RESUMO
Reactive astrocytes play critical roles in the occurrence of various neurological diseases such as multiple sclerosis. Activation of astrocytes is often accompanied by a glycolysis-dominant metabolic switch. However, the role and molecular mechanism of metabolic reprogramming in activation of astrocytes have not been clarified. Here, we found that PKM2, a rate-limiting enzyme of glycolysis, displayed nuclear translocation in astrocytes of EAE (experimental autoimmune encephalomyelitis) mice, an animal model of multiple sclerosis. Prevention of PKM2 nuclear import by DASA-58 significantly reduced the activation of mice primary astrocytes, which was observed by decreased proliferation, glycolysis and secretion of inflammatory cytokines. Most importantly, we identified the ubiquitination-mediated regulation of PKM2 nuclear import by ubiquitin ligase TRIM21. TRIM21 interacted with PKM2, promoted its nuclear translocation and stimulated its nuclear activity to phosphorylate STAT3, NF-κB and interact with c-myc. Further single-cell RNA sequencing and immunofluorescence staining demonstrated that TRIM21 expression was upregulated in astrocytes of EAE. TRIM21 overexpressing in mice primary astrocytes enhanced PKM2-dependent glycolysis and proliferation, which could be reversed by DASA-58. Moreover, intracerebroventricular injection of a lentiviral vector to knockdown TRIM21 in astrocytes or intraperitoneal injection of TEPP-46, which inhibit the nuclear translocation of PKM2, effectively decreased disease severity, CNS inflammation and demyelination in EAE. Collectively, our study provides novel insights into the pathological function of nuclear glycolytic enzyme PKM2 and ubiquitination-mediated regulatory mechanism that are involved in astrocyte activation. Targeting this axis may be a potential therapeutic strategy for the treatment of astrocyte-involved neurological disease.
Assuntos
Astrócitos , Encefalomielite Autoimune Experimental , Ribonucleoproteínas , Regulação para Cima , Animais , Astrócitos/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/genética , Camundongos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/genética , Proteínas de Ligação a Hormônio da Tireoide , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Camundongos Endogâmicos C57BL , Piruvato Quinase/metabolismo , Piruvato Quinase/genética , Transporte Ativo do Núcleo Celular , Feminino , Glicólise , Ubiquitinação , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Núcleo Celular/metabolismoRESUMO
Cardiac metabolism ensures a continuous ATP supply, primarily using fatty acids in a healthy state and favoring glucose in pathological conditions. Pyruvate kinase muscle (PKM) controls the final step of glycolysis, with PKM1 being the main isoform in the heart. PKM2, elevated in various heart diseases, has been suggested to play a protective role in cardiac stress, but its function in basal cardiac metabolism remains unclear. We examined hearts from global PKM2 knockout (PKM2-/-) mice and found reduced intracellular glucose. Isotopic tracing of U-13C glucose revealed a shift to biosynthetic pathways in PKM2-/- cardiomyocytes. Total ATP content was two-thirds lower in PKM2-/- hearts, and functional analysis indicated reduced mitochondrial oxygen consumption. Total reactive oxygen species (ROS) and mitochondrial superoxide were also increased in PKM2-/- cardiomyocytes. Intriguingly, PKM2-/- hearts had preserved ejection fraction compared to controls. Mechanistically, increased calcium/calmodulin-dependent kinase II activity and phospholamban phosphorylation may contribute to higher sarcoendoplasmic reticulum calcium ATPase 2 pump activity in PKM2-/- hearts. Loss of PKM2 led to altered glucose metabolism, diminished mitochondrial function, and increased ROS in cardiomyocytes. These data suggest that cardiac PKM2 acts as an important rheostat to maintain ATP levels while limiting oxidative stress. Although loss of PKM2 did not impair baseline contractility, its absence may make hearts more sensitive to environmental stress or injury.
Assuntos
Miócitos Cardíacos , Estresse Oxidativo , Animais , Miócitos Cardíacos/metabolismo , Camundongos , Camundongos Knockout , Glucose/metabolismo , Masculino , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Mitocôndrias Cardíacas/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Camundongos Endogâmicos C57BL , Piruvato Quinase/metabolismo , Piruvato Quinase/genética , Trifosfato de Adenosina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Miocárdio/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Sanqi oral solution (SQ) is a traditional Chinese patent medicine, widely used to treat chronic kidney diseases (CKD) in the clinic in China. Previous studies have confirmed its anti-renal fibrosis effect, but the specific pharmacological mechanism is still unclear. AIM OF THE STUDY: Focusing on energy metabolism in fibroblasts, the renoprotective mechanism of SQ was investigated in vitro and in vivo. METHODS: Firstly, the fingerprint of SQ was constructed and its elementary chemical composition was analyzed. In the 5/6Nx rats experiment, the efficacy of SQ on the kidney was evaluated by detecting serum and urine biochemical indexes and pathological staining of renal tissues. Lactic acid and pyruvic acid levels in serum and renal tissues were detected. PCNA protein expression in kidney tissue was detected by immunofluorescence assay and Western blot. Expression levels of HIF-1α, PKM2 and HK2 were determined by immunohistochemistry, Western blot or RT-qPCR assay. In addition, the effect of SQ intervention on cell proliferation and glycolysis was evaluated in TGF-ß1-induced NRK-49F cells, and the role of SQ exposure and HIF-1α/PKM2/glycolysis pathway were further investigated by silencing and overexpressing HIF-1α gene in NRK-49F cells. RESULTS: In 5/6 Nx rats, SQ effectively improved renal function and treated renal injury. It reduced the levels of lactic acid and pyruvic acid in kidney homogenates from CKD rats and decreased the expression levels of HIF-1α, PKM2, HK2, α-SMA, vimentin, collagen I and PCNA in kidney tissues. Similar results were observed in vitro. SQ inhibited NRK-49F cell proliferation, glycolysis and the expression levels of HIF-1α, PKM2 induced by TGF-ß1. Furthermore, we established NRK-49F cells transfected with siRNA or pDNA to silence or overexpress the HIF-1α gene. Overexpression of HIF-1α promoted cellular secretion of lactic acid and pyruvic acid in TGF-ß1-induced NRK-49F cells, however, this change was reversed by intervention with SQ or silencing the HIF-1α gene. Overexpression of HIF-1α can further induce increased PKM2 expression, while SQ intervention can reduce PKM2 expression. Moreover, PKM2 expression was also inhibited after silencing HIF-1α gene, and SQ was not effective even when given. CONCLUSION: The mechanism of action of SQ was explored from the perspective of energy metabolism, and it was found to regulate PKM2-activated glycolysis, inhibit fibroblast activation, and further ameliorate renal fibrosis in CKD by targeting HIF-1α.
Assuntos
Fibroblastos , Fibrose , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia , Rim , Ratos Sprague-Dawley , Insuficiência Renal Crônica , Proteínas de Ligação a Hormônio da Tireoide , Animais , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/metabolismo , Fibrose/tratamento farmacológico , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Masculino , Glicólise/efeitos dos fármacos , Ratos , Rim/efeitos dos fármacos , Rim/patologia , Rim/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Linhagem Celular , Piruvato Quinase/metabolismo , Piruvato Quinase/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Hormônios Tireóideos , Administração Oral , Proliferação de Células/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/genéticaRESUMO
Sirtuin 1 (SIRT1) is a histone deacetylase and plays diverse functions in various physiological events, from development to lifespan regulation. Here, in Parkinson's disease (PD) model mice, we demonstrated that SIRT1 ameliorates parkinsonism, while SIRT1 knockdown further aggravates PD phenotypes. Mechanistically, SIRT1 interacts with and deacetylates pyruvate kinase M2 (PKM2) at K135 and K206, thus leading to reduced PKM2 enzyme activity and lactate production, which eventually results in decreased glial activation in the brain. Administration of lactate in the brain recapitulates PD-like phenotypes. Furthermore, increased expression of PKM2 worsens PD symptoms, and, on the contrary, inhibition of PKM2 by shikonin or PKM2-IN-1 alleviates parkinsonism in mice. Collectively, our data indicate that excessive lactate in the brain might be involved in the progression of PD. By improving lactate homeostasis, SIRT1, together with PKM2, are likely drug targets for developing agents for the treatment of neurodegeneration in PD.
Assuntos
Encéfalo , Homeostase , Ácido Láctico , Piruvato Quinase , Sirtuína 1 , Sirtuína 1/metabolismo , Sirtuína 1/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Piruvato Quinase/metabolismo , Piruvato Quinase/genética , Camundongos , Ácido Láctico/metabolismo , Humanos , Acetilação/efeitos dos fármacos , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/genética , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos C57BL , Proteínas de Ligação a Hormônio da Tireoide , Hormônios Tireóideos/metabolismo , Naftoquinonas/farmacologiaRESUMO
Pyruvate kinase (PK) deficiency, a rare, congenital haemolytic anaemia caused by mutations in the PKLR gene, is associated with many clinical manifestations, but the full disease burden has yet to be characterised. The Peak Registry (NCT03481738) is an observational, longitudinal registry of adult and paediatric patients with PK deficiency. Here, we described comorbidities and complications in these patients by age at most recent visit and PKLR genotype. As of 13 May 2022, 241 patients were included in the analysis. In total, 48.3% had undergone splenectomy and 50.5% had received chelation therapy. History of iron overload (before enrolment/during follow-up) was common (52.5%), even in never-transfused patients (20.7%). Neonatal complications and symptoms included jaundice, splenomegaly and hepatomegaly, with treatment interventions required in 41.5%. Among adults, osteopenia/osteoporosis occurred in 19.0% and pulmonary hypertension in 6.7%, with median onset ages of 37, 33 and 22 years, respectively. Biliary events and bone health problems were common across PKLR genotypes. Among 11 patients who had thromboembolic events, eight had undergone prior splenectomy. Patients with PK deficiency may have many complications, which can occur early in and throughout life. Awareness of their high disease burden may help clinicians better provide appropriate monitoring and management of these patients.
Assuntos
Anemia Hemolítica Congênita não Esferocítica , Piruvato Quinase , Erros Inatos do Metabolismo dos Piruvatos , Sistema de Registros , Humanos , Piruvato Quinase/deficiência , Piruvato Quinase/genética , Masculino , Feminino , Adulto , Criança , Anemia Hemolítica Congênita não Esferocítica/genética , Anemia Hemolítica Congênita não Esferocítica/epidemiologia , Erros Inatos do Metabolismo dos Piruvatos/genética , Erros Inatos do Metabolismo dos Piruvatos/epidemiologia , Adolescente , Pré-Escolar , Lactente , Comorbidade , Pessoa de Meia-Idade , Esplenectomia , Adulto Jovem , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/epidemiologia , Sobrecarga de Ferro/etiologia , Sobrecarga de Ferro/epidemiologia , Doenças Ósseas Metabólicas/etiologia , Doenças Ósseas Metabólicas/epidemiologia , Recém-NascidoRESUMO
It is well established that pyruvate kinase M2 (PKM2) activity contributes to metabolic reprogramming in various cancers, including colorectal cancer (CRC). Estrogen or 17ß-estradiol (E2) signaling is also known to modulate glycolysis markers in cancer cells. However, whether the inhibition of PKM2 combined with E2 treatment could adversely affect glucose metabolism in CRC cells remains to be investigated. First, we confirmed the metabolic plasticity of CRC cells under varying environmental conditions. Next, we identified glycolysis markers that were upregulated in CRC patients and assessed in vitro mRNA levels following E2 treatment. We found that PKM2 expression, which is highly upregulated in CRC clinical samples, is not altered by E2 treatment in CRC cells. In this study, glucose uptake, generation of reactive oxygen species (ROS), lactate production, cell viability, and apoptosis were evaluated in CRC cells following E2 treatment, PKM2 silencing, or a combination of both. Compared to individual treatments, combination therapy resulted in a significant reduction in cell viability and enhanced apoptosis. Glucose uptake and ROS production were markedly reduced in PKM2-silenced E2-treated cells. The data presented here suggest that E2 signaling combined with PKM2 inhibition cumulatively targets glucose metabolism in a manner that negatively impacts CRC cell growth. These findings hold promise for novel therapeutic strategies targeting altered metabolic pathways in CRC.
Assuntos
Neoplasias Colorretais , Humanos , Neoplasias Colorretais/patologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/genética , Hormônios Tireóideos/metabolismo , Linhagem Celular Tumoral , Espécies Reativas de Oxigênio/metabolismo , Estrogênios/farmacologia , Proteínas de Ligação a Hormônio da Tireoide , Estradiol/farmacologia , Apoptose/efeitos dos fármacos , Glucose/metabolismo , Proteínas de Transporte/metabolismo , Piruvato Quinase/metabolismo , Piruvato Quinase/antagonistas & inibidores , Piruvato Quinase/genética , Glicólise/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , FemininoRESUMO
Glucose and lipid metabolism dysregulation in skeletal muscle contributes to the development of metabolic disorders. The efficacy of fucoxanthin in alleviating lipid metabolic disorders in skeletal muscle remains poorly understood. In this study, we systematically investigated the impact of fucoxanthin on mitigating lipid deposition and insulin resistance in skeletal muscle employing palmitic acid-induced lipid deposition in C2C12 cells and ob/ob mice. Fucoxanthin significantly alleviated PA-induced skeletal muscle lipid deposition and insulin resistance. In addition, fucoxanthin prominently upregulated the expression of lipid metabolism-related genes (Pparα and Cpt-1), promoting fatty acid ß-oxidation metabolism. Additionally, fucoxanthin significantly increased the expression of Pgc-1α and Tfam, elevated the mtDNA/nDNA ratio, and reduced ROS levels. Further, we identified pyruvate kinase muscle isozyme 1 (PKM1) as a high-affinity protein for fucoxanthin by drug affinity-responsive target stability and LC-MS and confirmed their robust interaction by CETSA, microscale thermophoresis, and circular dichroism. Supplementation with pyruvate, the product of PKM1, significantly attenuated the beneficial effects of fucoxanthin on lipid deposition and insulin resistance. Mechanistically, fucoxanthin reduced glucose glycolysis rate and enhanced mitochondrial biosynthesis and fatty acid ß-oxidation through inhibiting PKM1 activity, thereby alleviating lipid metabolic stress. These findings present a novel clinical strategy for treating metabolic diseases using fucoxanthin.
Assuntos
Resistência à Insulina , Metabolismo dos Lipídeos , Músculo Esquelético , Piruvato Quinase , Xantofilas , Animais , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacos , Xantofilas/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Piruvato Quinase/metabolismo , Piruvato Quinase/genética , Masculino , Humanos , Camundongos Endogâmicos C57BL , Dieta Hiperlipídica/efeitos adversosRESUMO
Most cancer cells reprogram their glucose metabolic pathway from oxidative phosphorylation to aerobic glycolysis for energy production. By reducing enzyme activity of pyruvate kinase M2 (PKM2), cancer cells attain a greater fraction of glycolytic metabolites for macromolecule synthesis needed for rapid proliferation. Here we demonstrate that hydrogen sulfide (H2S) destabilizes the PKM2 tetramer into monomer/dimer through sulfhydration at cysteines, notably at C326, leading to reduced PKM2 enzyme activity and increased PKM2-mediated transcriptional activation. Blocking PKM2 sulfhydration at C326 through amino acid mutation stabilizes the PKM2 tetramer and crystal structure further revealing the tetramer organization of PKM2-C326S. The PKM2-C326S mutant in cancer cells rewires glucose metabolism to mitochondrial respiration, significantly inhibiting tumor growth. In this work, we demonstrate that PKM2 sulfhydration by H2S inactivates PKM2 activity to promote tumorigenesis and inhibiting this process could be a potential therapeutic approach for targeting cancer metabolism.
Assuntos
Glucose , Sulfeto de Hidrogênio , Sulfeto de Hidrogênio/metabolismo , Humanos , Glucose/metabolismo , Animais , Linhagem Celular Tumoral , Camundongos , Piruvato Quinase/metabolismo , Piruvato Quinase/genética , Piruvato Quinase/química , Cisteína/metabolismo , Glicólise , Hormônios Tireóideos/metabolismo , Mutação , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Multimerização Proteica , Camundongos Nus , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Ligação a Hormônio da TireoideRESUMO
Pyruvate kinase M2 (PKM2) is a key glycolytic enzyme interacting with the inositol 1,4,5-trisphosphate receptor (IP3R). This interaction suppresses IP3R-mediated cytosolic [Ca2+] rises. As PKM2 exists in monomeric, dimeric and tetrameric forms displaying different properties including catalytic activity, we investigated the molecular determinants of PKM2 enabling its interaction with IP3Rs. Treatment of HeLa cells with TEPP-46, a compound stabilizing the tetrameric form of PKM2, increased both its catalytic activity and the suppression of IP3R-mediated Ca2+ signals. Consistently, in PKM2 knock-out HeLa cells, PKM2C424L, a tetrameric, highly active PKM2 mutant, but not inactive PKM2K270M or the less active PKM2K305Q, suppressed IP3R-mediated Ca2+ release. Surprisingly, however, in vitro assays did not reveal a direct interaction between purified PKM2 and either the purified Fragment 5 of IP3R1 (a.a. 1932-2216) or the therein located D5SD peptide (a.a. 2078-2098 of IP3R1), the presumed interaction sites of PKM2 on the IP3R. Moreover, on-nucleus patch clamp of heterologously expressed IP3R1 in DT40 cells devoid of endogenous IP3Rs did not reveal any functional effect of purified wild-type PKM2, mutant PKM2 or PKM1 proteins. These results indicate that an additional factor mediates the regulation of the IP3R by PKM2 in cellulo. Immunoprecipitation of GRP75 using HeLa cell lysates co-precipitated IP3R1, IP3R3 and PKM2. Moreover, the D5SD peptide not only disrupted PKM2:IP3R, but also PKM2:GRP75 and GRP75:IP3R interactions. Our data therefore support a model in which catalytically active, tetrameric PKM2 suppresses Ca2+ signaling via the IP3R through a multiprotein complex involving GRP75.
Assuntos
Receptores de Inositol 1,4,5-Trifosfato , Proteínas de Membrana , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Células HeLa , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Piruvato Quinase/metabolismo , Piruvato Quinase/genética , Sinalização do Cálcio , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/genética , Cálcio/metabolismo , Ligação Proteica , Multimerização ProteicaRESUMO
Aerobic glycolysis is a metabolic reprogramming of tumor cells that is essential for sustaining their phenotype of fast multiplication by continuously supplying energy and mass. Pyruvate kinase M2 (PKM2) has a vital role in this process, which has given it high interest as a target for anticancer drug development. With potent toxicity to many types of cancer cells, polyphyllin II (PP2), a steroidal saponin isolated from the herbaceous plant Rhizoma paridis, brought to our attention that it might interfere with the PKM2 activity. In this study, we discovered that PP2 was a novel agonist of PKM2. PP2 activated recombinant PKM2 and changed the protein's oligomeric state to activate intracellular PKM2. At the same time, PP2 suppressed its protein kinase function by decreasing the content of nuclear PKM2. The mRNA levels of its downstream genes, such as Glut1, LDHA, and MYC, were inhibited. In addition, PP2 induced oxidative stress by downregulating the expression and activity of antioxidant proteins such as NQO1, TrxR, and Trx in HT-1080 cells, which in turn led to mitochondrial dysfunction and ultimately induced apoptosis. Moreover, PP2 reduced the proliferation and migration of HT-1080 cells. Thus, targeting the glycolysis pathway offers an unprecedented mode of action for comprehending PP2's pharmacological impacts and advances PP2's further development in fibrosarcoma therapy.
Assuntos
Apoptose , Piruvato Quinase , Saponinas , Linhagem Celular Tumoral , Piruvato Quinase/antagonistas & inibidores , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Saponinas/farmacologia , Saponinas/toxicidade , Ativação Enzimática/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Fibrossarcoma , HumanosRESUMO
Nucleoside triphosphates are indispensable in numerous biological processes, with enzymes involved in their biogenesis playing pivotal roles in cell proliferation. Pyruvate kinase (PYK), commonly regarded as the terminal glycolytic enzyme that generates ATP in tandem with pyruvate, is also capable of synthesizing a wide range of nucleoside triphosphates from their diphosphate precursors. Despite their substrate promiscuity, some PYKs show preference towards specific nucleotides, suggesting an underlying mechanism for differentiating nucleotide bases. However, the thorough characterization of this mechanism has been hindered by the paucity of nucleotide-bound PYK structures. Here, we present crystal structures of Streptococcus pneumoniae PYK in complex with four different nucleotides. These structures facilitate direct comparison of the protein-nucleotide interactions and offer structural insights into its pronounced selectivity for GTP synthesis. Notably, this selectivity is dependent on a sequence motif in the nucleotide recognition site that is widely present among prokaryotic PYKs, particularly in Firmicutes species. We show that pneumococcal cell growth is significantly impaired when expressing a PYK variant with compromised GTP and UTP synthesis activity, underscoring the importance of PYK in maintaining nucleotide homeostasis. Our findings collectively advance our understanding of PYK biochemistry and prokaryotic metabolism.
Assuntos
Guanosina Trifosfato , Modelos Moleculares , Nucleotídeos , Piruvato Quinase , Streptococcus pneumoniae , Piruvato Quinase/metabolismo , Piruvato Quinase/química , Piruvato Quinase/genética , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Especificidade por Substrato , Cristalografia por Raios X , Nucleotídeos/metabolismo , Guanosina Trifosfato/metabolismo , Conformação Proteica , Sítios de Ligação , Ligação Proteica , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genéticaRESUMO
OBJECTIVE: To investigate the expression level of pyruvate kinase M1 (PKM1) in patients with acute myeloid leukaemia (AML) as well as its clinical significance. STUDY DESIGN: A case-control study. Place and Duration of the Study: Department of Haematology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China, from January 2013 to 2023. METHODOLOGY: The expression levels of PKM1 and pyruvate kinase m2 (PKM2) in the bone marrow of 65 AML patients (excluding M3) and 31 healthy volunteers were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), a method that measures fluorescence in real-time. The associations between PKM1, PKM2 expressions, clinical parameters, and the survival and prognosis of AML patients were analysed. RESULTS: AML patients showed higher PKM1 expression compared to controls. The area under the curve (AUC) of the receiver operating characteristics (ROC) was 0.65 (p = 0.017). PKM1 expression was correlated with peripheral blood leukocyte count (r = -0.276, p = 0.026), CCAAT enhancer-binding protein alpha CEBPA mutation (r = -0.306, p = 0.014), and chemotherapy-induced response (r = -0.292, p = 0.018). Patients with high PKM1 expression had a lower remission rate (p = 0.019) and long-term survival rate (p = 0.034) than those with low PKM1 expression. Patients with AML showed a rise in PKM2 levels; however, the variation was not statistically significant (p >0.05). CONCLUSION: PKM1 expression is upregulated in AML and patients with high PKM1 expression have a lower survival rate. KEY WORDS: PKM1, Acute myeloid leukaemia, Clinical prognosis.
Assuntos
Proteínas de Transporte , Leucemia Mieloide Aguda , Proteínas de Membrana , Proteínas de Ligação a Hormônio da Tireoide , Hormônios Tireóideos , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Estudos de Casos e Controles , China/epidemiologia , Leucemia Mieloide Aguda/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Prognóstico , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Hormônios Tireóideos/sangue , Hormônios Tireóideos/metabolismoRESUMO
The endogenous mitochondrial quality control (MQC) system serves to protect mitochondria against cellular stressors. Although mitochondrial dysfunction contributes to cardiac damage during many pathological conditions, the regulatory signals influencing MQC disruption during septic cardiomyopathy (SC) remain unclear. This study aimed to investigate the involvement of pyruvate kinase M2 (PKM2) and prohibitin 2 (PHB2) interaction followed by MQC impairment in the pathogenesis of SC. We utilized LPS-induced SC models in PKM2 transgenic (PKM2TG) mice, PHB2S91D-knockin mice, and PKM2-overexpressing HL-1 cardiomyocytes. After LPS-induced SC, cardiac PKM2 expression was significantly downregulated in wild-type mice, whereas PKM2 overexpression in vivo sustained heart function, suppressed myocardial inflammation, and attenuated cardiomyocyte death. PKM2 overexpression relieved sepsis-related mitochondrial damage via MQC normalization, evidenced by balanced mitochondrial fission/fusion, activated mitophagy, restored mitochondrial biogenesis, and inhibited mitochondrial unfolded protein response. Docking simulations, co-IP, and domain deletion mutant protein transfection experiments showed that PKM2 phosphorylates PHB2 at Ser91, preventing LPS-mediated PHB2 degradation. Additionally, the A domain of PKM2 and the PHB domain of PHB2 are required for PKM2-PHB2 binding and PHB2 phosphorylation. After LPS exposure, expression of a phosphorylation-defective PHB2S91A mutant negated the protective effects of PKM2 overexpression. Moreover, knockin mice expressing a phosphorylation-mimetic PHB2S91D mutant showed improved heart function, reduced inflammation, and preserved mitochondrial function following sepsis induction. Abundant PKM2 expression is a prerequisite to sustain PKM2-PHB2 interaction which is a key element for preservation of PHB2 phosphorylation and MQC, presenting novel interventive targets for the treatment of septic cardiomyopathy.
Assuntos
Cardiomiopatias , Mitocôndrias Cardíacas , Proibitinas , Piruvato Quinase , Proteínas Repressoras , Sepse , Animais , Humanos , Masculino , Camundongos , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias Cardíacas/metabolismo , Mitofagia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosforilação , Piruvato Quinase/metabolismo , Piruvato Quinase/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Sepse/metabolismoRESUMO
Rheostat positions, which can be substituted with various amino acids to tune protein function across a range of outcomes, are a developing area for advancing personalized medicine and bioengineering. Current methods cannot accurately predict which proteins contain rheostat positions or their substitution outcomes. To compare the prevalence of rheostat positions in homologs, we previously investigated their occurrence in two pyruvate kinase (PYK) isozymes. Human liver PYK contained numerous rheostat positions that tuned the apparent affinity for the substrate phosphoenolpyruvate (Kapp-PEP) across a wide range. In contrast, no functional rheostat positions were identified in Zymomonas mobilis PYK (ZmPYK). Further, the set of ZmPYK substitutions included an unusually large number that lacked measurable activity. We hypothesized that the inactive substitution variants had reduced protein stability, precluding detection of Kapp-PEP tuning. Using modified buffers, robust enzymatic activity was obtained for 19 previously-inactive ZmPYK substitution variants at three positions. Surprisingly, both previously-inactive and previously-active substitution variants all had Kapp-PEP values close to wild-type. Thus, none of the three positions were functional rheostat positions, and, unlike human liver PYK, ZmPYK's Kapp-PEP remained poorly tunable by single substitutions. To directly assess effects on stability, we performed thermal denaturation experiments for all ZmPYK substitution variants. Many diminished stability, two enhanced stability, and the three positions showed different thermal sensitivity to substitution, with one position acting as a "stability rheostat." The differences between the two PYK homologs raises interesting questions about the underlying mechanism(s) that permit functional tuning by single substitutions in some proteins but not in others.
Assuntos
Piruvato Quinase , Zymomonas , Humanos , Zymomonas/enzimologia , Zymomonas/genética , Zymomonas/química , Zymomonas/metabolismo , Piruvato Quinase/química , Piruvato Quinase/metabolismo , Piruvato Quinase/genética , Substituição de Aminoácidos , Estabilidade Proteica , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Estabilidade Enzimática , Fígado/enzimologia , Fígado/metabolismo , Fígado/química , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato/químicaRESUMO
Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle- and liver/erythrocyte-specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle-specific enzymes, PKM1 and PKM2, function in glycolysis, but PKM2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of PKM1 and PKM2 during myoblast differentiation. RNA-seq analysis revealed that PKM2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. DPF2 and BAF250a are subunits that identify a specific sub-family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for the activation of myogenic gene expression during differentiation. PKM2 also mediated the incorporation of DPF2 and BAF250a into the regulatory sequences controlling myogenic gene expression. PKM1 did not affect expression but was required for nuclear localization of DPF2. Additionally, PKM2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for PKM2 and a novel function for PKM1 in gene expression and chromatin regulation during myoblast differentiation.
Assuntos
Diferenciação Celular , Histonas , Mioblastos , Piruvato Quinase , Animais , Piruvato Quinase/metabolismo , Piruvato Quinase/genética , Camundongos , Fosforilação , Histonas/metabolismo , Histonas/genética , Mioblastos/metabolismo , Mioblastos/citologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ligação a Hormônio da Tireoide , Humanos , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Isoenzimas/metabolismo , Isoenzimas/genéticaRESUMO
BACKGROUND: The regenerative capacity of the adult mammalian hearts is limited. Numerous studies have explored mechanisms of adult cardiomyocyte cell-cycle withdrawal. This translational study evaluated the effects and underlying mechanism of rhCHK1 (recombinant human checkpoint kinase 1) on the survival and proliferation of cardiomyocyte and myocardial repair after ischemia/reperfusion injury in swine. METHODS AND RESULTS: Intramyocardial injection of rhCHK1 protein (1 mg/kg) encapsulated in hydrogel stimulated cardiomyocyte proliferation and reduced cardiac inflammation response at 3 days after ischemia/reperfusion injury, improved cardiac function and attenuated ventricular remodeling, and reduced the infarct area at 28 days after ischemia/reperfusion injury. Mechanistically, multiomics sequencing analysis demonstrated enrichment of glycolysis and mTOR (mammalian target of rapamycin) pathways after rhCHK1 treatment. Co-Immunoprecipitation (Co-IP) experiments and protein docking prediction showed that CHK1 (checkpoint kinase 1) directly bound to and activated the Serine 37 (S37) and Tyrosine 105 (Y105) sites of PKM2 (pyruvate kinase isoform M2) to promote metabolic reprogramming. We further constructed plasmids that knocked out different CHK1 and PKM2 amino acid domains and transfected them into Human Embryonic Kidney 293T (HEK293T) cells for CO-IP experiments. Results showed that the 1-265 domain of CHK1 directly binds to the 157-400 amino acids of PKM2. Furthermore, hiPSC-CM (human iPS cell-derived cardiomyocyte) in vitro and in vivo experiments both demonstrated that CHK1 stimulated cardiomyocytes renewal and cardiac repair by activating PKM2 C-domain-mediated cardiac metabolic reprogramming. CONCLUSIONS: This study demonstrates that the 1-265 amino acid domain of CHK1 binds to the 157-400 domain of PKM2 and activates PKM2-mediated metabolic reprogramming to promote cardiomyocyte proliferation and myocardial repair after ischemia/reperfusion injury in adult pigs.
Assuntos
Proliferação de Células , Quinase 1 do Ponto de Checagem , Modelos Animais de Doenças , Traumatismo por Reperfusão Miocárdica , Miócitos Cardíacos , Animais , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/genética , Quinase 1 do Ponto de Checagem/metabolismo , Quinase 1 do Ponto de Checagem/genética , Humanos , Piruvato Quinase/metabolismo , Piruvato Quinase/genética , Células HEK293 , Suínos , Reprogramação Celular , Proteínas de Ligação a Hormônio da Tireoide , Regeneração , Ligação Proteica , Sus scrofa , Remodelação Ventricular/fisiologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Metabolismo Energético/efeitos dos fármacos , Hormônios Tireóideos/metabolismo , Reprogramação MetabólicaRESUMO
Prior studies have emphasized a bioenergetic crisis in the retinal pigment epithelium (RPE) as a critical factor in the development of age-related macular degeneration (AMD). The isoforms Fructose-1,6-bisphosphate aldolase C (ALDOC) and pyruvate kinase M2 (PKM2) have been proposed to play a role in AMD pathogenesis. While PKM2 and ALDOC are crucial for aerobic glycolysis in the neural retina, they are not as essential for the RPE. In this study, we examined the expression and activity of PKM2 and ALDOC in both young and aged RPE cells, as well as in the retina and RPE tissue of mice, including an experimentally induced AMD mouse model. Our findings reveal an upregulation in PKM2 and ALDOC expression, accompanied by increased pyruvate kinase activity, in the aged and AMD mouse RPE. Conversely, there is a decrease in ALDOC expression but an increase in PKM2 expression and pyruvate kinase activity in the aged and AMD retina. Overall, our study indicates that aged and AMD RPE cells tend to favor aerobic glycolysis, while this tendency is diminished in the aged and AMD retina. These results underscore the significance of targeting PKM2 and ALDOC in the RPE as a promising therapeutic approach to address the bioenergetic crisis and prevent vision loss in AMD.
Assuntos
Frutose-Bifosfato Aldolase , Glicólise , Degeneração Macular , Piruvato Quinase , Epitélio Pigmentado da Retina , Animais , Piruvato Quinase/metabolismo , Piruvato Quinase/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Degeneração Macular/genética , Frutose-Bifosfato Aldolase/metabolismo , Frutose-Bifosfato Aldolase/genética , Camundongos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/enzimologia , Envelhecimento/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Modelos Animais de DoençasRESUMO
Protein post-translational modifications (PTMs) play an essential role in meat quality development. However, the effect of specific PTM sites on meat proteins has not been investigated yet. The characteristics of pyruvate kinase M (PKM) were found to exhibit a close correlation with final meat quality, and thus, serine 99 (S99) and lysine 137 (K137) in PKM were mutated to study their effect on PKM function. The structural and functional properties of five lamb PKM variants, including wild-type PKM (wtPKM), PKM_S99D (S99 phosphorylation), PKM_S99A (PKM S99 dephosphorylation), PKM_K137Q (PKM K137 acetylation), and PKM_K137R (PKM K137 deacetylation), were evaluated. The results showed that the secondary structure, tertiary structure, and polymer formation were affected among different PKM variants. In addition, the glycolytic activity of PKM_K137Q was decreased because of its weakened binding with phosphoenolpyruvate. In the PKM_K137R variant, the actin phosphorylation level exhibited a decrease, suggesting a low kinase activity of PKM_K137R. The results of molecular simulation showed a 42% reduction in the interface area between PKM_K137R and actin, in contrast to wtPKM and actin. These findings are significant for revealing the mechanism of how PTMs regulate PKM function and provide a theoretical foundation for the development of precise meat quality preservation technology.
Assuntos
Glicólise , Piruvato Quinase , Piruvato Quinase/metabolismo , Piruvato Quinase/genética , Piruvato Quinase/química , Fosforilação , Animais , Acetilação , Ovinos , Processamento de Proteína Pós-Traducional , Proteínas Quinases/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/química , Carne/análiseRESUMO
BACKGROUND: Glioma is a common intracranial tumor, exhibiting a high degree of aggressiveness and invasiveness. Pyruvate kinase M2 (PKM2) is overexpressed in glioma tissues. However, the biological role of PKM2 in glioma is unclear. METHODS: The qRT-PCR, CCK-8, Transwell, flow cytometry detection, western blot assays, ELISA assay, and pyruvate kinase activity assays were performed in glioma cells transfected with PKM2 shRNA to explore the function of PKM2 in glioma progression. Then, STRING website was used to predict the proteins that interacted with PKM2, and Co-IP assay was conducted to further validate their interaction. Subsequently, the above experiments were performed again to find the effect of catenin beta 1 (CTNNB1) overexpression on PKM2-deficient glioma cells. The transplanted tumor models were also established to further validate our findings. RESULTS: PKM2 was up-regulated in glioma cells and tissues. After inhibiting PKM2, the proliferation, migration, glycolysis, and EMT of glioma cells were significantly decreased, and the proportion of apoptosis was increased. The prediction results of STRING website showed that CTNNB1 and PKM2 had the highest interaction score. The correlation between CTNNB1 and PKM2 was further confirmed by Co-IP test. PKM2 knockdown suppressed glioma cell proliferation, migration, glycolysis, and EMT, while CTNNB1 overexpression rescued these inhibitory effects. Correspondingly, PKM2 knockdown inhibited glioma growth in vivo. CONCLUSION: In summary, these findings indicated that PKM2 promotes glioma progression by mediating CTNNB1 expression, providing a possible molecular marker for the clinical management of gliomas.
Assuntos
Neoplasias Encefálicas , Proliferação de Células , Progressão da Doença , Glioma , Proteínas de Ligação a Hormônio da Tireoide , Hormônios Tireóideos , beta Catenina , Glioma/patologia , Glioma/genética , Glioma/metabolismo , beta Catenina/metabolismo , beta Catenina/genética , Humanos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Animais , Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Camundongos Nus , Movimento Celular/fisiologia , Apoptose/fisiologia , Regulação Neoplásica da Expressão Gênica , Masculino , Piruvato Quinase/metabolismo , Piruvato Quinase/genéticaRESUMO
One of the hallmarks of cancer is metabolic reprogramming in tumor cells, and aerobic glycolysis is the primary mechanism by which glucose is quickly transformed into lactate. As one of the primary rate-limiting enzymes, pyruvate kinase (PK) M is engaged in the last phase of aerobic glycolysis. Alternative splicing is a crucial mechanism for protein diversity, and it promotes PKM precursor mRNA splicing to produce PKM2 dominance, resulting in low PKM1 expression. Specific splicing isoforms are produced in various tissues or illness situations, and the post-translational modifications are linked to numerous disorders, including cancers. hnRNPs are one of the main components of the splicing factor families. However, there have been no comprehensive studies on hnRNPs regulating PKM alternative splicing. Therefore, this review focuses on the regulatory network of hnRNPs on PKM pre-mRNA alternative splicing in tumors and clinical drug research. We elucidate the role of alternative splicing in tumor progression, prognosis, and the potential mechanism of abnormal RNA splicing. We also summarize the drug targets retarding tumorous splicing events, which may be critical to improving the specificity and effectiveness of current therapeutic interventions.