Mitochondrial Metabolic Enzymes Research Articles

Chemoproteomic Profiling of Signaling Metabolite Fructose-1,6-Bisphosphate Interacting Proteins in Living Cells.

Fructose-1,6-bisphosphate (FBP), a cellular endogenous sugar metabolite in the glycolytic pathway, has recently been reported to act as a signaling molecule to regulate various cellular events through the engagement of important proteins. Though tremendous progress has been made in identifying specific FBP-protein interactions, the comprehensive identification of FBP-interacting proteins and their regulatory mechanisms remains largely unexplored. Here, we describe a concise synthetic approach for the scalable preparation of a photoaffinity FBP probe that enables the quantitative chemoproteomic profiling of FBP-protein interactions based on photoaffinity labeling (PAL) directly in living cells. Using such a protocol, we captured known FBP targets including PKM2 and MDH2. Furthermore, among unknown FBP-interacting proteins, we identified a mitochondrial metabolic enzyme aldehyde dehydrogenase 2 (ALDH2), against which FBP showed inhibitory activity and resulted in cellular ROS upregulation accompanied by mitochondrial fragmentation. Our findings disclosed a new mode of glucose signaling mediating by the FBP-ALDH2-ROS axis.

The mitochondrial carboxylase PCCA interacts with Listeria monocytogenes phospholipase PlcB to modulate bacterial survival.

Mitochondria represent attractive targets for pathogenic bacteria seeking to modulate host cellular processes to promote their survival and replication. Our current study has uncovered mitochondrial carboxylase propionyl-coenzyme A carboxylase (PCCA) as a novel host cell protein that interacts with L. monocytogenes PlcB. The results demonstrate that PCCA plays a negative regulatory role in L. monocytogenes infection, as heightened PCCA levels are associated with reduced bacterial survival and persistence. However, L. monocytogenes may exploit the PlcB-PCCA interaction to maintain stable PCCA expression and establish a favorable intracellular milieu for bacterial infection. Our findings shed new light on the intricate interplay between bacterial pathogens and host cell mitochondria, while also highlighting the potential of mitochondrial metabolic enzymes as antimicrobial agents.

Serine synthesis and catabolism in starved lung cancer and primary bronchial epithelial cells

Serine and glycine give rise to important building blocks in proliferating cells. Both amino acids are either synthesized de novo or taken up from the extracellular space. In lung cancer, serine synthesis gene expression is variable, yet, expression of the initial enzyme, phosphoglycerate dehydrogenase (PHGDH), was found to be associated with poor prognosis. While the contribution of de novo synthesis to serine pools has been shown to be enhanced by serine starvation, the impact of glucose deprivation, a commonly found condition in solid cancers is poorly understood. Here, we utilized a stable isotopic tracing approach to assess serine and glycine de novo synthesis and uptake in different lung cancer cell lines and normal bronchial epithelial cells in variable serine, glycine, and glucose conditions. Under low glucose supplementation (0.2 mM, 3–5% of normal plasma levels), serine de novo synthesis was maintained or even activated. As previously reported, also gluconeogenesis supplied carbons from glutamine to serine and glycine under these conditions. Unexpectedly, low glucose treatment consistently enhanced serine to glycine conversion, along with an up-regulation of the mitochondrial one-carbon metabolism enzymes, serine hydroxymethyltransferase (SHMT2) and methylenetetrahydrofolate dehydrogenase (MTHFD2). The relative contribution of de novo synthesis greatly increased in low serine/glycine conditions. In bronchial epithelial cells, adaptations occurred in a similar fashion as in cancer cells, but serine synthesis and serine to glycine conversion, as assessed by label enrichments and gene expression levels, were generally lower than in (PHGDH positive) cancer cells. In summary, we found a variable contribution of glucose or non-glucose carbon sources to serine and glycine and a high adaptability of the downstream one-carbon metabolism pathway to variable glucose supply.

Comparative genomics analysis reveals sequence characteristics potentially related to host preference in Cryptosporidium xiaoi

Cryptosporidium spp. are important diarrhea-associated pathogens in humans and livestock. Among the known species, Cryptosporidium xiaoi, which causes cryptosporidiosis in sheep and goats, was previously recognized as a genotype of the bovine-specific Cryptosporidium bovis based on their high sequence identity in the ssrRNA gene. However, the lack of genomic data has limited characterization of the genetic differences between the two closely related species. In this study, we sequenced the genomes of two C. xiaoi isolates and performed comparative genomic analysis to identify the sequence uniqueness of this ovine-adapted species compared with other Cryptosporidium spp. Our results showed that C. xiaoi is genetically related to C. bovis as shown by their 95.8% genomic identity and similar gene content. Consistent with this, both C. xiaoi and C. bovis appear to have fewer genes encoding mitochondrial metabolic enzymes and invasion-related protein families. However, they appear to possess several species-specific genes. Further analysis indicates that the sequence differences between these two Cryptosporidium spp. are mainly in 24 highly polymorphic genes, half of which are located in the subtelomeric regions. Some of these subtelomeric genes encode secretory proteins that have undergone positive selection. In addition, the genomes of two C. xiaoi isolates, identified as subtypes XXIIIf and XXIIIh, share 99.9% nucleotide sequence identity, with six highly divergent genes encoding putative secretory proteins. Therefore, these species-specific genes and sequence polymorphism in subtelomeric genes probably contribute to the different host preference of C. xiaoi and C. bovis.

Liver metabolomics reveals potential mechanism of Jieduan-Niwan formula against acute-on-chronic liver failure (ACLF) by improving mitochondrial damage and TCA cycle

BackgroundAcute-on-chronic liver failure (ACLF) is a refractory disease with high mortality, which is characterized by a pathophysiological process of inflammation-related dysfunction of energy metabolism. Jieduan-Niwan formula (JDNWF) is a eutherapeutic Chinese medicine formula for ACLF. However, the intrinsic mechanism of its anti-ACLF effect still need to be studied systematically.PurposeThis study aimed to investigate the mechanism of JDNWF against ACLF based on altered substance metabolic profile in ACLF the expression levels of related molecules.Materials and methodsThe chemical characteristics of JDNWF were characterized using ultra performance liquid chromatography (UPLC) coupled with triple quadrupole mass spectrometry. Wistar rats subjected to a long-term CCL4 stimulation followed by a combination of an acute attack with LPS/D-GalN were used to establish the ACLF model. Liver metabolites were analyzed by LC–MS/MS and multivariate analysis. Liver function, coagulation function, histopathology, mitochondrial metabolic enzyme activity and mitochondrial damage markers were evaluated. The protein expression of mitochondrial quality control (MQC) was investigated by western blot.ResultsLiver function, coagulation function, inflammation, oxidative stress and mitochondrial enzyme activity were significantly improved by JDNWF. 108 metabolites are considered as biomarkers of JDNWF in treating ACLF, which were closely related to TCA cycle. It was further suggested that JDNWF alleviated mitochondrial damage and MQC may be potential mechanism of JDNWF improving mitochondrial function.ConclusionsMetabolomics revealed that TCA cycle was impaired in ACLF rats, and JDNWF had a regulatory effect on it. The potential mechanism may be improving the mitochondrial function through MQC pathway, thus restoring energy metabolism.

Structure and biochemical characterization of l-2-hydroxyglutarate dehydrogenase and its role in the pathogenesis of l-2-hydroxyglutaric aciduria

l-2-hydroxyglutarate dehydrogenase (L2HGDH) is a mitochondrial membrane-associated metabolic enzyme, which catalyzes the oxidation of l-2-hydroxyglutarate (l-2-HG) to 2-oxoglutarate (2-OG). Mutations in human L2HGDH lead to abnormal accumulation of l-2-HG, which causes a neurometabolic disorder named l-2-hydroxyglutaric aciduria (l-2-HGA). Here, we report the crystal structures of Drosophila melanogaster L2HGDH (dmL2HGDH) in FAD-bound form and in complex with FAD and 2-OG and show that dmL2HGDH exhibits high activity and substrate specificity for l-2-HG. dmL2HGDH consists of an FAD-binding domain and a substrate-binding domain, and the active site is located at the interface of the two domains with 2-OG binding to the re-face of the isoalloxazine moiety of FAD. Mutagenesis and activity assay confirmed the functional roles of key residues involved in the substrate binding and catalytic reaction and showed that most of the mutations of dmL2HGDH equivalent to l-2-HGA-associated mutations of human L2HGDH led to complete loss of the activity. The structural and biochemical data together reveal the molecular basis for the substrate specificity and catalytic mechanism of L2HGDH and provide insights into the functional roles of human L2HGDH mutations in the pathogeneses of l-2-HGA.

Isoquercitrin attenuates the progression of non-alcoholic steatohepatitis in mice by modulating galectin-3-mediated insulin resistance and lipid metabolism

BackgroundNon-alcoholic steatohepatitis (NASH) is a global health problem with no effective treatment. Isoquercitrin (IQ) alters hepatic lipid metabolism and inhibits adipocyte differentiation. The underlying regulatory mechanisms of IQ in regulating insulin resistance (IR) and lipid metabolism remain unclear. PurposeThis study was aimed at investigating the effects of IQ on NASH and deciphering whether the underlying mechanisms are via modulation of galectin-3 mediated IR and lipid metabolism. MethodsIR-HepG2 cell lines were used to demonstrate the ability of IQ to modulate galectin-3-mediated glucose disposal and lipid metabolism. A 20-week high-fat diet (HFD)-induced NASH model was established in C57BL/6J mice, and the protective effect of IQ on lipid disposal in the liver was verified. Further, the mRNA and protein levels of glucose and lipid metabolism were investigated, and lysophosphatidylcholine (LPC) and acylcarnitine (AC) profiling were performed to characterize the changes in endogenous substances associated with mitochondrial function and lipid metabolism in serum and cells. Furthermore, the pharmacokinetic features of IQ were explored in a rat model of NASH. ResultsIQ restored liver function and ameliorated inflammation and lipid accumulationin NASH model mice. Notably, significant regulation of the proteins included fatty acid-generating and transporting, cholesterol metabolism enzymes, nuclear transcription factors, mitochondrial metabolism, and IR-related enzymes was noted to be responsible for the therapeutic mechanisms of IQ against experimental NASH. Serum lipid metabolism-related metabolomic assay confirmed that LPC and AC biosynthesis mostly accounted for the therapeutic effect of IQ in mice with NASH and that IQ maintained the homeostasis of LPC and AC levels. ConclusionThis is the first study showing that IQ protects against of NASH by modulating galectin-3-mediated IR and lipid metabolism. The mechanisms responsible for liver protection and improved lipid metabolic disorder by IQ may be related to the suppression of IR and regulation of mitochondrial function and lipid metabolism. Galectin-3 down-regulation represents a potentially novel approach for the treatment and prevention of NASH.

Inflammation and mitochondria in the pathogenesis of chronic Chagas disease cardiomyopathy.

Chagas disease (CD), caused by the protozoan parasite Trypanosoma cruzi, is a neglected disease affecting around 6 million people. About 30% of CD patients develop chronic Chagas disease cardiomyopathy (CCC), an inflammatory cardiomyopathy that occurs decades after the initial infection, while most infected patients (60%) remain asymptomatic in the so-called indeterminate form (IF). Death results from heart failure or arrhythmia in a subset of CCC patients. Myocardial fibrosis, inflammation, and mitochondrial dysfunction are involved in the arrhythmia substrate and triggering events. Survival in CCC is worse than in other cardiomyopathies, which may be linked to a Th1-T cell rich myocarditis with abundant interferon (IFN)-γ and tumor necrosis factor (TNF)-α, selectively lower levels of mitochondrial energy metabolism enzymes in the heart, and reduced levels of high-energy phosphate, indicating poor adenosine triphosphate (ATP) production. IFN-γ and TNF-α signaling, which are constitutively upregulated in CD patients, negatively affect mitochondrial function in cardiomyocytes, recapitulating findings in CCC heart tissue. Genetic studies such as whole-exome sequencing (WES) in nuclear families with multiple CCC/IF cases has disclosed rare heterozygous pathogenic variants in mitochondrial and inflammatory genes segregating in CCC cases. In this minireview, we summarized studies showing how IFN-γ and TNF-α affect cell energy generation, mitochondrial health, and redox homeostasis in cardiomyocytes, in addition to human CD and mitochondria. We hypothesize that cytokine-induced mitochondrial dysfunction in genetically predisposed patients may be the underlying cause of CCC severity and we believe this mechanism may have a bearing on other inflammatory cardiomyopathies.

Cuproptosis-related prognostic signatures predict the prognosis and immunotherapy in HCC patients.

Cuproptosis, an unusual type of programmed cell death mechanism of cell death, involved the disruption of specific mitochondrial metabolic enzymes in the occurrence and development of tumors. However, it was still unclear how the relationship between cuproptosis-related genes (CRGs) may contribute to hepatocellular carcinoma (HCC) potential the prognosis of HCC remained limited. Here, the landscape of 14 CRGs in HCC was evaluated using the Cancer Genome Atlas and International Cancer Genome Consortium datasets. And then, 4 CRGs (ATP7A, MTF1, GLS, and CDKN2A) were screened for the construction of risk signatures for prognosis and drug therapy. The HCC patients with CRGs high-risk showed poor prognosis than those with low risk. Moreover, the CRGs risk signature was shown to be an independent prognostic factor and associated with the immune microenvironment in HCC. Meanwhile, we constructed and verified a prognostic model based on cuproptosis-related lncRNAs (Cr-lncRNAs). We obtained 291 Cr-lncRNAs and constructed Cr-lncRNA prognosis signature based on 3 key Cr-lncRNAs (AC026356.1, NRAV, AL031985.3). The Cr-lncRNA prognosis signature was also an independent prognostic factor and associated with the immune microenvironment in HCC. Finally, the drug sensitivity database showed that 8 candidate drugs related to CRGs signature and Cr-lncRNAs signature. In summary, we evaluated and validated the CRGs and Cr-lncRNAs as potential predictive markers for prognosis, immunotherapy, and drug candidate with the personalized diagnosis and treatment of HCC.

Mitochondrial one-carbon metabolic enzyme MTHFD2 facilitates mammary gland development during pregnancy

Mitochondrial one-carbon metabolism is crucial for embryonic development and tumorigenesis, as it supplies one-carbon units necessary for nucleotide synthesis and rapid cell proliferation. However, its contribution to adult tissue homeostasis remains largely unknown. To examine its role in adult tissue homeostasis, we specifically investigated mammary gland development during pregnancy, as it involves heightened cell proliferation. We discovered that MTHFD2, a mitochondrial one-carbon metabolic enzyme, is expressed in both luminal and basal/myoepithelial cell layers, with upregulated expression during pregnancy. Using the mouse mammary tumor virus (MMTV)-Cre recombinase system, we generated mice with a specific mutation of Mthfd2 in mammary epithelial cells. While the mutant mice were capable of properly nurturing their offspring, the pregnancy-induced expansion of mammary glands was significantly delayed. This indicates that MTHFD2 contributes to the rapid development of mammary glands during pregnancy. Our findings shed light on the role of mitochondrial one-carbon metabolism in facilitating rapid cell proliferation, even in the context of the adult tissue homeostasis.

A high-fat diet increases hepatic mitochondrial turnover through restricted acetylation in a NAFLD mouse model.

Lysine acetylation of proteins has emerged as a key posttranslational modification (PTM) that regulates mitochondrial metabolism. Acetylation may regulate energy metabolism by inhibiting and affecting the stability of metabolic enzymes and oxidative phosphorylation (OxPhos) subunits. Although protein turnover can be easily measured, due to the low abundance of modified proteins, it has been difficult to evaluate the effect of acetylation on the stability of proteins in vivo. We applied 2H2O-metabolic labeling coupled with immunoaffinity and high-resolution mass spectrometry method to measure the stability of acetylated proteins in mouse liver based on their turnover rates. As a proof-of-concept, we assessed the consequence of high-fat diet (HFD)-induced altered acetylation in protein turnover in LDL receptor-deficient (LDLR-/-) mice susceptible to diet-induced nonalcoholic fatty liver disease (NAFLD). HFD feeding for 12 wk led to steatosis, the early stage of NAFLD. A significant reduction in acetylation of hepatic proteins was observed in NAFLD mice, based on immunoblot analysis and label-free quantification with mass spectrometry. Compared with control mice on a normal diet, NAFLD mice had overall increased turnover rates of hepatic proteins, including mitochondrial metabolic enzymes (0.159 ± 0.079 vs. 0.132 ± 0.068 day-1), suggesting their reduced stability. Also, acetylated proteins had slower turnover rates (increased stability) than native proteins in both groups (0.096 ± 0.056 vs. 0.170 ± 0.059 day-1 in control, and 0.111 ± 0.050 vs. 0.208 ± 0.074 day-1 in NAFLD). Furthermore, association analysis revealed a relationship between the HFD-induced decrease in acetylation and increased turnover rates for hepatic proteins in NAFLD mice. These changes were associated with increased expressions of the hepatic mitochondrial transcriptional factor (TFAM) and complex II subunit without any changes to other OxPhos proteins, suggesting that enhanced mitochondrial biogenesis prevented restricted acetylation-mediated depletion of mitochondrial proteins. We conclude that decreased acetylation of mitochondrial proteins may contribute to adaptive improved hepatic mitochondrial function in the early stages of NAFLD.NEW & NOTEWORTHY This is the first method to quantify acetylome dynamics in vivo. This method revealed acetylation-mediated altered hepatic mitochondrial protein turnover in response to a high-fat diet in a mouse model of NAFLD.

Defective import of mitochondrial metabolic enzyme elicits ectopic metabolic stress

Deficiencies in mitochondrial protein import are associated with a number of diseases. However, although nonimported mitochondrial proteins are at great risk of aggregation, it remains largely unclear how their accumulation causes cell dysfunction. Here, we show that nonimported citrate synthase is targeted for proteasomal degradation by the ubiquitin ligase SCFUcc1. Unexpectedly, our structural and genetic analyses revealed that nonimported citrate synthase appears to form an enzymatically active conformation in the cytosol. Its excess accumulation caused ectopic citrate synthesis, which, in turn, led to an imbalance in carbon flux of sugar, a reduction of the pool of amino acids and nucleotides, and a growth defect. Under these conditions, translation repression is induced and acts as a protective mechanism that mitigates the growth defect. We propose that the consequence of mitochondrial import failure is not limited to proteotoxic insults, but that the accumulation of a nonimported metabolic enzyme elicits ectopic metabolic stress.

Effects of blueberry anthocyanins on perfluorooctane sulfonate-induced oxidative stress and mitochondrial dysfunction in dugesia japonica

The effects of blueberry anthocyanins against perfluorooctane sulfonate on antioxidant-related gene expression levels, antioxidase activities, malondialdehyde content, glutathione/oxidized glutathione ratios, mitochondrial metabolic enzyme activities, and mitochondrial permeability transition pore opening rates were examined in the planarian Dugesia japonica. Glutathione reductase, glutathione peroxidase, and glutathione S-transferase gene expression levels were up-regulated on day 1 and then down-regulated following exposure to 5 mg/L perfluorooctane sulfonate with those of the control. The activities of the three corresponding enzymes were inhibited by perfluorooctane sulfonate and activated by anthocyanins. Malondialdehyde contents and mitochondrial permeability transition pore opening rates as well as the activities of mitochondrial metabolic enzymes, also increased and the glutathione/oxidized glutathione ratio decreased. Anthocyanin treatments have an alleviating effect on the disorder caused by perfluorooctane sulfonate.

Paeoniflorin Protects against Acetaminophen-Induced Liver Injury in Mice via JNK Signaling Pathway

Drug-induced liver injury (DILI), represented by acetaminophen (APAP), is a common cause of acute liver failure in clinics. Paeoniflorin (PF) has been proven to demonstrate a significant hepatoprotective effect. However, it is still unclear whether it can be a potential agent against hepatotoxicity induced by APAP. This study aimed to explore the preventive and therapeutic effects and mechanisms of PF on APAP-induced liver injury. Different doses of PF (50, 100, and 200 mg/kg) were given to C57BL/6 male mice for five consecutive days. After 12 h of APAP (250 mg/kg i.p.) treatment, blood and liver tissues were collected and isolated for detection. The results showed that the therapeutic effects of PF on APAP mice were presented in the downregulation of the content of serum indices and significantly improved hepatic tissue edema and inflammatory infiltration. Meanwhile, PF reduces the level of the mitochondrial metabolic enzyme. Ulteriorly, it was found that PF has a downregulating effect on the apoptotic reaction and could inhibit the protein expression of CYP2E1/JNK signaling, which in turn reduces the damage of APAP. Our findings showed that PF acted as a protective agent against APAP-induced hepatotoxicity by inhibiting JNK-related signals, suggesting a novel insight into treating APAP-induced liver injury.

Abstract A017: Bidirectional signaling between mitochondria and the nucleus dictates epigenetic rewiring to drive tumorigenesis

Abstract Cancer cells have acquired the ability to sense and adapt to varying conditions of nutrient sources in the tumor microenvironment. Mitochondria lay at the core of cellular metabolism, whereas the nucleus integrates cellular and environmental signals to activate gene transcription that alter cell function and cell fate. However, it remains largely unexplored how mitochondrial metabolic enzymes and nuclear transcriptional machineries communicate to drive gene regulation in response to metabolic stress. Biochemical screening identified a TCA cycle enzyme aconitase (ACO2), which functions as a critical mitochondrial regulator of de novo lipogenesis to sustain prostate tumor growth. Metabolic isotope tracing experiments revealed that the ACO2 enzyme promotes citrate biosynthesis required for de novo lipogenesis by reductive carboxylation of alpha-ketoglutarate generated from glutamine. Proteomics studies identified an acetylation mark lysine258 on ACO2 that induces increased enzymatic activity, whereas acetylation deficient Lys258Arg-ACO2 mutant was inactive, and failed to promote lipogenesis and tumor growth. To uncover nuclear signals that may stimulate ACO2 activity, we identified transcriptional suppression of a mitochondrial deacetylase SIRT3 by AR and its coregulator SRC-2, thereby preserving enhanced K258Ac mark on ACO2 facilitating increased lipogenesis. Interestingly our spontaneous bone metastatic mouse studies revealed that increased ACO2 activity regulated by the SIRT3/SRC-2 axis functions as a predominant survival factor for prostate tumors in the early stages of homing and colonization during bone metastasis, suggesting this pathway may be essential for metabolic adaptation in hostile microenvironment. Next, to investigate the biochemical control of gene transcription that may support tumor cell adaptation in the hostile microenvironment, we found a potential metabolic pathway that may support acetyl-CoA synthesis in the nucleus. Reversible enzymes IDH2 and ACO2 which can generate citrate from alpha-ketoglutarate to produce acetyl CoA were found to be localized in the nucleus. Interestingly, ablation of ACO2 and/or IDH2 was sufficient to significantly reduce several histone acetylation marks such as H3K9ac and H3K14ac. In isolated intact nucleus, these marks could be increased by addition of alpha-ketoglutarate thereby increasing the overall chromatin accessibility, however loss of ACO2 or IDH2 abrogated the effect, implying these enzymes are required to generate acetyl CoA in the nucleus independent of mitochondrial contribution. ATAC-seq analysis further revealed genetic inhibition of ACO2 significantly reduced chromatin accessibility with an approximate loss of 16626 peaks and gain of only 486 peaks compared to control (WT) cells. Our findings indicate a potential metabolic control in the nucleus that may regulate chromatin accessibility required for gene activation to favor tumor cell adaptation in a hostile microenvironment. This work is supported by the funds from NIH (R01CA252092 and DP2CA260421) to S.D. Citation Format: Abhisha Sawant Dessai, Nadya Elhalawany, Eriko Katsuta, Christian Prechtl, Spencer Rosario, Bert W. O’Malley, Xiang H. Zhang, Subhamoy Dasgupta. Bidirectional signaling between mitochondria and the nucleus dictates epigenetic rewiring to drive tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A017.

The SMYD3-MTHFD1L-formate metabolic regulatory axis mediates mitophagy to inhibit M1 polarization in macrophages.

SMYD3 (protein 3 containing SET and MYND structural domains) belongs to the SMYD methylesterase family and is a histone lysine methyltransferase that promotes gene transcription mainly by catalysing the trimethylation of lysine at position 4 of histone subunit 3 (H3K4me3). Studies have shown that SMYD3 plays a key role in tumour cell proliferation and differentiation; however, its role in macrophage polarization is unclear. We screened the M1- and M2-polarized macrophage differential histone modifying enzyme using bioinformatics analysis. The SMYD3 overexpression plasmid was transfected into M1 macrophages, and the SMYD3-regulated target gene was analysed by RNA-seq and ChIP-Seq. The effect of knocking down MTHFD1L on M1 polarization and the change of the intracellular metabolite formic acid content were investigated. M1 macrophages were stimulated with different concentrations of formic acid (2, 10 and 40mM) to detect the expression of M1-related genes, ROS production, and changes in the expression of the mitophagy-related proteins LC3, PINK1 and p-Parkin. Here, we used bioinformatics to analyse SMYD3, a histone methyltransferase associated with M1 polarization; overexpression of SMYD3 significantly suppressed the LPS/IFN-γ-induced M1 phenotype in macrophages. RNA-seq analysis demonstrated that SMYD3 significantly activated the one-carbon folate metabolic pathway in M1 macrophages. In addition, we used ChIP-seq analysis to identify methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) as the target gene of the transcriptional activation by SMYD3 through H3K4me3 histone modification. Activation of MTHFD1L causes the accumulation of the intracellular metabolite formate. Exogenous stimulation with different concentrations of formate increased the expression of key genes involved in the mitochondrial respiratory chain complex, ROS production, and the expression of autophagy-related proteins LC3, PINK1, and p-Parkin and suppressed the expression of M1-related genes. Our study demonstrates that SMYD3 regulates the activity of the mitochondrial metabolic enzyme MTHFD1L through H3K4me3 histone methylation modification, promotes formate synthesis and induces mitophagy, which inhibits M1 polarization in macrophages.

Molecular subtypes based on cuproptosis regulators and immune infiltration in kidney renal clear cell carcinoma.

Copper toxicity involves the destruction of mitochondrial metabolic enzymes, triggering an unusual mechanism of cell death called cuproptosis, which proposes a novel approach using copper toxicity to treat cancer. However, the biological function of cuproptosis has not been fully elucidated in kidney renal clear cell carcinoma (KIRC). Using the expression profile of 13 cuproptosis regulators, we first identified two molecular subtypes related to cuproptosis defined as "hot tumor" and "cold tumor", having different levels of biological function, clinical prognosis, and immune cell infiltration. We obtained three gene clusters using the differentially expressed genes between the two cuproptosis-related subtypes, which were associated with different molecular activities and clinical characteristics. Next, we developed and validated a cuproptosis prognostic model that included two genes (FDX1 and DBT). The calculated risk score could divide patients into high- and low-risk groups. The high-risk group had a poorer prognosis, lower level of immune infiltration, higher frequency of gene alterations, and greater levels of FDX1 methylation and limited DBT methylation. The risk score was also an independent predictive factor for overall survival in KIRC. The established nomogram calculating the risk score achieved a high predictive ability for the prognosis of individual patients (area under the curve: 0.860). We then identified small molecular inhibitors as potential treatments and analyzed the sensitivity to chemotherapy of the signature genes. Tumor immune dysfunction and exclusion (TIDE) showed that the high-risk group had a higher level of TIDE, exclusion and dysfunction that was lower than the low-risk group, while the microsatellite instability of the high-risk group was significantly lower. The results of two independent immunotherapy datasets indicated that cuproptosis regulators could influence the response and efficacy of immunotherapy in KIRC. Our study provides new insights for individualized and comprehensive therapy of KIRC.

Bioinformatic profiling identifies the glutaminase to be a potential novel cuproptosis-related biomarker for glioma.

Glioma is the most common tumour of the central nervous system, with a poor prognosis and an increasing trend of incidence in recent years; it is also beginning to affect younger age groups more. Added to this, cuproptosis is a new form of cell death. Indeed, when a certain amount of copper accumulates in a cell, it affects specific mitochondrial metabolic enzymes in that cell and leads to cell death–a phenomenon known as cuproptosis. In this study, we applied bioinformatics analysis, and, according to the results of the study analysis and Gene Ontology (GO), as well as the Kyoto Encyclopedia of Genes and Genomes KyotoEncyclopediaofGenesandGenomes, the glutaminase (GLS) genes affect the prognosis and tumour mutation of glioma patients through cuproptosis. Interestingly, however, GLS is not involved in the immune escape of glioma. Glutaminase genes are a class of glucose metabolism-related genes that are involved in the tricarboxylic acid cycle of cells. At the same time, the expression of the glutaminase gene was positively correlated with the degree of immune cell infiltration and the expression of various immune cell markers, and thus affected the prognosis of glioma patients. Therefore, we believe that the cuproptosis-related glutaminase gene can be an important factor in determining the prognosis of glioma patients.

Targeting mitochondrial one-carbon enzyme MTHFD2 together with pemetrexed confers therapeutic advantages in lung adenocarcinoma

Metabolic remodeling is the fundamental molecular feature of malignant tumors. Cancer cells require sufficient energy supplies supporting their high proliferative rate. MTHFD2, a mitochondrial one-carbon metabolic enzyme, is dysregulated in several malignancies and may serve as a promising therapeutic candidate in cancer treatment. Here, our data confirmed that MTHFD2 gene and protein was upregulated in the cancerous tissues of LUAD patients and was correlated with a poor survival in LUAD. MTHFD2 was involved in lung cancer cell proliferation, migration, and apoptosis by mediating its downstream molecules, such as DNA helicases (MCM4 and MCM7), as well as ZEB1, Vimentin and SNAI1, which contributed to tumor cell growth and epithelial-to-mesenchymal transition (EMT) process. Moreover, we identified that miRNA-99a-3p appeared to be an upstream mediator directly regulating MTHFD2 and MCM4 expression. Moreover, specific inhibition of MTHFD2 functions by siRNA or a chemical compound, improved anti-tumor sensitivities induced by pemetrexed in LUAD. Taken together, our study revealed the underlying molecular mechanisms of MTHFD2 in regulating cell proliferation and identified a novel therapeutic strategy improving the treatment efficacies in LUAD.

Способ моделирования нормобарической хронической гипоксии у крыс самцов сток WISTAR

BACKGROUND: Hypoxia is a factor of development of many diseases, and, among other things, may be the cause of male infertility. The molecular basis of pathogenesis in hypoxia can serve a basis for the development of methods of treatment and correction. AIM: development of a model of chronic normobaric hypoxia in laboratory animals. MATERIALS AND METHODS: The study involved 32 mature male rats of Wistar line of 200–280 g mass. The animals were divided to 2 experimental groups and their control groups. In the first experimental model, a model of acute normobaric hypoxia with hypercapnia was used one time according to the method of M. V. Korableva and P. I. Lukienko (1976) in modification of N. D. Avseenko, in the second — a model of chronic hypoxia according to the method of acute normobaric hypoxic hypoxia with hypercapnia in our modification: the animals were placed in a hermetically closed chamber of 1.2 liter volume connected to the gas analyzer, and stayed there until the content of oxygen in the air decreased to 10%. Animals of the control groups were placed in ventilated chambers. The experiment was performed daily for 14 days. The activity of cytochrome oxidase (CO), lactate dehydrogenase (LDH) and superoxide dismutase (SOD) in the mitochondrial fraction of seminal vesicles and epididymis was determined. RESULTS: In modeling of acute normobaric hypoxia, the activity of the studied enzymes did not show statistically significant changes. In an experiment with chronic normobaric hypoxia, the activity of SOD and CO was significantly reduced in all the studied tissues, and the activity of LDH — only in the tissues of the epididymis head. CONCLUSION: Changes in the activity of key enzymes of mitochondrial metabolism indicate the adaptation to hypoxia at the cellular and subcellular levels, which proves the effectiveness of the described model for further use in research.