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Radiation Research May 2024In gene expression (GE) studies, housekeeping genes (HKGs) are required for normalization purposes. In large-scale inter-laboratory comparison studies, significant...
PUM1 and PGK1 are Favorable Housekeeping Genes over Established Biodosimetry-related Housekeeping Genes such as HPRT1, ITFG1, DPM1, MRPS5, 18S rRNA and Others after Radiation Exposure.
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.
Topics: Adult; Animals; Female; Humans; Male; Middle Aged; Dose-Response Relationship, Radiation; Genes, Essential; Radiation Exposure; Radiometry; RNA, Ribosomal, 18S; RNA-Binding Proteins; Macaca mulatta; Phosphoglycerate Kinase
PubMed: 38471523
DOI: 10.1667/RADE-23-00160.1 -
Biochimica Et Biophysica Acta. Proteins... Jan 2024Magnesium is an important divalent cation for the regulation of catalytic activity. Recently, we have described that the Mg binding through the PAS domain inhibits the...
Magnesium is an important divalent cation for the regulation of catalytic activity. Recently, we have described that the Mg binding through the PAS domain inhibits the phosphoglycerate kinase (PGK) activity in PAS domain-containing PGK from Leishmania major (LmPAS-PGK) at neutral pH 7.5, but PGK activity is derepressed at acidic pH 5.5. The acidic residue within the PAS domain of LmPAS-PGK is expected to bind the cofactor Mg ion at neutral pH, but which specific acidic residue(s) is/are responsible for the Mg binding is still unknown. To identify the residues, we exploited mutational studies of all acidic (twelve Asp/Glu) residues in the PAS domain for plausible Mg binding. Mg ion-dependent repression at pH 7.5 is withdrawn by substitution of Asp-4 with Ala, whereas other acidic residue mutants (D16A, D22A, D24A, D29A, D43A, D44A, D60A, D63A, D77A, D87A, and E107A) showed similar features compared to the wild-type protein. Fluorescence spectroscopic studies and isothermal titration calorimetry analysis showed that the Asp-4 is crucial for Mg binding in the absence of both PGK's substrates. These results suggest that Asp-4 residue in the regulatory (PAS) domain of wild type enzymes is required for Mg dependent repressed state of the catalytic PGK domain at neutral pH.
Topics: Phosphoglycerate Kinase; Leishmania major; Aspartic Acid; Calorimetry; Catalytic Domain
PubMed: 37726028
DOI: 10.1016/j.bbapap.2023.140964 -
JCI Insight May 2024This study lays the groundwork for future lentivirus-mediated gene therapy in patients with Diamond Blackfan anemia (DBA) caused by mutations in ribosomal protein S19...
This study lays the groundwork for future lentivirus-mediated gene therapy in patients with Diamond Blackfan anemia (DBA) caused by mutations in ribosomal protein S19 (RPS19), showing evidence of a new safe and effective therapy. The data show that, unlike patients with Fanconi anemia (FA), the hematopoietic stem cell (HSC) reservoir of patients with DBA was not significantly reduced, suggesting that collection of these cells should not constitute a remarkable restriction for DBA gene therapy. Subsequently, 2 clinically applicable lentiviral vectors were developed. In the former lentiviral vector, PGK.CoRPS19 LV, a codon-optimized version of RPS19 was driven by the phosphoglycerate kinase promoter (PGK) already used in different gene therapy trials, including FA gene therapy. In the latter one, EF1α.CoRPS19 LV, RPS19 expression was driven by the elongation factor alpha short promoter, EF1α(s). Preclinical experiments showed that transduction of DBA patient CD34+ cells with the PGK.CoRPS19 LV restored erythroid differentiation, and demonstrated the long-term repopulating properties of corrected DBA CD34+ cells, providing evidence of improved erythroid maturation. Concomitantly, long-term restoration of ribosomal biogenesis was verified using a potentially novel method applicable to patients' blood cells, based on ribosomal RNA methylation analyses. Finally, in vivo safety studies and proviral insertion site analyses showed that lentivirus-mediated gene therapy was nontoxic.
Topics: Anemia, Diamond-Blackfan; Humans; Genetic Therapy; Lentivirus; Ribosomal Proteins; Genetic Vectors; Hematopoietic Stem Cells; Animals; Mice; Male; Female; Ribosomes; Promoter Regions, Genetic; Mutation; Hematopoietic Stem Cell Transplantation
PubMed: 38775150
DOI: 10.1172/jci.insight.171650 -
European Journal of Medicinal Chemistry Mar 2024Our previous research has revealed phosphoglycerate kinase 1 (PGK1) enhances tumorigenesis and sorafenib resistance of kidney renal clear cell carcinoma (KIRC) by...
Our previous research has revealed phosphoglycerate kinase 1 (PGK1) enhances tumorigenesis and sorafenib resistance of kidney renal clear cell carcinoma (KIRC) by regulating glycolysis, so that PGK1 is a promising drug target. Herein we performed structure-based virtual screening and series of anticancer pharmaceutical experiments in vitro and in vivo to identify novel small-molecule PGK1-targeted compounds. As results, the compounds CHR-6494 and Z57346765 were screened and confirmed to specifically bind to PGK1 and significantly reduced the metabolic enzyme activity of PGK1 in glycolysis, which inhibited KIRC cell proliferation in a dose-dependent manner. While CHR-6494 showed greater anti-KIRC efficacy and fewer side effects than Z57346765 on nude mouse xenograft model. Mechanistically, CHR-9464 impeded glycolysis by decreasing the metabolic enzyme activity of PGK1 and suppressed histone H3T3 phosphorylation to inhibit KIRC cell proliferation. Z57346765 induced expression changes of genes related to cell metabolism, DNA replication and cell cycle. Overall, we screened two novel PGK1 inhibitors, CHR-6494 and Z57346765, for the first time and discovered their potent anti-KIRC effects by suppressing PGK1 metabolic enzyme activity in glycolysis.
Topics: Mice; Animals; Humans; Phosphoglycerate Kinase; Phosphorylation; Glycolysis; Carcinoma; Kidney; Cell Line, Tumor
PubMed: 38354523
DOI: 10.1016/j.ejmech.2024.116209 -
Discover Oncology May 2024Reprogramming of the serine synthesis pathway (SSP) is intricately linked to the progression of epithelial ovarian cancer (EOC). CBR-5884, a selective small-molecule...
Reprogramming of the serine synthesis pathway (SSP) is intricately linked to the progression of epithelial ovarian cancer (EOC). CBR-5884, a selective small-molecule inhibitor targeting phosphoglycerate dehydrogenase (PHGDH), effectively impedes the de novo synthesis of serine within cancer cells. This study aimed to evaluate the inhibitory effect of CBR-5884 on EOC cells and delineate its specific mechanism, thereby proposing a novel therapeutic approach for treating EOC. The suppression of serine biosynthesis after CBR-5884 treatment was evaluated using RNA sequencing and a serine assay kit, and the results showed that CBR-5884 effectively downregulated serine biosynthesis in EOC cells, particularly those expressing high levels of PHGDH. In vitro studies revealed that CBR-5884 demonstrated significant antitumor effects and suppressed migration and invasion of EOC cells through down-regulation of the integrin subunit beta 4 (ITGB4)/extracellular signal-regulated kinase (ERK)/epithelial-mesenchymal transition signal axis. Additionally, CBR-5884 mitigated the stemness of EOC cells and heightened their sensitivity to chemotherapy. Moreover, in vivo studies revealed that CBR-5884 significantly delayed tumor growth, with histological analysis indicating the safety profile of CBR-5884. Finally, the patient-derived organoid (PDO) models were utilized to explore the preclinical efficacy of CBR-5884 against EOC cells, and the results unveiled that CBR-5884 impeded proliferation and downregulated the expression of ITGB4 in EOC PDO models. Our findings supports the anticancer properties of CBR-5884 in EOC cells exhibiting high PHGDH expression, manifesting through the suppression of proliferation, migration, and invasion, while enhancing chemotherapy sensitivity, suggesting that CBR-5884 holds promise as an efficacious strategy for the treatment of EOC.
PubMed: 38733440
DOI: 10.1007/s12672-024-01013-0 -
Molecular and Cellular Biochemistry May 2024HOXC6 (Homeobox C6) and methyltransferase-like 3 (METTL3) have been shown to be involved in the progression of prostate cancer (PCa). However, whether HOXC6 performs...
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.
PubMed: 38822192
DOI: 10.1007/s11010-024-05023-y -
Seizure Apr 2024
Topics: Humans; Phosphoglycerate Kinase; Male; Joint Instability; Female; Spinal Diseases; Metabolism, Inborn Errors; Genetic Diseases, X-Linked
PubMed: 38432079
DOI: 10.1016/j.seizure.2024.02.010 -
Current Research in Food Science 2024Whitespotted conger () muscle proteins were susceptible to oxidative denaturation during frozen storage. The objective of this study was to investigate the alterations...
Whitespotted conger () muscle proteins were susceptible to oxidative denaturation during frozen storage. The objective of this study was to investigate the alterations in quality through physicochemical analysis and proteomics after whitespotted conger stored at temperatures of -18 °C and -60 °C. The microstructural observation revealed the noticeable variations such as increased interstitial space and fractured muscle fibre with extension of frozen storage time, and the muscle fibre of whitespotted conger stored at -60 °C were more intact than those stored at -18 °C. The raised TVB-N value indicated that the freshness of whitespotted conger decreased during 120-day frozen storage period. Analysis of myofibrillar protein content and SDS-PAGE demonstrated that compared to -18 °C, lower storage temperature (-60 °C) could better maintain the structure of whitespotted conger muscle by inhibiting protein degradation and oxidation. To reveal the mechanism of protein degradation, label-free quantitative proteomic analysis was performed through LC-MS/MS. The structural proteins including domain-associated proteins and actin-related proteins were up-regulated during frozen storage, but the phosphoglycerate kinase, phosphoglycerate mutase, and fructose-bisphosphate aldolase were down-regulated. Storage at -18 °C accelerated the up- or down-regulation of those differentially abundant proteins. According to KEGG analysis, up- or down-regulated pathways such as glycolysis/gluconeogenesis, carbon metabolism, biosynthesis of amino acids, and calcium signalling pathway mainly accounted for the protein degradation and quality reduction of whitespotted conger at low temperature. These results provided a theoretical basis for improving the quality stability of whitespotted conger during frozen storage.
PubMed: 38939611
DOI: 10.1016/j.crfs.2024.100779 -
Molecular Oncology Jun 2024Gemcitabine plus cisplatin (GC) combination chemotherapy is the primary treatment for advanced bladder cancer (BC) with unresectable or metastatic disease. However, most...
Gemcitabine plus cisplatin (GC) combination chemotherapy is the primary treatment for advanced bladder cancer (BC) with unresectable or metastatic disease. However, most cases develop resistance to this therapy. We investigated whether drug resistance could be targeted through metabolic reprogramming therapies. Metabolomics analyses in our lab's gemcitabine- and cisplatin-resistant cell lines revealed increased phosphoglycerate dehydrogenase (PHGDH) expression in gemcitabine-resistant cells compared with parental cells. Isocitrate dehydrogenase 2 (IDH2) gain of function stabilized hypoxia-inducible factor1α (HIF1α) expression, stimulating aerobic glycolysis. In gemcitabine-resistant cells, elevated fumaric acid suppressed prolyl hydroxylase domain-containing protein 2/Egl nine homolog 1 (PHD2) and stabilized HIF1α expression. PHGDH downregulation or inhibition in gemcitabine-resistant BC cells inhibited their proliferation, migration, and invasion. Cisplatin-resistant cells showed elevated fatty acid metabolism, upregulating fatty acid synthase (FASN) downstream of tyrosine kinase. Using the fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitor erdafitinib, we inhibited malonyl-CoA production, which is crucial for fatty acid synthesis, and thereby suppressed upregulated HIF1α expression. Combination treatment with NCT503 and erdafitinib synergistically suppressed tumor cell proliferation and induced apoptosis in vitro and in vivo. Understanding these mechanisms could enable innovative BC therapeutic strategies to be developed.
PubMed: 38874588
DOI: 10.1002/1878-0261.13684