RESUMO
Protein post-translational modifications (PTMs) are crucial for cancer cells to adapt to hypoxia; however, the functional significance of lysine crotonylation (Kcr) in hypoxia remains unclear. Herein we report a quantitative proteomics analysis of global crotonylome under normoxia and hypoxia, and demonstrate 128 Kcr site alterations across 101 proteins in MDA-MB231 cells. Specifically, we observe a significant decrease in K131cr, K156cr and K220cr of phosphoglycerate kinase 1 (PGK1) upon hypoxia. Enoyl-CoA hydratase 1 (ECHS1) is upregulated and interacts with PGK1, leading to the downregulation of PGK1 Kcr under hypoxia. Abolishment of PGK1 Kcr promotes glycolysis and suppresses mitochondrial pyruvate metabolism by activating pyruvate dehydrogenase kinase 1 (PDHK1). A low PGK1 K131cr level is correlated with malignancy and poor prognosis of breast cancer. Our findings show that PGK1 Kcr is a signal in coordinating glycolysis and the tricarboxylic acid (TCA) cycle and may serve as a diagnostic indicator for breast cancer.
Assuntos
Neoplasias da Mama , Ciclo do Ácido Cítrico , Glicólise , Fosfoglicerato Quinase , Fosfoglicerato Quinase/metabolismo , Fosfoglicerato Quinase/genética , Humanos , Glicólise/genética , Linhagem Celular Tumoral , Feminino , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Regulação para Baixo , Camundongos , Proteômica/métodos , Camundongos Nus , Regulação Neoplásica da Expressão Gênica , Mitocôndrias/metabolismo , Hipóxia Celular , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil/genéticaRESUMO
Although certain drivers of familial Parkinson's disease (PD) compromise mitochondrial integrity, whether metabolic deficits underly other idiopathic or genetic origins of PD is unclear. Here, we demonstrate that phosphoglycerate kinase 1 (PGK1), a gene in the PARK12 susceptibility locus, is rate limiting in neuronal glycolysis and that modestly increasing PGK1 expression boosts neuronal adenosine 5'-triphosphate production kinetics that is sufficient to suppress PARK20-driven synaptic dysfunction. We found that this activity enhancement depends on the molecular chaperone PARK7/DJ-1, whose loss of function significantly disrupts axonal bioenergetics. In vivo, viral expression of PGK1 confers protection of striatal dopamine axons against metabolic lesions. These data support the notion that bioenergetic deficits may underpin PD-associated pathologies and point to improving neuronal adenosine 5'-triphosphate production kinetics as a promising path forward in PD therapeutics.
Assuntos
Neurônios , Doença de Parkinson , Fosfoglicerato Quinase , Fosfoglicerato Quinase/metabolismo , Fosfoglicerato Quinase/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Animais , Neurônios/metabolismo , Camundongos , Humanos , Glicólise , Trifosfato de Adenosina/metabolismo , Metabolismo EnergéticoRESUMO
BACKGROUND: In prior research employing iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) technology, we identified a range of proteins in breast cancer tissues exhibiting high levels of acetylation. Despite this advancement, the specific functions and implications of these acetylated proteins in the context of cancer biology have yet to be elucidated. This study aims to systematically investigate the functional roles of these acetylated proteins with the objective of identifying potential therapeutic targets within breast cancer pathophysiology. METHODS: Acetylated targets were identified through bioinformatics, with their expression and acetylation subsequently confirmed. Proteomic analysis and validation studies identified potential acetyltransferases and deacetylases. We evaluated metabolic functions via assays for catalytic activity, glucose consumption, ATP levels, and lactate production. Cell proliferation and metastasis were assessed through viability, cycle analysis, clonogenic assays, PCNA uptake, wound healing, Transwell assays, and MMP/EMT marker detection. RESULTS: Acetylated proteins in breast cancer were primarily involved in metabolism, significantly impacting glycolysis and the tricarboxylic acid cycle. Notably, PGK1 showed the highest acetylation at lysine 323 and exhibited increased expression and acetylation across breast cancer tissues, particularly in T47D and MCF-7 cells. Notably, 18 varieties acetyltransferases or deacetylases were identified in T47D cells, among which p300 and Sirtuin3 were validated for their interaction with PGK1. Acetylation at 323 K enhanced PGK1's metabolic role by boosting its activity, glucose uptake, ATP production, and lactate output. This modification also promoted cell proliferation, as evidenced by increased viability, S phase ratio, clonality, and PCNA levels. Furthermore, PGK1-323 K acetylation facilitated metastasis, improving wound healing, cell invasion, and upregulating MMP2, MMP9, N-cadherin, and Vimentin while downregulating E-cadherin. CONCLUSION: PGK1-323 K acetylation was significantly elevated in T47D and MCF-7 luminal A breast cancer cells and this acetylation could be regulated by p300 and Sirtuin3. PGK1-323 K acetylation promoted cell glycolysis, proliferation, and metastasis, highlighting novel epigenetic targets for breast cancer therapy.
Assuntos
Neoplasias da Mama , Proliferação de Células , Glicólise , Lisina , Fosfoglicerato Quinase , Humanos , Fosfoglicerato Quinase/metabolismo , Fosfoglicerato Quinase/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Feminino , Acetilação , Lisina/metabolismo , Sirtuína 3/metabolismo , Células MCF-7 , Linhagem Celular Tumoral , Proteômica/métodos , Metástase Neoplásica , Movimento Celular , Regulação Neoplásica da Expressão GênicaRESUMO
Gastric ulcer is a highly prevalent digestive tract disease across the world, which is recurrent and hard to cure, sometimes transforming into gastric cancer if left untreated, posing great threat to human health. To develop new medicines for gastric ulcer, we ran a series of screens with ethanol stress model in GES-1 cells, and we uncovered that lamivudine rescued cells from ethanol toxicity. Then, we confirmed this discovery using the well-established ethanol-induced gastric ulcer model in mice and our findings suggest that lamivudine can directly activate phosphoglycerate kinase 1 (PGK1, EC 2.7.2.3), which binds and stimulates superoxide dismutase 1 (SOD1, EC 1.15.1.1) to inhibit ferroptosis and ultimately improve gastric ulcer. Moreover, AAV-PGK1 exhibited comparable gastroprotective effects to lamivudine. The findings are expected to offer novel therapeutic strategies for gastric ulcer, encompassing both lamivudine and AAV-PGK1.
Assuntos
Ferroptose , Lamivudina , Camundongos Endogâmicos C57BL , Fosfoglicerato Quinase , Úlcera Gástrica , Animais , Úlcera Gástrica/prevenção & controle , Úlcera Gástrica/induzido quimicamente , Úlcera Gástrica/tratamento farmacológico , Úlcera Gástrica/metabolismo , Úlcera Gástrica/patologia , Camundongos , Fosfoglicerato Quinase/metabolismo , Fosfoglicerato Quinase/genética , Ferroptose/efeitos dos fármacos , Ferroptose/fisiologia , Humanos , Lamivudina/farmacologia , Masculino , Etanol , Linhagem Celular , Superóxido Dismutase-1/metabolismo , Superóxido Dismutase-1/genéticaRESUMO
BACKGROUND: Acute hypobaric hypoxia-induced brain injury has been a challenge in the health management of mountaineers; therefore, new neuroprotective agents are urgently required. Meldonium, a well-known cardioprotective drug, has been reported to have neuroprotective effects. However, the relevant mechanisms have not been elucidated. We hypothesized that meldonium may play a potentially novel role in hypobaric hypoxia cerebral injury. METHODS: We initially evaluated the neuroprotection efficacy of meldonium against acute hypoxia in mice and primary hippocampal neurons. The potential molecular targets of meldonium were screened using drug-target binding Huprot™ microarray chip and mass spectrometry analyses after which they were validated with surface plasmon resonance (SPR), molecular docking, and pull-down assay. The functional effects of such binding were explored through gene knockdown and overexpression. RESULTS: The study clearly shows that pretreatment with meldonium rapidly attenuates neuronal pathological damage, cerebral blood flow changes, and mitochondrial damage and its cascade response to oxidative stress injury, thereby improving survival rates in mice brain and primary hippocampal neurons, revealing the remarkable pharmacological efficacy of meldonium in acute high-altitude brain injury. On the one hand, we confirmed that meldonium directly interacts with phosphoglycerate kinase 1 (PGK1) to promote its activity, which improved glycolysis and pyruvate metabolism to promote ATP production. On the other hand, meldonium also ameliorates mitochondrial damage by PGK1 translocating to mitochondria under acute hypoxia to regulate the activity of TNF receptor-associated protein 1 (TRAP1) molecular chaperones. CONCLUSION: These results further explain the mechanism of meldonium as an energy optimizer and provide a strategy for preventing acute hypobaric hypoxia brain injury at high altitudes.
Assuntos
Lesões Encefálicas , Fosfoglicerato Quinase , Animais , Fosfoglicerato Quinase/metabolismo , Fosfoglicerato Quinase/genética , Camundongos , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Masculino , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Hipocampo/metabolismo , Hipóxia/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismoRESUMO
BACKGROUND: Diallyl trisulfide (DATS) has a direct antioxidant capacity and emerges as a promising neuroprotective agent. This study was designed to investigate the role of DATS in traumatic brain injury (TBI). METHODS: TBI mouse models were established using the controlled cortical impact, followed by DATS administration. The effects of DATS on neurological deficit, brain damage, inflammation and phosphoglycerate kinase 1 (PGK1) expression were detected using mNSS test, histological analysis, TUNEL assay, enzyme-linked immunosorbent assay and immunofluorescence. PC12 cells were subjected to H2O2-induced oxidative injury after pre-treatment with DATS, followed by cell counting kit-8 assay, flow cytometry and ROS production detection. Apoptosis-related proteins and the PGK1/nuclear factor erythroid-2 related factor 2 (Nrf2) pathway were examined using Western blot. RESULTS: DATS ameliorated the cerebral cortex damage, neurological dysfunction and apoptosis, as well as decreased PGK1 expression and expressions of pro-inflammatory cytokines (IL-6, IL-1ß, TNF-α) in mice after TBI. DATS also enhanced viability, blocked apoptosis and inhibited ROS production in H2O2-induced PC12 cells. DATS downregulated Cleaved-Caspase3, Bax and PGK1 levels, and upregulated Bcl-2 and Nrf2 levels in TBI mouse models and the injured cells. CONCLUSION: DATS regulates PGK1/Nrf2 expression and inflammation to alleviate neurological damage in mice after TBI.
Assuntos
Compostos Alílicos , Apoptose , Lesões Encefálicas Traumáticas , Fator 2 Relacionado a NF-E2 , Fosfoglicerato Quinase , Sulfetos , Animais , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/patologia , Sulfetos/farmacologia , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Fosfoglicerato Quinase/metabolismo , Compostos Alílicos/farmacologia , Células PC12 , Masculino , Apoptose/efeitos dos fármacos , Inflamação/metabolismo , Inflamação/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Ratos , Estresse Oxidativo/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Antioxidantes/farmacologiaRESUMO
Ubiquitination plays a pivotal regulatory role in tumor progression. Among the components of the ubiquitin-proteasome system (UPS), ubiquitin-protein ligase E3 has emerged as a key molecule. Nevertheless, the biological functions of E3 ubiquitin ligases and their potential mechanisms orchestrating glycolysis in gastric cancer (GC) remain to be elucidated. In this study, we conducted a comprehensive transcriptomic analysis to identify the core E3 ubiquitin ligases in GC, followed by extensive validation of the expression patterns and clinical significance of Tripartite motif-containing 50 (TRIM50) both in vitro and in vivo. Remarkably, we found that TRIM50 was downregulated in GC tissues, associated with malignant progression and poor patient survival. Functionally, overexpression of TRIM50 suppressed GC cell proliferation and indirectly mitigated the invasion and migration of GC cells by inhibiting the M2 polarization of tumor-associated macrophages (TAMs). Mechanistically, TRIM50 inhibited the glycolytic pathway by ubiquitinating Phosphoglycerate Kinase 1 (PGK1), thereby directly suppressing GC cell proliferation. Simultaneously, the reduction in lactate led to diminished M2 polarization of TAMs, indirectly inhibiting the invasion and migration of GC cells. Notably, the downregulation of TRIM50 in GC was mediated by the METTL3/YTHDF2 axis in an m6A-dependent manner. In our study, we definitively identified TRIM50 as a tumor suppressor gene (TSG) that effectively inhibits glycolysis and the malignant progression of GC by ubiquitinating PGK1, thus offering novel insights and promising targets for the diagnosis and treatment of GC.
Assuntos
Glicólise , Fosfoglicerato Quinase , Neoplasias Gástricas , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Ubiquitinação , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Neoplasias Gástricas/genética , Humanos , Fosfoglicerato Quinase/metabolismo , Fosfoglicerato Quinase/genética , Linhagem Celular Tumoral , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Proliferação de Células/genética , Animais , Camundongos , Camundongos Nus , Progressão da Doença , Movimento Celular/genética , Camundongos Endogâmicos BALB C , Regulação Neoplásica da Expressão Gênica , Metiltransferases/metabolismo , Metiltransferases/genéticaRESUMO
Acute lung injury (ALI) is characterized by an unregulated inflammatory reaction, often leading to severe morbidity and ultimately death. Excessive inflammation caused by M1 macrophage polarization and pyroptosis has been revealed to have a critical role in ALI. Recent study suggests that glycolytic reprogramming is important in the regulation of macrophage polarization and pyroptosis. However, the particular processes underlying ALI have yet to be identified. In this study, we established a Lipopolysaccharide(LPS)-induced ALI model and demonstrated that blocking glycolysis by using 2-Deoxy-D-glucose(2-DG) significantly downregulated the expression of M1 macrophage markers and pyroptosis-related genes, which was consistent with the in vitro results. Furthermore, our research has revealed that Phosphoglycerate Kinase 1(PGK1), an essential enzyme in the glycolysis pathway, interacts with NOD-, LRR- and pyrin domain-containing protein 3(NLRP3). We discovered that LPS stimulation improves the combination of PGK1 and NLRP3 both in vivo and in vitro. Interestingly, the absence of PGK1 reduces the phosphorylation level of NLRP3. Based on in vitro studies with mice bone marrow-derived macrophages (BMDMs), we further confirmed that siPGK1 plays a protective role by inhibiting macrophage pyroptosis and M1 macrophage polarization. The PGK1 inhibitor NG52 suppresses the occurrence of excessive inflammation in ALI. In general, it is plausible to consider a therapeutic strategy that focuses on modulating the relationship between PGK1 and NLRP3 as a means to mitigate the activation of inflammatory macrophages in ALI.
Assuntos
Lesão Pulmonar Aguda , Macrófagos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR , Fosfoglicerato Quinase , Piroptose , Piroptose/fisiologia , Piroptose/efeitos dos fármacos , Animais , Fosfoglicerato Quinase/metabolismo , Fosfoglicerato Quinase/genética , Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/enzimologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Camundongos , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Glicólise/fisiologia , Glicólise/efeitos dos fármacos , Masculino , Lipopolissacarídeos/toxicidade , Camundongos Knockout , Células CultivadasRESUMO
Ischemic stroke is acknowledged as one of the most frequent causes of death and disability, in which neuroinflammation plays a critical role. Emerging evidence supports that the PGK1/Nrf2/HO-1 signaling can modulate inflammation and oxidative injury. Albiflorin (ALB), a main component of Radix paeoniae Alba, possesses anti-inflammatory and antioxidative properties. However, how it exerts a protective role still needs further exploration. In our study, the middle cerebral artery occlusion (MCAO) model was established, and the Longa score was applied to investigate the degree of neurological impairment. Dihydroethidium (DHE) staining and Malondialdehyde (MDA) assay were used to detect the level of lipid peroxidation. 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining was used to measure the infarct area. Evans blue staining was employed to observe the integrality of the blood-brain barrier (BBB). The injury of brain tissue in each group was observed via HE staining. Immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA) and western blot assay were used for the measurement of inflammatory factors and protein levels. We finally observed that ALB relieved cerebral infarction symptoms, attenuated oxidative damage in brain tissues, and reduced neuroinflammation and cell injury in MCAO rats. The overexpression of PGK1 abrogated the protective effect of ALB after experimental cerebral infarction. ALB promoted PGK1 degradation and induced Nrf2 signaling cascade activation for subsequent anti-inflammatory and antioxidant damage. Generally speaking, ALB exerted a protective role in treating cerebral ischemia, and it might target at PGK1/Nrf2/HO-1 signaling. Thus, ALB might be a potential therapeutic agent to alleviate neuroinflammation and protect brain cells after cerebral infarction.
Assuntos
Anti-Inflamatórios , Infarto da Artéria Cerebral Média , Fator 2 Relacionado a NF-E2 , Fosfoglicerato Quinase , Ratos Sprague-Dawley , Transdução de Sinais , Animais , Fator 2 Relacionado a NF-E2/metabolismo , Masculino , Transdução de Sinais/efeitos dos fármacos , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/patologia , Ratos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Fosfoglicerato Quinase/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Doenças Neuroinflamatórias/tratamento farmacológico , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Modelos Animais de Doenças , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Encéfalo/metabolismo , Humanos , Heme Oxigenase (Desciclizante)/metabolismo , Hidrocarbonetos Aromáticos com PontesRESUMO
Etomidate (ETO), a hypnotic agent used for anesthesia induction, has been shown to induce long-lasting cognitive deficits. In the present study, we investigated whether ETO could activate the HIF1A/PGK1 pathway to antagonize oxidative damage in mice with postoperative cognitive dysfunction (POCD). A mouse model of ETO-mediated POCD was established, and pathological changes, apoptosis, and inflammatory factors in mouse hippocampal tissues were analyzed by HE staining, TUNEL assay, and ELISA. ETO was revealed to cause cognitive dysfunction in mice. Integrated database mining was conducted to screen out transcription factors that are both related to ETO and POCD. Hypoxia-inducible factor 1-alpha (HIF1A) was overexpressed in mice with POCD, and downregulation of HIF1A alleviated cognitive dysfunction in mice. HIF1A downregulation inhibited the transcription of phosphoglycerate kinase 1 (PGK1). Overexpression of PGK1 abated the alleviating effects of HIF1A knockdown on oxidative stress in mice with POCD. In addition, HIF1A activation of PGK1 induced oxidative stress and apoptosis in HT-22 cells while inhibiting cell viability. Taken together, we demonstrated that HIF1A activation of PGK1 induced oxidative stress in ETO-mediated POCD.
Assuntos
Etomidato , Subunidade alfa do Fator 1 Induzível por Hipóxia , Estresse Oxidativo , Fosfoglicerato Quinase , Complicações Cognitivas Pós-Operatórias , Animais , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Fosfoglicerato Quinase/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Complicações Cognitivas Pós-Operatórias/metabolismo , Etomidato/farmacologia , Masculino , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Disfunção Cognitiva/metabolismo , Modelos Animais de DoençasRESUMO
HOXC6 (Homeobox C6) and methyltransferase-like 3 (METTL3) have been shown to be involved in the progression of prostate cancer (PCa). However, whether HOXC6 performs oncogenic effects in PCa via METTL3-mediated N6-methyladenosine (m6A) modification is not yet reported. The Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell, scratch, sphere formation assays were applied for cell growth, invasion, migration and stemness analyses. Glycolysis was evaluated by measuring glucose consumption, lactate generation and ATP/ADP ratio. The N6-methyladenine (m6A) modification profile was determined by RNA immunoprecipitation (Me-RIP) assay. The proteins that interact with PGK1 (phosphoglycerate kinase 1) were confirmed by Co-immunoprecipitation assay. Tumor formation experiments in mice were conducted for in vivo assay. PCa tissues and cells showed highly expressed HOXC6 and METTL3. Functionally, the silencing of HOXC6 or METTL3 suppresses PCa cell proliferation, invasion, migration, stemness, and glycolysis. Moreover, METTL3-induced HOXC6 m6A modification to stabilize its expression. In addition, the m6A reader IGF2BP2 directly recognized and bound to HOXC6 mRNA, and maintained its stability, and was involved in the regulation of HOXC6 expression by METTL3. Furthermore, IGF2BP2 knockdown impaired PCa cell proliferation, invasion, migration, stemness, and glycolysis by regulating HOXC6. Besides that HOXC6 interacted with the glycoytic enzyme PGK1 in PCa cells. In vivo assays further showed that METTL3 silencing reduced the expression of HOXC6 and PGK1, and impeded PCa growth. METTL3 promoted PCa progression by maintaining HOXC6 expression in an m6A-IGF2BP2-dependent mechanism.
Assuntos
Adenosina , Proteínas de Homeodomínio , Metiltransferases , Neoplasias da Próstata , Proteínas de Ligação a RNA , Metiltransferases/metabolismo , Metiltransferases/genética , Masculino , Humanos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Animais , Camundongos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Progressão da Doença , Fosfoglicerato Quinase/metabolismo , Fosfoglicerato Quinase/genética , Linhagem Celular Tumoral , Glicólise , Movimento Celular , Camundongos NusRESUMO
The delay in making a correct diagnosis of Candida auris causes concern in the healthcare system setting, and immunoproteomics studies are important to identify immunoreactive proteins for new diagnostic strategies. In this study, immunocompetent murine systemic infections caused by non-aggregative and aggregative phenotypes of C. auris and by Candida albicans and Candida haemulonii were carried out, and the obtained sera were used to study their immunoreactivity against C. auris proteins. The results showed higher virulence, in terms of infection signs, weight loss, and histopathological damage, of the non-aggregative isolate. Moreover, C. auris was less virulent than C. albicans but more than C. haemulonii. Regarding the immunoproteomics study, 13 spots recognized by sera from mice infected with both C. auris phenotypes and analyzed by mass spectrometry corresponded to enolase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate mutase. These four proteins were also recognized by sera obtained from human patients with disseminated C. auris infection but not by sera obtained from mice infected with C. albicans or Aspergillus fumigatus. Spot identification data are available via ProteomeXchange with the identifier PXD049077. In conclusion, this study showed that the identified proteins could be potential candidates to be studied as new diagnostic or even therapeutic targets for C. auris.
Assuntos
Candida , Candidíase , Imunoglobulina G , Animais , Camundongos , Candida/imunologia , Candida/patogenicidade , Humanos , Candidíase/imunologia , Candidíase/microbiologia , Candidíase/sangue , Imunoglobulina G/sangue , Antígenos de Fungos/imunologia , Antígenos de Fungos/sangue , Proteômica/métodos , Candida albicans/imunologia , Candida albicans/patogenicidade , Proteínas Fúngicas/imunologia , Fosfoglicerato Mutase/imunologia , Fosfoglicerato Quinase/imunologia , Gliceraldeído-3-Fosfato Desidrogenases/imunologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Anticorpos Antifúngicos/sangue , Anticorpos Antifúngicos/imunologia , Feminino , VirulênciaRESUMO
In gene expression (GE) studies, housekeeping genes (HKGs) are required for normalization purposes. In large-scale inter-laboratory comparison studies, significant differences in dose estimates are reported and divergent HKGs are employed by the teams. Among them, the 18S rRNA HKG is known for its robustness. However, the high abundance of 18S rRNA copy numbers requires dilution, which is time-consuming and a possible source of errors. This study was conducted to identify the most promising HKGs showing the least radiation-induced GE variance after radiation exposure. In the screening stage of this study, 35 HKGs were analyzed. This included selected HKGs (ITFG1, MRPS5, and DPM1) used in large-scale biodosimetry studies which were not covered on an additionally employed pre-designed 96-well platform comprising another 32 HKGs used for different exposures. Altogether 41 samples were examined, including 27 ex vivo X-ray irradiated blood samples (0, 0.5, 4 Gy), six X-irradiated samples (0, 0.5, 5 Gy) from two cell lines (U118, A549), as well as eight non-irradiated tissue samples to encompass multiple biological entities. In the independent validation stage, the most suitable candidate genes were examined from another 257 blood samples, taking advantage of already stored material originating from three studies. These comprise 100 blood samples from ex vivo X-ray irradiated (0-4 Gy) healthy donors, 68 blood samples from 5.8 Gy irradiated (cobalt-60) Rhesus macaques (RM) (LD29/60) collected 0-60 days postirradiation, and 89 blood samples from chemotherapy-(CTx) treated breast tumor patients. CTx and radiation-induced GE changes in previous studies appeared comparable. RNA was isolated, converted into cDNA, and GE was quantified employing TaqMan assays and quantitative RT-PCR. We calculated the standard deviation (SD) and the interquartile range (IQR) as measures of GE variance using raw cycle threshold (Ct) values and ranked the HKGs accordingly. Dose, time, age, and sex-dependent GE changes were examined employing the parametrical t-test and non-parametrical Kruskal Wallis test, as well as linear regression analysis. Generally, similar ranking results evolved using either SD or IQR GE measures of variance, indicating a tight distribution of GE values. PUM1 and PGK1 showed the lowest variance among the first ten most suitable genes in the screening phase. MRPL19 revealed low variance among the first ten most suitable genes in the screening phase only for blood and cells, but certain comparisons indicated a weak association of MRPL19 with dose (P = 0.02-0.09). In the validation phase, these results could be confirmed. Here, IQR Ct values from, e.g., X-irradiated blood samples were 0.6 raw Ct values for PUM1 and PGK1, which is considered to represent GE differences as expected due to methodological variance. Overall, when compared, the GE variance of both genes was either comparable or lower compared to 18S rRNA. Compared with the IQR GE values of PUM1 and PGKI, twofold-fivefold increased values were calculated for the biodosimetry HKG HPRT1, and comparable values were calculated for biodosimetry HKGs ITFG1, MRPS5, and DPM1. Significant dose-dependent associations were found for ITFG1 and MRPS5 (P = 0.001-0.07) and widely absent or weak (P = 0.02-0.07) for HPRT1 and DPM1. In summary, PUM1 and PGK1 appeared most promising for radiation exposure studies among the 35 HKGs examined, considering GE variance and adverse associations of GE with dose.
Assuntos
Genes Essenciais , Fosfoglicerato Quinase , Proteínas de Ligação a RNA , Exposição à Radiação , Adulto , Animais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Relação Dose-Resposta à Radiação , Genes Essenciais/efeitos da radiação , Exposição à Radiação/efeitos adversos , Radiometria , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/efeitos da radiação , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/efeitos da radiação , Macaca mulatta , Fosfoglicerato Quinase/genética , Fosfoglicerato Quinase/efeitos da radiaçãoRESUMO
BACKGROUND: Hypoxia is a pathological hallmark in most cancers, including glioblastoma (GBM). Hypoxic signaling activation and post-translational modification (PTM) of oncogenic proteins are well-studied in cancers. Accumulating studies indicate glycolytic enzyme PGK1 plays a crucial role in tumorigenesis, yet the underlying mechanisms remain unknown. METHODS: We first used ChIP assays to uncover the crosstalk between HIF1α and ATF3 and their roles in P4HA1 regulation. Protein degradation analysis, LC-MS/MS, and in vitro succinate production assays were performed to examine the effect of protein succinylation on GBM pathology. Seahorse assay measured the effects of PGK1 succinylation at K191/K192 or its mutants on glucose metabolism. We utilized an in vivo intracranial mouse model for biochemical studies to elucidate the impact of ATF3 and P4HA1 on aerobic glycolysis and the tumor immune microenvironment. RESULTS: We demonstrated that HIF1α and ATF3 positively and negatively regulate the transcription of P4HA1, respectively, leading to an increased succinate production and increased activation of HIF1α signaling. P4HA1 expression elevated the succinate concentration, resulting in the enhanced succinylation of PGK1 at the K191 and K192 sites. Inhibition of proteasomal degradation of PGK1 by succinylation significantly increased aerobic glycolysis to generate lactate. Furthermore, ATF3 overexpression and P4HA1 knockdown reduced succinate and lactate levels in GBM cells, inhibiting immune responses and tumor growth. CONCLUSIONS: Together, our study demonstrates that HIF1α/ATF3 participated in P4HA1/succinate signaling, which is the major regulator of succinate biosynthesis and PGK1 succinylation at K191 and K192 sites in GBM. The P4HA1/succinate pathway might be a novel and promising target for aerobic glycolysis in GBM.
Assuntos
Fator 3 Ativador da Transcrição , Neoplasias Encefálicas , Glioblastoma , Subunidade alfa do Fator 1 Induzível por Hipóxia , Fosfoglicerato Quinase , Transdução de Sinais , Ácido Succínico , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/genética , Fosfoglicerato Quinase/metabolismo , Fosfoglicerato Quinase/genética , Animais , Fator 3 Ativador da Transcrição/metabolismo , Fator 3 Ativador da Transcrição/genética , Camundongos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Ácido Succínico/metabolismo , Regulação Neoplásica da Expressão Gênica , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proliferação de CélulasRESUMO
BACKGROUND: Circular RNAs (circRNAs) have been proved to play crucial roles in the development of various cancers. However, the molecular mechanism of circGLIS3 involved in gastric cancer (GC) tumorigenesis has not been elucidated. METHODS: The higher expression level of circGLIS3 was identified in GC through RNA sequencing and subsequent tissue verification using Quantitative real-time PCR (qRT-PCR). A series of functional experiments in vitro and in vivo were performed to evaluated the effects of circGLIS3 on tumor growth and metastasis in GC. The interaction and regulation of circGLIS3/miR-1343-3p/PGK1 axis was confirmed by RNA pulldown, western blot, and rescue experiments. RIP and western blot were performed to demonstrate the role of circGLIS3 in regulating phosphorylation of VIMENTIN. We then used qRT-PCR and co culture system to trace circGLIS3 transmission via exosomal communication and identify the effect of exosomal circGLIS3 on gastric cancer and macrophages. Finally, RIP experiments were used to determine that EIF4A3 regulates circGLIS3 expression. RESULTS: CircGLIS3(hsa_circ_0002874) was significantly upregulated in GC tissues and high circGLIS3 expression was associated with advanced TNM stage and lymph node metastasis in GC patients. We discovered that overexpression of circGLIS3 promoted GC cell proliferation, migration, invasion in vitro and in vivo, while suppression of circGLIS3 exhibited the opposite effect. Mechanistically, circGLIS3 could sponge miR-1343-3p and up-regulate the expression of PGK1 to promote GC tumorigenesis. We also found that circGLIS3 reduced the phosphorylation of VIMENTIN at ser 83 site by binding with VIMENTIN. Moreover, it was proven that exosomal circGLIS3 could promote gastric cancer metastasis and the M2 type polarization of macrophages. In the final step, the mechanism of EIF4A3 regulating the generation of circGLIS3 was determined. CONCLUSION: Our findings demonstrate that circGLIS3 promotes GC progression through sponging miR-1343-3p and regulating VIMENTIN phosphorylation. CircGLIS3 is a potential therapeutic target for GC patients.
Assuntos
MicroRNAs , Neoplasias Gástricas , Humanos , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células/genética , Transformação Celular Neoplásica , RNA Helicases DEAD-box , Fator de Iniciação 4A em Eucariotos , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Fosfoglicerato Quinase , Fosforilação , Neoplasias Gástricas/genética , Vimentina/genéticaRESUMO
Neuronal energy metabolism dysregulation is involved in various pathologies of Ischemia-reperfusion (I/R), yet the role of RGMA in neuronal metabolic reprogramming has not been reported. In this study, we found that RGMA expression significantly increased after I/R, and compared to control mice, mice with MCAO/R showed an increase in glycolytic metabolic products and the expression of glycolytic pathway proteins. Furthermore, RGMA levels are closely related to neuronal energy metabolism. We discovered that knockdown of RGMA can shift neuronal energy metabolism towards oxidative phosphorylation and the pentose phosphate pathway, thereby protecting mice from ischemic reperfusion injury. Mechanistically, knockdown of RGMA can downregulate PGK1 expression, reducing the increase in glycolytic flux following ischemia reperfusion. Moreover, we found that knockdown of RGMA can reduce the interaction between USP10 and PGK1, thus affecting the ubiquitination degradation of PGK1. In summary, our data suggest that RGMA may regulate neuronal energy metabolism by inhibiting the USP10-mediated deubiquitination of PGK1, thus protecting it from I/R injury. This study provides new ideas for clarifying the intrinsic mechanism of neuronal damage after I/R.
Assuntos
Metabolismo Energético , AVC Isquêmico , Neurônios , Fosfoglicerato Quinase , Traumatismo por Reperfusão , Animais , Humanos , Masculino , Camundongos , Modelos Animais de Doenças , Metabolismo Energético/genética , Técnicas de Silenciamento de Genes , Glicólise/genética , AVC Isquêmico/metabolismo , AVC Isquêmico/genética , AVC Isquêmico/patologia , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Neurônios/patologia , Fosforilação Oxidativa , Via de Pentose Fosfato/genética , Fosfoglicerato Quinase/metabolismo , Fosfoglicerato Quinase/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , UbiquitinaçãoRESUMO
Liquid-liquid phase separation (LLPS) plays a crucial role in various biological processes in eukaryotic organisms, including immune responses in mammals. However, the specific function of LLPS in immune responses in Drosophila melanogaster remains poorly understood. Cactin, a highly conserved protein in eukaryotes, is involved in a non-canonical signaling pathway associated with Nuclear factor-κB (NF-κB)-related pathways in Drosophila. In this study, we investigated the role of Cactin in LLPS and its implications for immune response modulation. We discovered that Cactin undergoes LLPS, forming droplet-like particles, primarily mediated by its intrinsically disordered region (IDR). Utilizing immunoprecipitation and mass spectrometry analysis, we identified two phosphorylation sites at serine residues 99 and 104 within the IDR1 domain of Cactin. Co-immunoprecipitation and mass spectrometry further revealed phosphoglycerate kinase (PGK) as a Cactin-interacting protein responsible for regulating its phosphorylation. Phosphorylation of Cactin by PGK induced a transition from stable aggregates to dynamic liquid droplets, enhancing its ability to interact with other components in the cellular environment. Overexpression of PGK inhibited Drosophila C virus (DCV) replication, while PGK knockdown increased replication. DCV infection also increased Cactin phosphorylation. We also found that phosphorylation enhances the antiviral ability of Cactin by promoting liquid-phase droplet formation. These findings demonstrate the role of Cactin-phase separation in regulating DCV replication and highlight the modulation of its antiviral function through phosphorylation, providing insights into the interplay between LLPS and antiviral defense mechanisms. IMPORTANCE: Liquid-liquid phase separation (LLPS) plays an integral role in various biological processes in eukaryotic organisms. Although several studies have highlighted its crucial role in modulating immune responses in mammals, its function in immune responses in Drosophila melanogaster remains poorly understood. Our study investigated the role of Cactin in LLPS and its implications for immune response modulation. We identified that phosphoglycerate kinase (PGK), an essential enzyme in the glycolytic pathway, phosphorylates Cactin, facilitating its transition from a relatively stable aggregated state to a more dynamic liquid droplet phase during the phase separation process. This transformation allows Cactin to rapidly interact with other cellular components, enhancing its antiviral properties and ultimately inhibiting virus replication. These findings expand our understanding of the role of LLPS in the antiviral defense mechanism, shedding light on the intricate mechanisms underlying immune responses in D. melanogaster.
Assuntos
Proteínas de Transporte , Proteínas de Drosophila , Drosophila melanogaster , Drosophila , Fosfoglicerato Quinase , Animais , Separação de Fases , FosforilaçãoRESUMO
Phosphoglycerate kinase 1 (PGK1) serves as a pivotal enzyme in the cellular glycolysis pathway, facilitating adenosine-triphosphate (ATP) production in tumor cells and driving the Warburg effect. PGK1 generates ATP through the reversible phosphorylation reaction of 1,3-bisphosphoglycerate (1,3-BPG) to Mg-adenosine-5'-diphosphate (Mg-ADP). In addition to its role in regulating cellular metabolism, PGK1 plays a pivotal role in autophagy induction, regulation of the tricarboxylic acid cycle (TCA), and various mechanisms including tumor cell drug resistance, and so on. Given its multifaceted functions within cells, the involvement of PGK1 in many types of cancer, including breast cancer, astrocytoma, metastatic colon cancer, and pancreatic ductal adenocarcinoma, is intricate. Notably, PGK1 can function as an intracellular protein kinase to coordinate tumor growth, migration, and invasion via posttranslational modifications (PTMs). Furthermore, elevated expression levels of PGK1 have been observed in cancer tissues, indicating its association with unfavorable treatment outcomes and prognosis. This review provides a comprehensive summary of PGK1's expression pattern, structural features, functional properties, involvement in PTMs, and interaction with tumors. Additionally highlighted are the prospects for developing and applying related inhibitors that confirm the indispensable value of PGK1 in tumor progression.
Assuntos
Neoplasias do Colo , Fosfoglicerato Quinase , Humanos , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Fosfoglicerato Quinase/genética , Fosfoglicerato Quinase/metabolismo , FosforilaçãoRESUMO
BACKGROUND: Genetic underpinnings in Parkinson's disease (PD) and parkinsonian syndromes are challenging, and recent discoveries regarding their genetic pathways have led to potential gene-specific treatment trials. CASES: We report 3 X-linked levodopa (l-dopa)-responsive parkinsonism-epilepsy syndrome cases due to a hemizygous variant in the phosphoglycerate kinase 1 (PGK1) gene. The likely pathogenic variant NM_000291.4 (PGK1):c.950G > A;p.(Gly317Asp) was identified in a hemizygous state. LITERATURE REVIEW: Only 8 previous cases have linked this phenotype to PGK1, a gene more commonly associated with hemolytic anemia and myopathy. The unusual association of epilepsy, psychiatric symptoms, action tremor, limb dystonia, cognitive symptoms, and l-dopa-responsive parkinsonism must draw attention to PGK1 mutations, especially because this gene is absent from most commercial hereditary parkinsonism panels. CONCLUSIONS: This report aims to shed light on an overlooked gene that causes hereditary parkinsonian syndromes. Further research regarding genetic pathways in PD may provide a better understanding of its pathophysiology and open possibilities for new disease-modifying trials, such as SNCA, LRRK2, PRKN, PINK1, and DJ-1 genes.