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Cell Discovery Feb 2024Inflammasome activation and pyroptotic cell death are known to contribute to the pathogenesis of cardiovascular diseases, such as myocardial ischemia-reperfusion (I/R)...
Inflammasome activation and pyroptotic cell death are known to contribute to the pathogenesis of cardiovascular diseases, such as myocardial ischemia-reperfusion (I/R) injury, although the underlying regulatory mechanisms remain poorly understood. Here we report that expression levels of the E3 ubiquitin ligase membrane-associated RING finger protein 2 (MARCH2) were elevated in ischemic human hearts or mouse hearts upon I/R injury. Genetic ablation of MARCH2 aggravated myocardial infarction and cardiac dysfunction upon myocardial I/R injury. Single-cell RNA-seq analysis suggested that loss of MARCH2 prompted activation of NLRP3 inflammasome in cardiomyocytes. Mechanistically, phosphoglycerate mutase 5 (PGAM5) was found to act as a novel regulator of MAVS-NLRP3 signaling by forming liquid-liquid phase separation condensates with MAVS and fostering the recruitment of NLRP3. MARCH2 directly interacts with PGAM5 to promote its K48-linked polyubiquitination and proteasomal degradation, resulting in reduced PGAM5-MAVS co-condensation, and consequently inhibition of NLRP3 inflammasome activation and cardiomyocyte pyroptosis. AAV-based re-introduction of MARCH2 significantly ameliorated I/R-induced mouse heart dysfunction. Altogether, our findings reveal a novel mechanism where MARCH2-mediated ubiquitination negatively regulates the PGAM5/MAVS/NLRP3 axis to protect against cardiomyocyte pyroptosis and myocardial I/R injury.
PubMed: 38409220
DOI: 10.1038/s41421-023-00622-3 -
Annals of Hepatology Feb 2024Acute liver injury (ALI) is characterized by massive hepatocyte death with high mortality and poor prognosis. Hepatocyte pyroptosis plays a key role in the...
INTRODUCTION AND OBJECTIVES
Acute liver injury (ALI) is characterized by massive hepatocyte death with high mortality and poor prognosis. Hepatocyte pyroptosis plays a key role in the physiopathological processes of ALI, which can damage mitochondria and release NLRP3 inflammasome particles, causing systemic inflammatory responses. Z-DNA Binding Protein 1 (ZBP1) is a sensor that induces cell death. Here, we investigated whether ZBP1 participates in hepatocyte pyroptosis and explored the possible pathogenesis of ALI.
MATERIALS AND METHODS
Hepatocyte pyrotosis was induced with lipopolysaccharide (LPS) and nigericin (Nig), and the expression of Zbp1 (ZBP1) was examined by western blot analysis and RT-qPCR. Further, we transfected AML-12 (LO2 and HepG2) cell lines with Zbp1 (ZBP1) siRNA. After ZBP1 was silenced, LDH release and flow cytometry were used to measure the cell death; Western blot analysis and RT-qPCR were used to detect the marker of NLRP3 inflammasome activation and pyroptosis. We also detected the expression of mitochondrial linear rupture marker phosphoglycerate mutase family member 5 (PGAM5) using western blot analysis and reactive oxygen species (ROS) using the DCFH-DA method.
RESULTS
The expression of ZBP1 was up-regulated in LPS/Nig-induced hepatocytes. Si-Zbp1 (Si-ZBP1) inhibited NLRP3 inflammasome activation and pyroptosis in LPS/Nig-induced hepatocytes. Moreover, ZBP1 silencing inhibited the expression of PGAM5 by reducing ROS production.
CONCLUSIONS
ZBP1 promotes hepatocellular pyroptosis by modulating mitochondrial damage, which facilitates the extracellular release of ROS.
PubMed: 38331384
DOI: 10.1016/j.aohep.2024.101475 -
BioRxiv : the Preprint Server For... Dec 2023Upon nutrient starvation, serovar L2 (CTL) shifts from its normal growth to a non-replicating form, termed persistence. It is unclear if persistence is an adaptive...
Upon nutrient starvation, serovar L2 (CTL) shifts from its normal growth to a non-replicating form, termed persistence. It is unclear if persistence is an adaptive response or lack of it. To understand that transcriptomics data were collected for nutrient-sufficient and nutrient-starved CTL. Applying machine learning approaches on transcriptomics data revealed a global transcriptomic rewiring of CTL under stress conditions without having any global stress regulator. This indicated that CTL's stress response is due to lack of an adaptive response mechanism. To investigate the impact of this on CTL metabolism, we reconstructed a genome-scale metabolic model of CTL (CTL278) and contextualized it with the collected transcriptomics data. Using the metabolic bottleneck analysis on contextualized CTL278, we observed phosphoglycerate mutase () regulates the entry of CTL to the persistence. Later, was found to have the highest thermodynamics driving force and lowest enzymatic cost. Furthermore, CRISPRi-driven knockdown of and tryptophan starvation experiments revealed the importance of this gene in inducing persistence. Hence, this work, for the first time, introduced thermodynamics and enzyme-cost as tools to gain deeper understanding on CTL persistence.
PubMed: 38187683
DOI: 10.1101/2023.12.18.572198 -
Clinical and Translational Medicine Dec 2023Hepatocellular carcinoma (HCC) cells undergo reprogramming of glucose metabolism to support uncontrolled proliferation, of which the intrinsic mechanism still merits...
BACKGROUND
Hepatocellular carcinoma (HCC) cells undergo reprogramming of glucose metabolism to support uncontrolled proliferation, of which the intrinsic mechanism still merits further investigation. Although regulatory factor X6 (RFX6) is aberrantly expressed in different cancers, its precise role in cancer development remains ambiguous.
METHODS
Microarrays of HCC tissues were employed to investigate the expression of RFX6 in tumour and adjacent non-neoplastic tissues. Functional assays were employed to explore the role of RFX6 in HCC development. Chromatin immunoprecipitation, untargeted metabolome profiling and sequencing were performed to identify potential downstream genes and pathways regulated by RFX6. Metabolic assays were employed to investigate the effect of RFX6 on glycolysis in HCC cells. Bioinformatics databases were used to validate the above findings.
RESULTS
HCC tissues exhibited elevated expression of RFX6. High RFX6 expression represented as an independent hazard factor correlated to poor prognosis in patients with HCC. RFX6 deficiency inhibited HCC development in vitro and in vivo, while its overexpression exerted opposite functions. Mechanistically, RFX6 bound to the promoter area of phosphoglycerate mutase 1 (PGAM1) and upregulated its expression. The increased PGAM1 protein levels enhanced glycolysis and further promoted the development of HCC.
CONCLUSIONS
RFX6 acted as a novel driver for HCC development by promoting aerobic glycolysis, disclosing the potential of the RFX6-PGAM1 axis for therapeutic targeting.
Topics: Humans; Carcinoma, Hepatocellular; Cell Proliferation; Glycolysis; Liver Neoplasms; Phosphoglycerate Mutase
PubMed: 38093528
DOI: 10.1002/ctm2.1511 -
CNS Neuroscience & Therapeutics Apr 2024Despite extensive work to identify diagnostic plasma markers for Parkinson's disease (PD), there are still no accepted and validated surrogate biomarkers....
BACKGROUND
Despite extensive work to identify diagnostic plasma markers for Parkinson's disease (PD), there are still no accepted and validated surrogate biomarkers. Mitophagy-associated proteins (MAPs), including PTEN-induced putative kinase 1 (PINK1), Parkin, phosphoglycerate mutase 5 (PGAM5), BCL2 interacting protein 3 (BNIP3), and phosphorylated-TBK1 (p-TBK1), are, to our best knowledge, not well studied as a panel of biomarkers of neurodegeneration in PD.
METHODS
The study population comprised 116 age-matched controls (HC), 179 PD patients, alongside and 90 PD syndromes (PDs) divided between two cohorts: (i) the modeling cohort (cohort 1), including 150 PD, 97 HC, and 80 PDs; and (ii) the validated cohort (cohort 2), including 29 PD, 19 HC, and 10 PDs.
RESULTS
MAPs are elevated in the plasma of PD patients. PINK1, Parkin, and PGAM5 displayed the top three measurable increase trends in amplitude compared to BNIP3 and p-TBK1. Moreover, the area under the curve (AUC) values of PINK1, PGAM5, and Parkin were ranked the top three MAP candidates in diagnosis accuracy for PD from HC, but the MAPs make it hard to differentiate PD from PDs. In addition, there are higher plasma PINK1-Parkin levels and prominent diagnostic accuracy in A-synuclein (+) subjects than in A-synuclein (-) subjects.
CONCLUSIONS
These results uncover that plasma MAPs (PINK1, Parkin, and PGAM5) may be potentially useful diagnostic biomarkers for PD diagnosis. Studies on larger cohorts would be required to test whether elevated plasma MAP levels are related to PD risk or prognosis.
Topics: Humans; Parkinson Disease; Male; Female; Biomarkers; Aged; Middle Aged; Mitophagy; Protein Kinases; Ubiquitin-Protein Ligases; Cohort Studies; Mitochondrial Proteins; Membrane Proteins; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Phosphoprotein Phosphatases
PubMed: 37990436
DOI: 10.1111/cns.14532 -
Biomolecules Sep 2023Hypoxic-ischaemic encephalopathy (HIE) is an important cause of morbidity and mortality globally. Although mild therapeutic hypothermia (TH) may improve outcomes in...
Hypoxic-ischaemic encephalopathy (HIE) is an important cause of morbidity and mortality globally. Although mild therapeutic hypothermia (TH) may improve outcomes in selected babies, the mechanism of action is not fully understood. A proteomics discovery study was carried out to analyse proteins in the plasma of newborns with HIE. Proteomic analysis of plasma from 22 newborns with moderate-severe HIE that had initially undergone TH, and relative controls including 10 newborns with mild HIE who did not warrant TH and also cord blood from 10 normal births (non-HIE) were carried out using the isobaric Tandem Mass Tag (TMT) 10plex labelling with tandem mass spectrometry. A total of 7818 unique peptides were identified in all TMT10plex samples, translating to 3457 peptides representing 405 proteins, after applying stringent filter criteria. Apart from the unique protein signature from normal cord blood, unsupervised analysis revealed several significantly regulated proteins in the TH-treated moderate-severe HIE group. GO annotation and functional clustering revealed various proteins associated with glucose metabolism: the enzymes fructose-bisphosphate aldolase A, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase 1, phosphoglycerate kinase 1, and pyruvate kinase PKM were upregulated in newborns with favourable (sHIE+) outcomes compared to newborns with unfavourable (sHIE-) outcomes. Those with favourable outcomes had normal MR imaging or mild abnormalities not predictive of adverse outcomes. However, in comparison to mild HIE and the sHIE- groups, the sHIE+ group had the additional glucose metabolism-related enzymes upregulated, including triosephosphate isomerase, α-enolase, 6-phosphogluconate dehydrogenase, transaldolase, and mitochondrial glutathione reductase. In conclusion, our plasma proteomic study demonstrates that TH-treated newborns with favourable outcomes have an upregulation in glucose metabolism. These findings may open new avenues for more effective neuroprotective therapy.
Topics: Infant; Humans; Infant, Newborn; Asphyxia; Proteomics; Carbohydrate Metabolism; Tandem Mass Spectrometry; Peptides
PubMed: 37892154
DOI: 10.3390/biom13101471 -
Cancers Sep 2023Phosphoglycerate mutase 5 (PGAM5) is a Ser/His/Thr phosphatase responsible for regulating mitochondrial homeostasis. Overexpression of PGAM5 is correlated with a poor...
Phosphoglycerate mutase 5 (PGAM5) is a Ser/His/Thr phosphatase responsible for regulating mitochondrial homeostasis. Overexpression of PGAM5 is correlated with a poor prognosis in hepatocellular carcinoma, colon cancer, and melanoma. In hepatocellular carcinoma, silencing of PGAM5 reduces growth, which has been attributed to decreased mitophagy and enhanced apoptosis. Yet in colon cancer, PGAM5's pro-tumor survival effect is correlated to lipid metabolism. We sought to identify whether deletion of PGAM5 modulated lipid droplet accrual in hepatocellular carcinoma. HepG2 and Huh7 knockout cell lines generated using CRISPR/Cas9 technology were used to measure cell growth, cellular ATP, and long-chain fatty acid uptake. Expression of hepatocellular fatty acid transporters, cluster of differentiation 36 (CD36), solute carrier family 27 member 2 (SLC27A2), solute carrier family 27 member 5 (SLC27A5), and fatty acid binding protein 1 (FABP1) was measured by quantitative PCR and Western blot. We found that deletion of PGAM5 attenuates hepatocellular carcinoma cell growth and ATP production. Further, knockout ameliorates palmitate-induced steatosis and reduces expression of FABP1 in HepG2 and Huh7 cell lines. PGAM5's role in hepatocellular carcinoma includes regulation of fatty acid metabolism, which may be related to expression of the fatty acid transporter, FABP1.
PubMed: 37835490
DOI: 10.3390/cancers15194796 -
International Journal of Biological... 2023Numerous mitochondrial abnormalities are reported to result from excessive inflammation during endotoxemia. Prohibitin 2 (PHB2) and phosphoglycerate mutase 5 (Pgam5)...
Numerous mitochondrial abnormalities are reported to result from excessive inflammation during endotoxemia. Prohibitin 2 (PHB2) and phosphoglycerate mutase 5 (Pgam5) have been associated with altered mitochondrial homeostasis in several cardiovascular diseases; however, their role in endotoxemia-related myocardial dysfunction has not been explored. Our experiments were aimed to evaluate the potential contribution of Pgam5 and PHB2 to endotoxemia-induced mitochondrial dysfunction in cardiomyocytes, with a focus on two endogenous protective programs that sustain mitochondrial integrity, namely mitophagy and the mitochondrial unfolded protein response (UPR). We found that PHB2 transgenic mice are resistant to endotoxemia-mediated myocardial depression and mitochondrial damage. Our assays indicated that PHB2 overexpression activates mitophagy and the UPR, which maintains mitochondrial metabolism, prevents oxidative stress injury, and enhances cardiomyocyte viability. Molecular analyses further showed that Pgam5 binds to and dephosphorylates PHB2, resulting in cytosolic translocation of mitochondrial PHB2. Silencing of Pgam5 or transfection of a phosphorylated PHB2 mutant in mouse HL-1 cardiomyocytes prevented the loss of mitochondrially-localized PHB2 and activated mitophagy and UPR in the presence of LPS. Notably, cardiomyocyte-specific deletion of Pgam5 attenuated LPS-mediated myocardial dysfunction and preserved cardiomyocyte viability. These findings suggest that Pgam5/PHB2 signaling and mitophagy/UPR are potential targets for the treatment of endotoxemia-related cardiac dysfunction.
Topics: Animals; Mice; Endotoxemia; Lipopolysaccharides; Mitophagy; Phosphoprotein Phosphatases; Prohibitins; Unfolded Protein Response
PubMed: 37781037
DOI: 10.7150/ijbs.85767 -
Frontiers in Immunology 2023Macrophages play a critical role in the regulation of inflammation and tissue homeostasis. In addition to their vital functions for cell survival and physiology,...
Macrophages play a critical role in the regulation of inflammation and tissue homeostasis. In addition to their vital functions for cell survival and physiology, mitochondria play a crucial role in innate immunity as a platform for the induction of inflammatory responses by regulating cell signaling and dynamics. Dynamin-related protein 1 (Drp1) plays a role in the induction of inflammatory responses and the subsequent development of various diseases. PGAM5 (phosphoglycerate mutase member 5) is a mitochondrial outer membrane phosphatase that dephosphorylates its substrate, Drp1. Previous studies showed that PGAM5 regulates the phosphorylation of Drp1 for the activation of NKT cells and T cells. However, it is not clear how PGAM5 regulates Drp1 activity for the induction of inflammation in macrophages. Here, we demonstrate that PGAM5 activity regulates the dephosphorylation of Drp1 in macrophages, leading to the induction of proinflammatory responses in macrophages. In TLR signaling, PGAM5 regulates the expression and production of inflammatory cytokines by regulating the activation of downstream signaling pathways, including the NF-κB and MAPK pathways. Upon LPS stimulation, PGAM5 interacts with Drp1 to form a complex, leading to the production of mtROS. Furthermore, PGAM5-Drp1 signaling promotes the polarization of macrophages toward a proinflammatory phenotype. Our study further demonstrates that PGAM5-Drp1 signaling promotes metabolic reprogramming by upregulating glycolysis and mitochondrial metabolism in macrophages. Altogether, PGAM5 signaling is a linker between alterations in Drp1-mediated mitochondrial dynamics and inflammatory responses in macrophages and may be a target for the treatment of inflammatory diseases.
Topics: Humans; Dynamins; Inflammation; Macrophages; Mitochondrial Proteins; Phosphoprotein Phosphatases; Signal Transduction; Animals
PubMed: 37771598
DOI: 10.3389/fimmu.2023.1243548 -
IScience Oct 2023Mitophagy is critical for maintaining proper cellular functions, and it contributes to the onset and progression of osteoarthritis (OA). A recent study showed that...
Mitophagy is critical for maintaining proper cellular functions, and it contributes to the onset and progression of osteoarthritis (OA). A recent study showed that focused low-intensity pulsed ultrasound (FLIPUS) could activate mitophagy, but the molecular mechanism remains unclear. This study aimed to elucidate the chondroprotective effects of FLIPUS in OA and the regulatory effects on FUN14-domain containing 1 (FUNDC1-mediated mitophagy. , FLIPUS improved inflammatory response, anabolism, and catabolism in interleukin (IL)-1β-induced OA chondrocytes. The chondroprotective effects of FLIPUS were attributed to promoting the expression of phosphoglycerate mutase 5 (PGAM5) and the dephosphorylation of FUNDC1 at serine 13 (Ser13), as well as promoting the mitophagy process. , FLIPUS reduced the cartilage degeneration and apoptosis and reversed the change of anabolic- and catabolic-related proteins in destabilized medial meniscus (DMM)-induced mouse model. Thus, the study indicates that FLIPUS exhibits a chondroprotective effect via activating impaired FUNDC1-mediated mitophagy.
PubMed: 37720103
DOI: 10.1016/j.isci.2023.107772