Polyamines, often elevated in cancer cells, have been shown to promote cell growth and proliferation. Whether polyamines regulate other cell functions remains unclear. Here, we explore whether and ...how polyamines affect genome integrity. When DNA double-strand break (DSB) is induced in hair follicles by ionizing radiation, reduction of cellular polyamines augments dystrophic changes with delayed regeneration. Mechanistically, polyamines facilitate homologous recombination-mediated DSB repair without affecting repair via non-homologous DNA end-joining and single-strand DNA annealing. Biochemical reconstitution and functional analyses demonstrate that polyamines enhance the DNA strand exchange activity of RAD51 recombinase. The effect of polyamines on RAD51 stems from their ability to enhance the capture of homologous duplex DNA and synaptic complex formation by the RAD51-ssDNA nucleoprotein filament. Our work demonstrates a novel function of polyamines in the maintenance of genome integrity via homology-directed DNA repair.
Cysteine oxidation, either biologically reversible or irreversible, is the main posttranslational modification associated with redox signaling and oxidative stress. Maleimide‐polyethylene glycol ...(m‐PEG) has been used to detect reversibly oxidized proteins by reacting to the reduced cysteine residues leading to mobility shift in immunoblots; a method called PEG‐switch. With PEG‐switch, both reduced and oxidized proteins can be observed on the same immunoblot simultaneously, providing a simple quantitative measurement for protein thiol modifications. In this report, we optimized the assay conditions and exploited the applications of PEG‐switch in quantitation of the extent of protein thiol oxidation in cells in response to H2O2 and insulin. In addition, we have proposed a redox scoring system for measuring the redox status of any given protein from the m‐PEG immunoblot. Our results provided quantitative data showing that two cysteine residues of protein tyrosine phosphatase 1B are prone to oxidation following insulin treatment in cultured HeLa cells.
The combination of EGF, CHIR99021, A83-01, SB431542, VPA, and Y27632 (EGF/CASVY) facilitates the derivation of trophoblast stem (TS) cells from human blastocysts and first-trimester, but not term, ...cytotrophoblasts. The mechanism underlying this chemical induction of TS cells remains elusive. Here we demonstrate that the induction efficiency of cytotrophoblast is determined by functional antagonism of the placental transcription factor GCM1 and the stemness regulator ΔNp63α. ΔNp63α reduces GCM1 transcriptional activity, whereas GCM1 inhibits ΔNp63α oligomerization and autoregulation. EGF/CASVY cocktail activates ΔNp63α, thereby partially inhibiting GCM1 activity and reverting term cytotrophoblasts into stem cells. By applying hypoxia condition, we can further reduce GCM1 activity and successfully induce term cytotrophoblasts into TS cells. Consequently, we identify mitochondrial creatine kinase 1 (CKMT1) as a key GCM1 target crucial for syncytiotrophoblast differentiation and reveal decreased CKMT1 expression in preeclampsia. Our study delineates the molecular underpinnings of trophoblast stemness and differentiation and an efficient method to establish TS cells from term placentas.
Cancer cell migration plays a crucial role during the metastatic process. Reversible tyrosine phosphorylation by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) have been ...implicated in the regulation of cancer cell migration and invasion. However, the underlying mechanisms have not been fully elucidated. Here, we show that depletion of the FERM and PDZ domain-containing protein tyrosine phosphatase PTPN3 enhances lung cancer cell migration/invasion and metastasis by promoting actin filament assembly and focal adhesion dynamics. We further identified Src and DAAM1 (dishevelled associated activator of morphogenesis 1) as interactors of PTPN3. DAAM1 is a formin-like protein involved in the regulation of actin cytoskeletal remodeling. PTPN3 inhibits Src activity and Src-mediated phosphorylation of Tyr652 on DAAM1. The tyrosine phosphorylation of DAAM1 is essential for DAAM1 homodimer formation and actin polymerization. Ectopic expression of a DAAM1 phosphodeficient mutant inhibited F-actin assembly and suppressed lung cancer cell migration and invasion. Our findings reveal a novel mechanism by which reversible tyrosine phosphorylation of DAAM1 by Src and PTPN3 regulates actin dynamics and lung cancer invasiveness.
In this study, we used a systems biology approach to investigate changes in the proteome and metabolome of shrimp hemocytes infected by the invertebrate virus WSSV (white spot syndrome virus) at the ...viral genome replication stage (12 hpi) and the late stage (24 hpi). At 12 hpi, but not at 24 hpi, there was significant up-regulation of the markers of several metabolic pathways associated with the vertebrate Warburg effect (or aerobic glycolysis), including glycolysis, the pentose phosphate pathway, nucleotide biosynthesis, glutaminolysis and amino acid biosynthesis. We show that the PI3K-Akt-mTOR pathway was of central importance in triggering this WSSV-induced Warburg effect. Although dsRNA silencing of the mTORC1 activator Rheb had only a relatively minor impact on WSSV replication, in vivo chemical inhibition of Akt, mTORC1 and mTORC2 suppressed the WSSV-induced Warburg effect and reduced both WSSV gene expression and viral genome replication. When the Warburg effect was suppressed by pretreatment with the mTOR inhibitor Torin 1, even the subsequent up-regulation of the TCA cycle was insufficient to satisfy the virus's requirements for energy and macromolecular precursors. The WSSV-induced Warburg effect therefore appears to be essential for successful viral replication.
Current clinical trials of combined EGFR-tyrosine kinase inhibitors (TKI) and immune checkpoint blockade (ICB) therapies show no additional effect. This raises questions regarding whether EGFR-TKIs ...attenuate ICB-enhanced CD8
T lymphocyte function. Here we show that the EGFR-TKI afatinib suppresses CD8
T lymphocyte proliferation, and we identify CAD, a key enzyme of
pyrimidine biosynthesis, to be a novel afatinib target. Afatinib reduced tumor-infiltrating lymphocyte numbers in Lewis lung carcinoma (LLC)-bearing mice. Early afatinib treatment inhibited CD8
T lymphocyte proliferation in patients with non-small cell lung cancer, but their proliferation unexpectedly rebounded following long-term treatment. This suggests a transient immunomodulatory effect of afatinib on CD8
T lymphocytes. Sequential treatment of afatinib with anti-PD1 immunotherapy substantially enhanced therapeutic efficacy in MC38 and LLC-bearing mice, while simultaneous combination therapy showed only marginal improvement over each single treatment. These results suggest that afatinib can suppress CD8
T lymphocyte proliferation by targeting CAD, proposing a timing window for combined therapy that may prevent the dampening of ICB efficacy by EGFR-TKIs. SIGNIFICANCE: This study elucidates a mechanism of afatinib-mediated immunosuppression and provides new insights into treatment timing for combined targeted therapy and immunotherapy. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3270/F1.large.jpg.
•Iodoacetanilide reacts with H2S and thiol metabolites.•The derivatization improves the LC-ESI–MS detection.•Factors affecting H2S and thiol metabolites quantification are ...optimized.•13C6-iodoacetanilide labeling enhances accurate identification and quantification.•Differential analysis with isotopic derivatives becomes feasible.
Hydrogen sulfide (H2S), previously known as a toxic gas, is now recognized as a gasotransmitter along with nitric oxide and carbon monoxide. However, only few methods are available for quantitative determination of H2S in biological samples. 2-Iodoacetanilide (2-IAN), a thiol-reacting agent, has been used to tag the reduced cysteine residues of proteins for quantitative proteomics and for detection of cysteine oxidation modification. In this article, we proposed a new method for quantitative analyses of H2S and thiol metabolites using the procedure of pre-column 2-IAN derivatization coupled with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI–MS). 13C6-Labeled and label-free 2-IAN efficiently react with H2S and thiol compounds at pH 9.5 and 65 °C. The derivatives exhibit excellent stability at alkaline conditions, high resolution on reverse phase liquid chromatography and great sensitivity for ESI–MS detection. The measurement of H2S, l-cysteine, glutathione, and DL-homocysteine derivatives was validated using 13C6-labeled standard in LC-ESI–MS analyses and exhibited 10 nM–1 μM linear ranges for DL-homocysteine and glutathione and 1 nM–1 μM linear ranges for l-cysteine and H2S. In addition, the sequence of derivatization and extraction of metabolites is important in the quantification of thiol metabolites suggesting the presence of matrix effects. Most importantly, labeling with 2-IAN and 13C6-2-IAN isotopologues could achieve quantitative and matched sample comparative analyses with minimal bias using our extraction and labeling procedures before LC–MS analysis.
Diabetes is characterized by chronic hyperglycemia, which in turn facilitates the formation of advanced glycation end products (AGEs). AGEs activate signaling proteins such as Src, Akt and ERK1/2. ...However, the mechanisms by which AGEs activate these kinases remain unclear. We examined the effect of AGEs on Akt activation in 3T3-L1 preadipocytes. Addition of AGEs to 3T3-L1 cells activated Akt in a dose- and time-dependent manner. The AGEs-stimulated Akt activation was blocked by a PI3-kinase inhibitor LY 294002, Src inhibitor PP2, an antioxidant NAC, superoxide scavenger Tiron, or nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase inhibitor DPI, suggesting the involvement of Src and NAD(P)H oxidase in the activation of PI3-kinase-Akt pathway by AGEs. AGEs-stimulated Src tyrosine phosphorylation was inhibited by NAC, suggesting that Src is downstream of NAD(P)H oxidase. The AGEs-stimulated Akt activity was sensitive to Insulin-like growth factor 1 receptor (IGF-1R) kinase inhibitor AG1024. Furthermore, AGEs induced phosphorylation of IGF-1 receptorβsubunit (IGF-1Rβ) on Tyr1135/1136, which was sensitive to PP2, indicating that AGEs stimulate Akt activity by transactivating IGF-1 receptor. In addition, the AGEs-stimulated Akt activation was attenuated by β-methylcyclodextrin that abolishes the structure of caveolae, and by lowering caveolin-1 (Cav-1) levels with siRNAs. Furthermore, addition of AGEs enhanced the interaction of phospho-Cav-1 with IGF-1Rβ and transfection of 3T3-L1 cells with Cav-1 Y14F mutants inhibited the activation of Akt by AGEs. These results suggest that AGEs activate NAD(P)H oxidase and Src which in turn phosphorylates IGF-1 receptor and Cav-1 leading to activation of IGF-1 receptor and the downstream Akt in 3T3-L1 cells. AGEs treatment promoted the differentiation of 3T3-L1 preadipocytes and addition of AG1024, LY 294002 or Akt inhibitor attenuated the promoting effect of AGEs on adipogenesis, suggesting that IGF-1 receptor, PI3-Kinase and Akt are involved in the facilitation of adipogenesis by AGEs.
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