Intracellular Serine Research Articles

Debridement efficacy of serine protease and formulated cream by In Vitro assessment against artificial wound eschar

Chronic wounds typically comprise of necrotic tissue and dried secretions, often culminating in the formation of a thick and tough layer of dead skin known as eschar. Removal of eschar is imperative to facilitate wound healing. Conventional approach for eschar removal involves surgical excision and grafting, which can be traumatic and frequently leads to viable tissue damage. There has been growing interest in the use of enzymatic agents for a gentler approach to debridement, utilizing proteolytic enzymes. In this study, a purified intracellular recombinant serine protease from Bacillus sp. (SPB) and its cream formulation were employed to evaluate their ability to degrade artificial wound eschar; composed of collagen, fibrin, and elastin. Degradation was assessed based on percentage weight reduction of eschar biomass, analysis via sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and scanning electron microscopy (SEM). Both SPB and its cream formulation were able to degrade up to 50 % artificial wound eschar, with the SPB cream maintaining its degradation efficiency for up to 24 hours. Additionally, the SPB-based cream demonstrated the ability to hydrolyze proteinaceous components of eschars individually (fibrin and collagen) as determined through qualitative assessment. These findings suggest that SPB holds promise for the debridement of wound eschar.

Bacterial biofilm growth and perturbation by serine protease from Bacillus sp.

In nature, bacteria are ubiquitous and can be categorized as beneficial or harmless to humans, but most bacteria have one thing in common which is their ability to produce biofilm. Biofilm is encased within an extracellular polymeric substance (EPS) which provides resistance against antimicrobial agents. Protease enzymes have the potential to degrade or promote the growth of bacterial biofilms. In this study, the effects of a recombinant intracellular serine protease from Bacillus sp. (SPB) on biofilms from Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa were analyzed. SPB was purified using HisTrap HP column and concentrated using Amicon 30 ultra-centrifugal filter. SPB was added with varying enzyme activity and assay incubation period after biofilms were formed in 96-well plates. SPB was observed to have contrasting effects on different bacterial biofilms, where biofilm degradations were observed for both 7-day-old A. baumannii (37.26%) and S. aureus (71.51%) biofilms. Meanwhile, SPB promoted growth of P. aeruginosa biofilm up to 176.32%. Compatibility between protein components in S. aureus biofilm with SPB as well as a simpler membrane structure morphology led to higher biofilm degradation for S. aureus compared to A. baumannii. However, SPB promoted growth of P. aeruginosa biofilm due likely to its degrading protein factors that are responsible for biofilm detachment and dispersion, thus resulting in more multi-layered biofilm formation. Commercial protease Savinase which was used as a comparison showed degradation for all three bacterial biofilms. The results obtained are unique and will expand our understanding on the effects that bacterial proteases have toward biofilms.

Multiomics Reveals Induction of Neuroblastoma SK-N-BE(2)C Cell Death by Mitochondrial Division Inhibitor 1 through Multiple Effects.

Mitochondrial division inhibitor 1 (Mdivi-1) is a well-known synthetic compound aimed at inhibiting dynamin-related protein 1 (Drp1) to suppress mitochondrial fission, making it a valuable tool for studying mitochondrial dynamics. However, its specific effects beyond Drp1 inhibition remain to be confirmed. In this study, we employed integrative proteomics and phosphoproteomics to delve into the molecular responses induced by Mdivi-1 in SK-N-BE(2)C cells. A total of 3070 proteins and 1945 phosphorylation sites were identified, with 880 of them represented as phosphoproteins. Among these, 266 proteins and 97 phosphorylation sites were found to be sensitive to the Mdivi-1 treatment. Functional enrichment analysis unveiled their involvement in serine biosynthesis and extrinsic apoptotic signaling pathways. Through targeted metabolomics, we observed that Mdivi-1 enhanced intracellular serine biosynthesis while reducing the production of C24:1-ceramide. Within these regulated phosphoproteins, dynamic dephosphorylation of proteasome subunit alpha type 3 serine 250 (PSMA3-S250) occurred after Mdivi-1 treatment. Further site-directed mutagenesis experiments revealed that the dephosphorylation-deficient mutant PSMA3-S250A exhibited a decreased cell survival. This research confirms that Mdivi-1's inhibition of mitochondrial division leads to various side effects, ultimately influencing cell survival, rather than solely targeting Drp1 inhibition.

OR11-04 Activatable Glucocorticoid Receptor Agonists As Treatments For The Consequences Of Preterm Birth

Abstract Disclosure: R.S. Dhavan: None. K.L. Short: None. J. Ng: None. M.J. Wallace: None. P. Monaghan-Nichols: None. D. DeFranco: None. T.J. Cole: None. Glucocorticoid (GC) signaling is essential for normal fetal organ development. During late gestation a surge of endogenous GCs contributes to organ maturation, particularly important for the lung where thinning the mesenchymal tissue cause an increased alveolar-gas exchange surface area and decreased gas-exchange diffusion distance that is critical for lung function after birth. Currently potent synthetic GCs such as betamethasone (Bet) or dexamethasone (Dex) are administered antenatally to accelerate fetal lung maturation and reduce the risk of respiratory distress with preterm birth. There are however growing concerns that systemic exposure to powerful synthetic GCs such as Dex is associated with detrimental side effects, particularly in the developing fetal brain. We are currently assessing novel activatable and potentially partially selective agonists of the glucocorticoid receptor (GR) as new antenatal steroid treatments of preterm birth. One such GR agonist is a steroid prodrug called ciclesonide (Cic) that is activated in vivo to the GR agonist Des-Cic by a family of intracellular serine esterase enzymes, called carboxylesterases (Ces). We have previously demonstrated that postnatal administration of Cic and Dex drive similar stimulation of important preterm lung-specific biomarkers but in contrast to Dex, Cic produced no postnatal growth retardation, no reduction in brain weight, and no alteration in levels of neural myelination. In this current study we show by western blot analysis and immunofluorescence that the CES enzymes are expressed strongly in epithelial cells and the mesenchyme of peripheral organs such as the lung and kidney but are expressed at very low levels in the fetal and adult mouse brain, providing a basis for the preferential activation of Cic to Des-Cic only in peripheral organs. To examine the transcriptome changes induced by Cic vs Dex in the fetal mouse lung we have assessed cultured primary mouse fetal fibroblasts (the primary cell target for GCs during lung development) stimulated by 1μM Dex and Cic, for 6 hours. Changes in the fibroblast transcriptome was assessed by microarray analysis and relevant targets validated by real-time-qPCR. Analysis of the top 30 induced and repressed gene targets, including Fkbp5, Crispld2, Tgm2 and Zbtb16, demonstrated that Dex and Cic have almost identical activity profiles. Induction of transcriptional changes was completely lost in GR-null- derived fetal lung fibroblasts demonstrating specificity via the GR. The data suggests that Cic is able to regulate a respiratory genomic signature similar to Dex. Further studies will examine the effects of Cic vs Beta and Dex in mouse models of preterm birth, particularly in the context of unwanted side-effects in neural development. Presentation: Friday, June 16, 2023

Viral SERPINS-A Family of Highly Potent Immune-Modulating Therapeutic Proteins.

Serine protease inhibitors, SERPINS, are a highly conserved family of proteins that regulate serine proteases in the central coagulation and immune pathways, representing 2-10% of circulating proteins in the blood. Serine proteases form cascades of sequentially activated enzymes that direct thrombosis (clot formation) and thrombolysis (clot dissolution), complement activation in immune responses and also programmed cell death (apoptosis). Virus-derived serpins have co-evolved with mammalian proteases and serpins, developing into highly effective inhibitors of mammalian proteolytic pathways. Through interacting with extracellular and intracellular serine and cysteine proteases, viral serpins provide a new class of highly active virus-derived coagulation-, immune-, and apoptosis-modulating drug candidates. Viral serpins have unique characteristics: (1) function at micrograms per kilogram doses; (2) selectivity in targeting sites of protease activation; (3) minimal side effects at active concentrations; and (4) the demonstrated capacity to be modified, or fine-tuned, for altered protease targeting. To date, the virus-derived serpin class of biologics has proven effective in a wide range of animal models and in one clinical trial in patients with unstable coronary disease. Here, we outline the known viral serpins and review prior studies with viral serpins, considering their potential for application as new sources for immune-, coagulation-, and apoptosis-modulating therapeutics.

Multi-Omics Analysis Revealed Increased De Novo Synthesis of Serine and Lower Activity of the Methionine Cycle in Breast Cancer Cell Lines.

A pipeline for metabolomics, based on UPLC-ESI-MS, was tested on two malignant breast cancer cell lines of the sub-types ER(+), PR(+), and HER2(3+) (MCF-7 and BCC), and one non-malignant epithelial cancer cell line (MCF-10A). This allowed us to quantify 33 internal metabolites, 10 of which showed a concentration profile associated with malignancy. Whole-transcriptome RNA-seq was also carried out for the three mentioned cell lines. An integrated analysis of metabolomics and transcriptomics was carried out using a genome-scale metabolic model. Metabolomics revealed the depletion of several metabolites that have homocysteine as a precursor, which was consistent with the lower activity of the methionine cycle caused by lower expression of the AHCY gene in cancer cell lines. Increased intracellular serine pools in cancer cell lines appeared to result from the over-expression of PHGDH and PSPH, which are involved in intracellular serine biosynthesis. An increased concentration of pyroglutamic acid in malignant cells was linked to the overexpression of the gene CHAC1.

Abstract 695: Serine Hydroxymethyltransferase 2 Regulates Mitochondrial Function And Ceramide Metabolism In Macrophages During Atherosclerosis

Increasing evidence suggests a critical role for amino acid metabolism in cardiovascular disease, highlighting potential therapeutic targets and our need for further understanding. Plasma serine has been found to be a biomarker for cardiovascular disease, while glycine is negatively correlated with coronary artery disease. Furthermore, attenuated plasma glycine:serine is associated with unstable atherosclerotic plaques. Mitochondrial Serine hydroxymethyl transferase (SHMT) catalyzes the bulk formation of glycine from serine in most cell types and drives one-carbon metabolism in the cytosol (SHMT1) or mitochondria (SHMT2). SHMT2 is necessary for mitochondrial protein translation and critical in development since global knockout mice are nonviable. We found LPS-treated macrophages have reduced SHMT2 expression concomitant with attenuated intracellular glycine:serine. Deletion of macrophage SHMT2 in vitro enhanced intracellular serine, which contributes to the rate-limiting step of ceramide de novo biosynthesis. C16:0 ceramide is highly abundant in atherosclerotic plaques and is positively associated with plaque severity, and SHMT2-deleted macrophages treated with C16:0 fatty acid enhances intracellular ceramide. Furthermore, deletion of SHMT2 reduced mitochondrial respiration and enhanced glycolysis similar to LPS-treated macrophages. Moreover, we found SHMT2 is highly expressed in plaque-associated macrophages in both human and mouse atherosclerotic plaques. We therefore hypothesized that SHMT2 regulates macrophage inflammatory phenotype in atherosclerosis through alterations in mitochondrial function and ceramide biosynthesis. To determine whether macrophage-expressed SHMT2 contributes to atherosclerosis, we developed a novel Shmt2 -floxed mouse crossed with LysM-Cre ( Shmt2 MKO ) and Apoe -/- mice. After 12 weeks on Western diet, we observed no differences in plasma glycine:serine or circulating lipids. Nevertheless, Shmt2 MKO / Apoe -/- mice had significantly enhanced plaque area compared to littermate controls. Our findings, representing the first investigation of conditional SHMT2 knockout mice, suggest a novel immunometabolic role for SHMT2 in macrophages in atherosclerosis.

In situ visualization of opioid and cannabinoid drug effects using phosphosite-specific GPCR antibodies

G protein-coupled receptors (GPCRs) are important signal transducers that are phosphorylated upon activation at intracellular serine and threonine residues. Although antibodies that specifically recognize the phosphorylation state of GPCRs have been available for many years, efficient immunolocalization of phosphorylated receptors in their tissues of origin has not been possible. Here, we show that phosphorylation of receptors is highly unstable during routine immunohistochemical procedures, requiring the use of appropriate phosphatase inhibitors particular during tissue perfusion, post-fixation, and cryoprotection but not during immunostaining of tissue sections. We provide proof of concept using phosphorylation state-specific μ-opioid receptor (MOP) and cannabinoid receptor 1 (CB1) antibodies. Indeed, three of four well-characterized phosphosite-specific MOP antibodies, including pS375-MOP, pT376-MOP, and pT379-MOP, showed robust neuronal immunostaining in brain and spinal cord sections of opioid-treated mice only after inclusion of phosphatase inhibitors. We then extended this approach to the CB1 receptor and demonstrated that one of three newly-generated phosphosite-specific CB1 antibodies, namely pS425-CB1, showed striking staining of fibers and varicosities in brain slices from cannabinoid-treated mice. Although subsequent experiments showed that phospho-CB1 immunostaining was less sensitive to phosphatases, we conclude that the use of phosphatase inhibitors should always be considered in the development of immunohistochemical procedures for new phosphosite-specific GPCR antibodies. In summary, we anticipate that this improved protocol will facilitate the widespread use of phosphorylation state-specific antibodies to monitor the activation of endogenous GPCRs under physiological and pharmacological conditions. Our approach may also prove useful to confirm target engagement of GPCR drug candidates in native tissues.

Phosphoglycerate dehydrogenase activates PKM2 to phosphorylate histone H3T11 and attenuate cellular senescence

Vascular endothelial cells (ECs) senescence correlates with the increase of cardiovascular diseases in ageing population. Although ECs rely on glycolysis for energy production, little is known about the role of glycolysis in ECs senescence. Here, we report a critical role for glycolysis-derived serine biosynthesis in preventing ECs senescence. During senescence, the expression of serine biosynthetic enzyme PHGDH is significantly reduced due to decreased transcription of the activating transcription factor ATF4, which leads to reduction of intracellular serine. PHGDH prevents premature senescence primarily by enhancing the stability and activity of pyruvate kinase M2 (PKM2). Mechanistically, PHGDH interacts with PKM2, which prevents PCAF-catalyzed PKM2 K305 acetylation and subsequent degradation by autophagy. In addition, PHGDH facilitates p300-catalyzed PKM2 K433 acetylation, which promotes PKM2 nuclear translocation and stimulates its activity to phosphorylate H3T11 and regulate the transcription of senescence-associated genes. Vascular endothelium-targeted expression of PHGDH and PKM2 ameliorates ageing in mice. Our findings reveal that enhancing serine biosynthesis could become a therapy to promote healthy ageing.

Characterization of a novel cold-adapted intracellular serine protease from the extremophile Planococcus halocryophilus Or1.

The enzymes of microorganisms that live in cold environments must be able to function at ambient temperatures. Cold-adapted enzymes generally have less ordered structures that convey a higher catalytic rate, but at the cost of lower thermodynamic stability. In this study, we characterized P355, a novel intracellular subtilisin protease (ISP) derived from the genome of Planococcus halocryophilus Or1, which is a bacterium metabolically active down to -25°C. P355's stability and activity at varying pH values, temperatures, and salt concentrations, as well as its temperature-dependent kinetics, were determined and compared to an uncharacterized thermophilic ISP (T0099) from Parageobacillus thermoglucosidasius, a previously characterized ISP (T0034) from Planococcus sp. AW02J18, and Subtilisin Carlsberg (SC). The results showed that P355 was the most heat-labile of these enzymes, closely followed by T0034. P355 and T0034 exhibited catalytic constants (k cat ) that were much higher than those of T0099 and SC. Thus, both P355 and T0034 demonstrate the characteristics of the stability-activity trade-off that has been widely observed in cold-adapted proteases.

DPP8/9 are not Required to Cleave Most Proline-Containing Peptides.

Small molecule inhibitors of the intracellular serine peptidases DPP8 and DPP9 (DPP8/9) activate the NLRP1 and CARD8 inflammasomes, but the key DPP8/9 substrates have not yet been identified. DPP8/9 cleave after proline to remove N-terminal dipeptides from peptides or proteins, and studies using pseudo-peptide reporter substrates have suggested that these enzymes may play key roles in the catabolism of many proline-containing peptides generated by the proteasome. Here, we evaluated the degradation of a wide array of actual peptides in cell lysates, and discovered that DPP8/9 are not in fact involved in the processing of the vast majority of proline-containing peptides. Overall, these results indicate that DPP8/9 have a much more limited substrate scope than previously thought, and likely specifically cleave some critically important, but as yet unknown, intracellular peptide or protein that regulates inflammasome activation.

Study of high selenium interfering with glucose and one-carbon metabolism in hepatocytes in vitro

To investigate the effects of high selenium environment on the expression of selenoproteins and enzymes related to glucose and one-carbon metabolism in normal human hepatocytes. Ten different concentrations of selenomethionine(SeMet, 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 μmol/L) was added into the normal human hepatocyts and incubated for 48 hours. The expressions of selenoprotein(GPX1 and SELENOP1) and metabolic enzymes(PHGDH, SHMT1, MTHFR and MS) were analyzed by Western blot. When the concentration of SeMet was 0-10 μmol/L, the expression trend of selenoprotein(GPX1 and SELENOP1) is similar, which first increases and then decreases. There is a slight difference between the inflection points of GPX1 and SELENOP1, which are respectively 0.5 μmol/L and 0.1 μmol/L. The expression trend of serine de novo synthesis pathway key enzymes(PHGDH) and folate cycle metabolizing enzymes(SHMT1, MTHFR and MS) is similar to that of selenoproteins, which also increases first and then decreases, but the inflection points are different, which are respectively 0.1 μmol/L(PHGDH and SHMT1) and 0.01 μmol/L(MTHFR and MS). Under the high selenium environment, the glycolytic bypass-serine de novo synthesis pathway is activated to synthesize endogenous serine due to the insufficient intracellular serine supply, causing abnormal glucose metabolism, which is an important extension to the hypothesis of the molecular mechanism of high selenium causing IR.

P731: INTERPLAY BETWEEN INFLAMMATION AND EPIGENETICS IN TUMOR REPROGRAMMING OF MESENCHYMAL STROMAL CELLS (MSCS) IN MYELODYSPLASTIC SYNDROMES (MDS)

Background: The alteration of hematopoiesis in MDS patients is deeply associated with microenvironment alterations, in particular in MSCs. Stromal cells are epigenetically reprogrammed to function in cooperation with leukemic cells and propagate the disease as a “tumor unit”. Notably, dysfunctions of MDS-MSCs persist following expansion ex vivo suggesting a hereditable epigenetic dysregulation which endures despite removal of disease-associated microenvironment factors. Aims: Here, we investigated the role of histone H2A variant MacroH2A1 (mH2A1) in promoting pro-tumorigenic inflammation and metabolic reprogramming of MSCs. Methods: MSCs were collected from BM of MDS patients (n=18) and matched healthy controls (n=8; HC). HS-5 cells were used as model of healthy MSCs. Real time, western blot, immunohistochemistry and immunofluorescence were used. Proteomic and metabolic analysis were performed. Results: MDS patients had significant higher signature of mH2A1 in bone marrow slides compared to age-matched control. Investigating mH2A1 expression in stromal cells, MDS-MSCs had increased levels of expression in respect of HC-MSCs. In accordance with the aberrant inflammation described in MDS microenvironment, MDS-MSCs showed higher levels of the sum of NO2+ and NO3+ associated to increased GSH and NADP+/NAPH. TLR4 also was upregulated and positively correlated to mH2A1 expression. To better investigate the relationship between mH2A1 and TLR4 in MDS-MSCs, we induced mH2A1 overexpression in HS-5 cells (mH2A1-OE) by mH2A1-CT-MYC plasmid. Our data showed that mH2A1-OE upregulated TLR4 and increased NFkB nuclear translocation compared to cells transfected with empty vector (CTL). Proteomic analysis confirmed upregulation of intracellular serine protease inhibitors (SerpinB2, B8, B6) strongly induced during inflammation and important for the maintenance of TLR4 activation. Moreover, proteomic approach identified upregulation of several proteins associated to hypermethylation of DNA and histones in mH2A1-OE. In particular, S-adenosylhomocysteine hydrolase which regulates the concentration of S-adenosylhomocysteine (SAH), a strong inhibitor of methyltransferase reactions and of the methyl donor S-adenosyl-methionine (SAM), resulted overexpressed. HPLC analysis showed higher SAM/SAH ratio associated to a significant reduction of SAH in mH2A1-OE, confirming the increase of the methylation index. In addition, the higher levels of CBX3, a suppressive epigenetic mark which recognizes histone H3K9me3, contributes to the maintenance of the heterochromatin. The higher levels of H3K9me3 in mh2A1-OE were confirmed by western blot analysis. Overexpression of mH2A1 also induced metabolic reprogramming with the acquisition of a more glycolytic metabolism characterized by decreased levels of NAD+/NADH, upregulation of LDHA and MCT4. As LDHA could translocate into the nucleus, we evaluated if it used a noncanonical enzymatic activity in mh2A1-OE. Data showed increased nuclear localization of LDHA producing histone H3K79 hypermethylation. Nuclear LDHA was also observed in MDS-MSC. Finally, we treated ex-vivo HC- and MDS-MSCs with azacytidine founding a significant reduction both of mH2A1 and TLR4 associated to lower levels of nuclear NFKB. Summary/Conclusion: Our data provide a key role of mH2A1in driving the crosstalk between inflammation, metabolism and epigenetic signatures in MDS-MSC. Future experiments will define the contribution of mH2A to ineffective hematopoiesis and leukemic evolution.

De novo serine synthesis regulates chondrocyte proliferation during bone development and repair

The majority of the mammalian skeleton is formed through endochondral ossification starting from a cartilaginous template. Cartilage cells, or chondrocytes, survive, proliferate and synthesize extracellular matrix in an avascular environment, but the metabolic requirements for these anabolic processes are not fully understood. Here, using metabolomics analysis and genetic in vivo models, we show that maintaining intracellular serine homeostasis is essential for chondrocyte function. De novo serine synthesis through phosphoglycerate dehydrogenase (PHGDH)-mediated glucose metabolism generates nucleotides that are necessary for chondrocyte proliferation and long bone growth. On the other hand, dietary serine is less crucial during endochondral bone formation, as serine-starved chondrocytes compensate by inducing PHGDH-mediated serine synthesis. Mechanistically, this metabolic flexibility requires ATF4, a transcriptional regulator of amino acid metabolism and stress responses. We demonstrate that both serine deprivation and PHGDH inactivation enhance ATF4 signaling to stimulate de novo serine synthesis and serine uptake, respectively, and thereby prevent intracellular serine depletion and chondrocyte dysfunction. A similar metabolic adaptability between serine uptake and de novo synthesis is observed in the cartilage callus during fracture repair. Together, the results of this study reveal a critical role for PHGDH-dependent serine synthesis in maintaining intracellular serine levels under physiological and serine-limited conditions, as adequate serine levels are necessary to support chondrocyte proliferation during endochondral ossification.

Impact of serine and serine synthesis genes on H2S release in Saccharomyces cerevisiae during wine fermentation

Excessive hydrogen sulfide (H2S) during fermentation causes undesirable sensory properties in wine. In yeast, serine functions as a precursor in the biosynthesis of S-containing compounds, which facilitates H2S consumption. To investigate the effect of serine on H2S release and the underlying mechanism, extracellular and intracellular serine levels were separately increased under fermentation conditions. The results show that, although the abundance of extracellular serine was ineffective in decreasing H2S levels, increased levels of intracellular serine levels from SER1 and SER2 overexpression reduced H2S release through increased consumption of sulfur metabolites. In contrast, SER33 overexpression significantly increased H2S release, and the metabolites and gene expression profile of the sulfur assimilation pathway indicates that SER33 regulated MET17, which mediated H2S release. Our study revealed valuable insights on the relationship between serine levels and H2S release, and may be helpful in understanding the H2S regulation mechanism in yeast during fermentation.

Platelet-activating factor acetyl hydrolase IB2 dysregulated cell proliferation in ovarian cancer

BackgroundOvarian cancer is the leading cause of death from gynaecologic illnessed worldwide. Platelet-activating factor acetyl hydrolase IB2 (PAF-AH IB2) is an intracellular serine esterase that hydrolyzes platelet-activating factor, a G-protein-like trimer with two catalytic subunits and one regulatory subunit. The regulatory role of PAF-AH IB2 in the oncogenesis of ovarian cancer is not well understood.MethodsIn this study, the TCGA dataset and clinical cancer tissue microarray were utilized to investigate abnormal overexpression of PAF-AH IB2 in ovarian cancer. To investigate the impact on the cell proliferation, migration, and tumorigenicity in vitro, PAF-AH IB2 stable knocking down (KD) ovarian cancer cells were established by ShRNA. The whole transcription profiling, tyrosine kinase profiling and standard cell functional assays were integrated to explore the biological importance and mechanism of PAF-AH IB2 modulated in ovarian cancer.ResultsPAF-AH IB2 was identified significantly overexpression in four subtypes of ovarian cancer. In vitro, PAF-AH IB2 KD significantly inhibited cancer cell proliferation, migration, and tumorigenicity, activated caspases and caused cell cycle arrest, and made the cells more sensitive to PAF. PAF-AH 1B2 KD cells down-regulated several key regulators of the multiple tyrosine kinases-mediated signaling pathway, suggesting a novel interaction network between the growth factor receptors pathway and PAF-AH 1B2 mediated PAF signalling.ConclusionsThese findings revealed a previously undiscovered role for PAF-AH IB2 as a potenial therapy target and essential signaling mediators in ovarian cancer pathogenesis, as well as new possible preventive and therapeutic strategies to inhibit this enzyme in clinical treatment for ovarian cancer.

ROLE OF GSK-3 IN Wnt/β-CATENIN SIGNALING PATHWAY IN OBESITY

The complexity of the adipogenesis mechanism results from the impact of multiple cues, among which an important place is held by the components of the Wnt signaling pathway. The search for potential markers of the development of diseases related to obesity aroused an interest in the study of GSK-3 (glycogen synthase kinase), β-catenin. GSK-3β is an intracellular serine / threonine kinase found in the cytoplasm, nucleus, mitochondria, synthesized in all body tissues and involved in regulating metabolic processes, cell proliferation, apoptosis etc. The first of the discovered functions of GSK-3β was the regulation of glycogen synthesis. Active GSK-3β phosphorylates and thereby inhibits glycogen synthase. As a result of the insulin binding to the cell receptor via inositol-3-phosphate, protein kinase B (Akt1) is activated, which, in turn, phosphorylates and inhibits GSK-3β. In addition, GSK-3β is involved in the regulating glucose metabolism. The most important function of GSK-3β is the inhibition of the β-catenin protein. In a resting cell, GSK-3β in complex with the APC and Axin proteins binds and phosphorylates the β-catenin transcription factor, which leads to its ubiquitination and degradation. When Wnt proteins act on the cell, the Dvl protein is activated, which, by binding to GSK-3β, releases β-catenin, preventing its degradation, however, the role of GSK3α/β in the adipocyte inflammatory response has not yet been fully investigated, therefore it seems promising to study the role of GSK-3 in the Wnt/β-catenin signaling pathway in obesityThe aim of the study was to assess the activity of the components of the Wnt signaling pathway in obese patients by measuring the serum level of GSK-3 and β-catenin. There were enrolled 32 patients with progressive forms of I-III degree obesity in the absence of diabetes mellitus. The concentration of serum GSK-3α, GSK-3β, and β-catenin was measured by enzyme-linked immunoassay. Data are presented as absolute and relative (%) number of patients; arithmetic mean; medians, 1 and 3 quartiles – Ме (Q0.25-Q0.75). Obese patients contained a 7.5-fold increased serum level of GSK-3α (785 (371-1317.5) pg/ml) compared to healthy individuals 105 (102.5-110) pg/ml, (p < 0.001), paralleled with increased amount of GSK-3β, which level in obese patients was 295 (190-695) pg/ml, which is by 18.3% higher than those in healthy individuals 241 (218.75-287.5) pg/ml, p = 0.111. Amount of GSK-3 depending on the degree of obesity tended to increase, most often coupled to decreased β-catenin level which is consistent with the literature data and can be considered as a prognostic criterion for the course of pathological processes in obesity.

ATF3 promotes the serine synthesis pathway and tumor growth under dietary serine restriction.

SUMMARYThe serine synthesis pathway (SSP) involving metabolic enzymes phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH) drives intracellular serine biosynthesis and is indispensable for cancer cells to grow in serine-limiting environments. However, how SSP is regulated is not well understood. Here, we report that activating transcription factor 3 (ATF3) is crucial for transcriptional activation of SSP upon serine deprivation. ATF3 is rapidly induced by serine deprivation via a mechanism dependent on ATF4, which in turn binds to ATF4 and increases the stability of this master regulator of SSP. ATF3 also binds to the enhancers/promoters of PHGDH, PSAT1, and PSPH and recruits p300 to promote expression of these SSP genes. As a result, loss of ATF3 expression impairs serine biosynthesis and the growth of cancer cells in the serine-deprived medium or in mice fed with a serine/glycine-free diet. Interestingly, ATF3 expression positively correlates with PHGDH expression in a subset of TCGA cancer samples.

Study on degranulation of mast cells under C48/80 treatment by electroporation-assisted and ultrasound-assisted surface-enhanced Raman spectrascopy

Both electroporation-assisted and ultrasound-assisted delivery methods can rapidly deliver nanoparticles into living cells for surface-enhanced Raman scattering (SERS) detection, but these two methods have never been compared. In this study, electroporation-assisted SERS and ultrasound-assisted SERS were employed to detect the biochemical changes of degranulated mast cells induced by mast cell stimulator (C48/80). The results showed that the cell damage of electroporation based on controllable electric pulse was smaller than that of ultrasound based on cavitation. Transmission electron microscope images of cells indicated that the nanoparticles delivered by electroporation were mainly distributed in the cytoplasm, while ultrasound could transport nanoparticles to the cytoplasm and nucleus. Therefore, electroporation-assisted SERS mainly detects the biochemical information of cytoplasm, while ultrasound-assisted SERS gets more spectral signals of nucleic acid. Both methods can obtain high quality SERS signal of cells. With drug treatment, the SERS peak intensity of 733 cm−1 attributed to phosphatidylserine decreased significantly, which may be due to the activation of mast cell degranulation pathway stimulated by C48/80 agonist, resulting in a large amount of intracellular serine being used to synthesize tryptase, while the production of phosphatidylserine decreased. Further, based on principal component analysis and linear discriminant analysis (PCA-LDA approach), ultrasound-assisted SERS could achieve better sensitivity, specificity and accuracy in the discrimination and identification of drug-treated degranulated mast cells than electroporation assisted SERS. This exploratory work is helpful to realize the real-time dynamic SERS detection of intracellular biochemical components, and it also has great potential in intracellular SERS analysis, such as the cytotoxicity assay of anti-tumor drugs or cancer cell screening.

Discovery of novel inhibitors of human phosphoglycerate dehydrogenase by activity-directed combinatorial chemical synthesis strategy

Serine, the source of the one-carbon units essential for de novo purine and deoxythymidine synthesis plays a crucial role in the growth of cancer cells. Phosphoglycerate dehydrogenase (PHGDH) which catalyzes the first, rate-limiting step in de novo serine biosynthesis has become a promising target for the cancer treatment. Here we identified H-G6 as a potential PHGDH inhibitor from the screening of an in-house small molecule library based on the enzymatic assay. We adopted activity-directed combinatorial chemical synthesis strategy to optimize this hit compound. Compound b36 was found to be the noncompetitive and the most promising one with IC50 values of 5.96 ± 0.61 μM against PHGDH. Compound b36 inhibited the proliferation of human breast cancer and ovarian cancer cells, reduced intracellular serine synthesis, damaged DNA synthesis, and induced cell cycle arrest. Collectively, our results suggest that b36 is a novel PHGDH inhibitor, which could be a promising modulator to reprogram the serine synthesis pathway and might be a potential anticancer lead worth further exploration.