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Journal of the American Chemical Society Nov 2023DNA-encoded chemical library (DEL) has been extensively used for lead compound discovery for decades in academia and industry. Incorporating an electrophile warhead into...
DNA-encoded chemical library (DEL) has been extensively used for lead compound discovery for decades in academia and industry. Incorporating an electrophile warhead into DNA-encoded compounds recently permitted the discovery of covalent ligands that selectively react with a particular cysteine residue. However, noncysteine residues remain underexplored as modification sites of covalent DELs. Herein, we report the design and utility of tyrosine-targeting DELs of 67 million compounds. Proteome-wide reactivity analysis of tyrosine-reactive sulfonyl fluoride (SF) covalent probes suggested three enzymes (phosphoglycerate mutase 1, glutathione s-transferase 1, and dipeptidyl peptidase 3) as models of tyrosine-targetable proteins. Enrichment with SF-functionalized DELs led to the identification of a series of tyrosine-targeting covalent inhibitors of the model enzymes. In-depth mechanistic investigation revealed their novel modes of action and reactive ligand-accessible hotspots of the enzymes. Our strategy of combining activity-based proteome profiling and covalent DEL enrichment (ABPP-CoDEL), which generated selective covalent binders against a variety of target proteins, illustrates the potential use of this methodology in further covalent drug discovery.
Topics: Proteome; Tyrosine; Drug Discovery; Small Molecule Libraries; Ligands; DNA
PubMed: 37857329
DOI: 10.1021/jacs.3c08852 -
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 -
Frontiers in Oncology 2023(wormwood) exhibits anticancer properties by inhibiting proliferation and causing cell death in breast cancer. Targeted drug delivery of nanoformulation using...
INTRODUCTION
(wormwood) exhibits anticancer properties by inhibiting proliferation and causing cell death in breast cancer. Targeted drug delivery of nanoformulation using N-isopropyl acrylamide, N-vinyl pyrrolidone, and acrylic acid-based polymeric nanoparticles (NVA-AA NPs) was ensured by utilizing features of the tumor microenvironment, although their mechanism of action involved in cytotoxicity remains unknown.
METHODS
The present study employed nano LC-MS/MS to identify differences in secretory protein expression associated with the treatment of breast cancer cell lines (MCF-7; MDA-MB-231) by NVA-AA NPs for the determination of affected pathways and easily accessible therapeutic targets. Different bioinformatics tools were used to identify signature differentially expressed proteins (DEPs) using survival analysis by GENT2 and correlation analysis between their mRNA expressions and sensitivity toward small-molecule drugs as well as immune cell infiltration by GSCA.
RESULTS
Analysis by GENT2 revealed 22 signature DEPs with the most significant change in their expression regulation, namely, gelsolin, alpha-fetoprotein, complement component C3, C7, histone H2B type 1-K, histone H2A.Z, H2AX, heat shock cognate 71 kDa protein, heat shock 70 kDa protein 1-like, cytochrome c somatic, GTP-binding nuclear protein Ran, tubulin beta chain, tubulin alpha-1B chain, tubulin alpha-1C chain, phosphoglycerate mutase 1, kininogen 1, carboxypeptidase N catalytic chain, fibulin-1, peroxiredoxins 4, lactate dehydrogenase C, SPARC, and SPARC-like protein 1. Correlation analysis between their mRNA expressions versus immune cell infiltrates showed a positive correlation with antitumor immune response elicited by these NPs as well as a correlation with drug response shown by the GDSC and CTRP drugs in different cancer cells.
DISCUSSION
Our results suggest that NVA-AA NPs were able to invade the tumor microenvironment; transformed the communication network between the cancer cells; affected potential drivers of microtubular integrity, nucleosome assembly, and cell cycle; and eventually caused cell death.
PubMed: 37719007
DOI: 10.3389/fonc.2023.1209168 -
Biochemistry and Biophysics Reports Sep 2023A growing body of evidence indicated that Alantolactone (ALT) promotes Reactive Oxygen Species (ROS) generation exclusively in cancer cells. Therefore, the aim of this...
INTRODUCTION
A growing body of evidence indicated that Alantolactone (ALT) promotes Reactive Oxygen Species (ROS) generation exclusively in cancer cells. Therefore, the aim of this study was to investigate the effect of ALT on the molecular mechanism of oxeiptosis, as a novel cell death pathway due to the high levels of intracellular ROS in ovarian cancer.
METHODS
MTT assay was used to evaluate the effect of ALT on SKOV3 cell viability. mRNA and protein expression levels of Nrf2 (nuclear factor erythroid 2-related factor 2), KEAP1 (Kelch-like ECH-associated protein 1), PGAM5 (phosphoglycerate mutase family member 5), AIFM1 (Mitochondrial Apoptosis-Inducing Factor), Glutathione synthetase (GSS) and glutathione peroxidase (GPX) were analyzed by real time PCR and western blotting methods respectively.
RESULTS
Our findings showed that ALT inhibits the proliferation of skov3 cells in a time and dose dependent manner and IC50 was 32 μM at 24h.A significant down-regulation of Nrf2, GSH and GPX mRNA levels was seen in skov3 cells incubated with 32 and 64 μM of ALT in comparison with control group, while, mRNA expression levels of PGAM5 and KEAP1 were increased.Western blot analysis showed that ALT significantly decreases protein levels of Nrf2 and increases PGAM5 and KEAP1.ALT dephosphorylated PS116-AIFM1 and total AIFM1 protein level was elevated.
CONCLUSION
Our results provided evidence that ALT could be a potential option for ovarian cancer treatment by ROS-mediated oxeiptosis.
PubMed: 37712005
DOI: 10.1016/j.bbrep.2023.101537 -
Advanced Science (Weinheim,... Oct 2023The combination of immunotherapy and molecular targeted therapy exhibits promising therapeutic efficacy in hepatocellular carcinoma (HCC), but the underlying mechanism...
The combination of immunotherapy and molecular targeted therapy exhibits promising therapeutic efficacy in hepatocellular carcinoma (HCC), but the underlying mechanism is still unclear. Here, phosphoglycerate mutase 1 (PGAM1) is identified as a novel immunometabolic target by using a bioinformatic algorithm based on multiple HCC datasets. PGAM1 is highly expressed in HCC and associated with a poor prognosis and a poor response to immunotherapy. In vitro and in vivo experiments indicate that targeting PGAM1 inhibited HCC cell growth and promoted the infiltration of CD8 T-cells due to decreased enzymatic activity. Mechanistically, inhibition of PGAM1 promotes HCC cell ferroptosis by downregulating Lipocalin (LCN2) by inducing energy stress and ROS-dependent AKT inhibition, which can also downregulate Programmed death 1-ligand 1 (PD-L1). Moreover, an allosteric PGAM1 inhibitor (KH3) exhibits good antitumor effects in patient-derived xenograft (PDX) models and enhanced the efficacy of anti-PD-1 immunotherapy in subcutaneous and orthotopic HCC models. Taken together, the findings demonstrate that PGAM1 inhibition exerts an antitumor effect by promoting ferroptosis and CD8 T-cell infiltration and can synergize with anti-PD-1 immunotherapy in HCC. Targeting PGAM1 can be a promising new strategy of "killing two birds with one stone" for HCC treatment.
Topics: Humans; Carcinoma, Hepatocellular; Liver Neoplasms; Phosphoglycerate Mutase; CD8-Positive T-Lymphocytes; Ferroptosis; Immunotherapy
PubMed: 37705495
DOI: 10.1002/advs.202301928 -
Acta Pharmacologica Sinica Jan 2024Acute kidney injury (AKI) is a worldwide public health problem characterized by the massive loss of tubular cells. However, the precise mechanism for initiating tubular...
Acute kidney injury (AKI) is a worldwide public health problem characterized by the massive loss of tubular cells. However, the precise mechanism for initiating tubular cell death has not been fully elucidated. Here, we reported that phosphoglycerate mutase 5 (PGAM5) was upregulated in renal tubular epithelial cells during ischaemia/reperfusion or cisplatin-induced AKI in mice. PGAM5 knockout significantly alleviated the activation of the mitochondria-dependent apoptosis pathway and tubular apoptosis. Apoptosis inhibitors alleviated the activation of the mitochondria-dependent apoptosis pathway. Mechanistically, as a protein phosphatase, PGAM5 could dephosphorylate Bax and facilitate Bax translocation to the mitochondrial membrane. The translocation of Bax to mitochondria increased membrane permeability, decreased mitochondrial membrane potential and facilitated the release of mitochondrial cytochrome c (Cyt c) into the cytoplasm. Knockdown of Bax attenuated PGAM5 overexpression-induced Cyt c release and tubular cell apoptosis. Our results demonstrated that the increase in PGAM5-mediated Bax dephosphorylation and mitochondrial translocation was implicated in the development of AKI by initiating mitochondrial Cyt c release and activating the mitochondria-dependent apoptosis pathway. Targeting this axis might be beneficial for alleviating AKI.
Topics: Mice; Animals; Cytochromes c; Phosphoglycerate Mutase; bcl-2-Associated X Protein; Apoptosis; Mitochondria; Acute Kidney Injury; Carrier Proteins; Phosphoprotein Phosphatases
PubMed: 37684381
DOI: 10.1038/s41401-023-01151-1 -
Cancer Research Nov 2023The next-generation androgen receptor (AR) inhibitor enzalutamide is the mainstay treatment for metastatic prostate cancer. Unfortunately, resistance occurs rapidly in...
UNLABELLED
The next-generation androgen receptor (AR) inhibitor enzalutamide is the mainstay treatment for metastatic prostate cancer. Unfortunately, resistance occurs rapidly in most patients, and once resistance occurs, treatment options are limited. Therefore, there is an urgent need to identify effective targets to overcome enzalutamide resistance. Here, using a genome-wide CRISPR-Cas9 library screen, we found that targeting a glycolytic enzyme, phosphoglycerate mutase PGAM2, significantly enhanced the sensitivity of enzalutamide-resistant prostate cancer cells to enzalutamide both in vivo and in vitro. Inhibition of PGAM2 together with enzalutamide treatment triggered apoptosis by decreasing levels of the antiapoptotic protein BCL-xL and increasing activity of the proapoptotic protein BAD. Mechanistically, PGAM2 bound to 14-3-3ζ and promoted its interaction with phosphorylated BAD, resulting in activation of BCL-xL and subsequent resistance to enzalutamide-induced apoptosis. In addition, high PGAM2 expression, which is transcriptionally regulated by AR, was associated with shorter survival and rapid development of enzalutamide resistance in patients with prostate cancer. Together, these findings provide evidence of a nonmetabolic function of PGAM2 in promoting enzalutamide resistance and identify PGAM2 inhibition as a promising therapeutic strategy for enzalutamide-resistant prostate cancer.
SIGNIFICANCE
PGAM2 promotes resistance to enzalutamide by activating antiapoptotic BCL-xL and suppressing apoptosis, indicating that PGAM2 is a potential target for overcoming enzalutamide resistance in prostate cancer.
Topics: Humans; Male; 14-3-3 Proteins; Androgen Receptor Antagonists; Cell Line, Tumor; Drug Resistance, Neoplasm; Prostatic Neoplasms, Castration-Resistant; Proto-Oncogene Proteins c-bcl-2; Receptors, Androgen
PubMed: 37676279
DOI: 10.1158/0008-5472.CAN-23-0308