The expression of PKM2 declines in some tissues following embryogenesis, while other pyruvate kinase isozymes are upregulated. However, PKM2 is highly expressed in cancer cells and is believed to play a role in supporting anabolic processes during tumour formation. In this study, PKM2 was identified as an inositol 1,4,5-trisphosphate receptor (IP3R)-interacting protein by mass spectrometry. The PKM2:IP3R interaction was further characterized by pull-down and co-immunoprecipitation assays, which showed that PKM2 interacted with all three IP3R isoforms. Moreover, fluorescence microscopy indicated that both IP3R and PKM2 localized at the endoplasmic reticulum. PKM2 binds to IP3R at a highly conserved 21-amino acid site (corresponding to amino acids 2078-2098 in mouse type 1 IP3R isoform). Synthetic peptides (denoted 'TAT-D5SD' and 'D5SD'), based on the amino acid sequence at this site, disrupted the PKM2:IP3R interaction and potentiated IP3R-mediated Ca2+ release both in intact cells (TAT-D5SD peptide) and in a unidirectional 45Ca2+ flux assay on permeabilized cells (D5SD peptide). The TAT-D5SD peptide did not affect the enzymatic activity of PKM2. Reducing PKM2 protein expression using siRNA increased IP3R-mediated Ca2+ signalling in intact cells without altering the ER Ca2+ content. These data identify PKM2 as an IP3R-interacting protein that inhibits intracellular Ca2+ signalling. The elevated expression of PKM2 in cancer cells is therefore not solely connected to its canonical role in glycolytic metabolism, rather PKM2 also has a novel non-canonical role in regulating intracellular signalling.


Assuntos
Sinalização do Cálcio , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Piruvato Quinase/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Linhagem Celular , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/química , Linfócitos/citologia , Linfócitos/metabolismo , Camundongos , Ligação Proteica , Domínios Proteicos , Isoformas de Proteínas/metabolismo , Piruvato Quinase/antagonistas & inibidores , Piruvato Quinase/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo
18.
BMB Rep ; 55(2): 98-103, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35000669

RESUMO

Increased mRNA levels of cancer upregulated gene (CUG)2 have been detected in many different tumor tissues using Affymetrix microarray. Oncogenic capability of the CUG2 gene has been further reported. However, the mechanism by which CUG2 overexpression promotes cancer stem cell (CSC)-like phenotypes remains unknown. With recent studies showing that pyruvate kinase muscle 2 (PKM2) is overexpressed in clinical tissues from gastric, lung, and cervical cancer patients, we hypothesized that PKM2 might play an important role in CSC-like phenotypes caused by CUG2 overexpression. The present study revealed that PKM2 protein levels and translocation of PKM2 into the nucleus were enhanced in CUG2-overexpressing lung carcinoma A549 and immortalized bronchial BEAS-2B cells than in control cells. Expression levels of c-Myc, CyclinD1, and PKM2 were increased in CUG2-overexpressing cells than in control cells. Furthermore, EGFR and ERK inhibitors as well as suppression of Yap1 and NEK2 expression reduced PKM2 protein levels. Interestingly, knockdown of ß-catenin expression failed to reduce PKM2 protein levels. Furthermore, reduction of PKM2 expression with its siRNA hindered CSC-like phenotypes such as faster wound healing, aggressive transwell migration, and increased size/number of sphere formation. The introduction of mutant S37A PKM2-green fluorescence protein (GFP) into cells without ability to move to the nucleus did not confer CSC-like phenotypes, whereas forced expression of wild-type PKM2 promoted such phenotypes. Overall, CUG2-induced increase in the expression of nuclear PKM2 contributes to CSC-like phenotypes by upregulating c-Myc and CyclinD1 as a co-activator. [BMB Reports 2022;55(2): 98-103].


Assuntos
Proteínas de Transporte/genética , Proteínas Cromossômicas não Histona , Proteínas de Membrana/genética , Neoplasias , Piruvato Quinase , Hormônios Tireóideos/genética , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Musculares/genética , Quinases Relacionadas a NIMA/genética , Quinases Relacionadas a NIMA/metabolismo , Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Transdução de Sinais/genética
19.
Sci Rep ; 12(1): 1459, 2022 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-35087114

RESUMO

Glycolysis is the metabolic pathway that converts glucose into pyruvate. Central nervous system (CNS) pathologies, such as spinal cord injury (SCI) and ischemia, are accompanied by an increase of the glycolytic pathway in the damaged areas as part of the inflammatory response. Pyruvate kinase is a key glycolytic enzyme that converts phosphoenolpyruvate and ADP to pyruvate and ATP. The protein has two isoforms, PKM1 and PKM2, originated from the same gene. As a homodimer, PKM2 loses the pyruvate kinase activity and acts as a transcription factor that regulates the expression of target genes involved in glycolysis and inflammation. After SCI, resident microglia and hematogenous macrophages are key inducers of the inflammatory response with deleterious effects. Activation of the bile acid receptor TGR5 inhibits the pro-inflammatory NFκB pathway in microglia and macrophages. In the present study we have investigated whether bile acids affect the expression of glycolytic enzymes and their regulation by PKM2. Bacterial lipopolysaccharide (LPS) induced the expression of PKM1, PKM2 and its target genes in primary cultures of microglial and Raw264.7 macrophage cells. SCI caused an increase of PKM2 immunoreactivity in macrophages after SCI. Pretreatment with tauroursodeoxycholic acid (TUDCA) or taurolithocholic acid (TLCA) reduced the expression of PKM2 and its target genes in cell cultures. Similarly, after SCI, TUDCA treatment reduced the expression of PKM2 in the lesion center. These results confirm the importance of PKM2 in the inflammatory response in CNS pathologies and indicate a new mechanism of bile acids as regulators of PKM2 pathway.


Assuntos
Ácidos e Sais Biliares/metabolismo , Microglia/patologia , Piruvato Quinase/metabolismo , Traumatismos da Medula Espinal/imunologia , Animais , Modelos Animais de Doenças , Glicólise , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos , Masculino , Camundongos , Microglia/imunologia , Cultura Primária de Células , Piruvato Quinase/genética , Células RAW 264.7 , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/patologia
20.
Blood ; 139(8): 1234-1245, 2022 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-34529778

RESUMO

There is a critical need for cerebro-protective interventions to improve the suboptimal outcomes of patients with ischemic stroke who have been treated with reperfusion strategies. We found that nuclear pyruvate kinase muscle 2 (PKM2), a modulator of systemic inflammation, was upregulated in neutrophils after the onset of ischemic stroke in both humans and mice. Therefore, we determined the role of PKM2 in stroke pathogenesis by using murine models with preexisting comorbidities. We generated novel myeloid cell-specific PKM2-/- mice on wild-type (PKM2fl/flLysMCre+) and hyperlipidemic background (PKM2fl/flLysMCre+Apoe-/-). Controls were littermate PKM2fl/flLysMCre- or PKM2fl/flLysMCre-Apoe-/- mice. Genetic deletion of PKM2 in myeloid cells limited inflammatory response in peripheral neutrophils and reduced neutrophil extracellular traps after cerebral ischemia and reperfusion, suggesting that PKM2 promotes neutrophil hyperactivation in the setting of stroke. In the filament and autologous clot and recombinant tissue plasminogen activator stroke models, irrespective of sex, deletion of PKM2 in myeloid cells in either wild-type or hyperlipidemic mice reduced infarcts and enhanced long-term sensorimotor recovery. Laser speckle imaging revealed improved regional cerebral blood flow in myeloid cell-specific PKM2-deficient mice that was concomitant with reduced post-ischemic cerebral thrombo-inflammation (intracerebral fibrinogen, platelet [CD41+] deposition, neutrophil infiltration, and inflammatory cytokines). Mechanistically, PKM2 regulates post-ischemic inflammation in peripheral neutrophils by promoting STAT3 phosphorylation. To enhance the translational significance, we inhibited PKM2 nuclear translocation using a small molecule and found significantly reduced neutrophil hyperactivation and improved short-term and long-term functional outcomes after stroke. Collectively, these findings identify PKM2 as a novel therapeutic target to improve brain salvage and recovery after reperfusion.


Assuntos
Trombose Intracraniana/enzimologia , AVC Isquêmico/enzimologia , Ativação de Neutrófilo , Neutrófilos/enzimologia , Piruvato Quinase/metabolismo , Animais , Feminino , Inflamação/enzimologia , Inflamação/genética , Trombose Intracraniana/genética , AVC Isquêmico/genética , Masculino , Camundongos , Camundongos Knockout para ApoE , Piruvato Quinase/genética
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