Moonlighting Proteins and Cardiopathy in the Spatial Response of MCF‐7 Breast Cancer Cells to Tamoxifen

Background: The purpose of this study is to apply quantitative high‐throughput proteomics methods to investigate dynamic aspects of protein changes in nucleocytoplasmic distribution of proteins and of total protein abundance for MCF‐7 cells exposed to tamoxifen (Tam) in order to reveal the agonistic and antagonistic roles of the drug. / Experimental design: The MS‐based global quantitative proteomics with the analysis of fractions enriched in target subcellular locations is applied to measure the changes in total abundance and in the compartmental abundance/distribution between the nucleus and cytoplasm for several thousand proteins differentially expressed in MCF‐7 cells in response to Tam stimulation. / Results: The response of MCF‐7 cells to the Tam treatment shows significant changes in subcellular abundance rather than in their total abundance. The bioinformatics study reveals the relevance of moonlighting proteins and numerous pathways involved in Tam response of MCF‐7 including some of which may explain the agonistic and antagonistic roles of the drug. / Conclusions: The results indicate possible protective role of Tam against cardiovascular diseases as well as its involvement in G‐protein coupled receptors pathways that enhance breast tissue proliferation

Molecular basis for the reversible ADP-ribosylation of guanosine bases

Modification of nucleic acids by ADP-ribosylation is catalyzed by various ADP-ribosyltransferases, including the DarT enzyme. The latter is part of the bacterial toxin-antitoxin (TA) system DarTG, which was shown to provide control of DNA replication and bacterial growth as well as protection against bacteriophages. Two subfamilies have been identified, DarTG1 and DarTG2, which are distinguished by their associated antitoxins. While DarTG2 catalyzes reversible ADP-ribosylation of thymidine bases employing a macrodomain as antitoxin, the DNA ADP-ribosylation activity of DarTG1 and the biochemical function of its antitoxin, a NADAR domain, are as yet unknown. Using structural and biochemical approaches, we show that DarT1-NADAR is a TA system for reversible ADP-ribosylation of guanosine bases. DarT1 evolved the ability to link ADP-ribose to the guanine amino group, which is specifically hydrolyzed by NADAR. We show that guanine de-ADP-ribosylation is also conserved among eukaryotic and non-DarT-associated NADAR members, indicating a wide distribution of reversible guanine modifications beyond DarTG systems

Allosteric activation of T cell antigen receptor signaling by quaternary structure relaxation

The mechanism of T cell antigen receptor (TCR-CD3) signaling remains elusive. Here, we identify mutations in the transmembrane region of TCRβ or CD3ζ that augment peptide T cell antigen receptor (pMHC)-induced signaling not explicable by enhanced ligand binding, lateral diffusion, clustering, or co-receptor function. Using a biochemical assay and molecular dynamics simulation, we demonstrate that the gain-of-function mutations loosen the interaction between TCRαβ and CD3ζ. Similar to the activating mutations, pMHC binding reduces TCRαβ cohesion with CD3ζ. This event occurs prior to CD3ζ phosphorylation and at 0°C. Moreover, we demonstrate that soluble monovalent pMHC alone induces signaling and reduces TCRαβ cohesion with CD3ζ in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCRαβ to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to license CD3ζ phosphorylation and initiate signal propagation

Regulation of Hemolysin Expression and Virulence of Staphylococcus aureus by a Serine/Threonine Kinase and Phosphatase

Exotoxins, including the hemolysins known as the alpha (α) and beta (β) toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1) were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1) increased expression. Transcription of the hla gene encoding α toxin was decreased in a Δstp1 mutant strain and increased in a Δstk1 strain. Microarray analysis of a Δstk1 mutant revealed increased transcription of additional exotoxins. A Δstp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Δstk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU), serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE) and a hypothetical protein (NWMN_1123) were present in the wild type and not in the Δstk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence

Rapid aggregation and assembly in aqueous solution of A beta (25-35) peptide

The highly toxic Aβ(25-35) is a peculiar peptide that differs from all the other commonly studied β-amyloid peptides because of its extremely rapid aggregation properties and enhanced neurotoxicity. We investigated Aβ(25-35) aggregation in H2O at pH 3.0 and at pH 7.4 by means of in-solution analyses. Adopting UV spectroscopy, Congo red spectrophotometry and thiofl avin T fl uorimetry, we were able to quantify, in water, the very fast assembling time necessary for Aβ(25-35) to form stable insoluble aggregates and their ability to seed or not seed fi bril growth. Our quantitative results, which confi rm a very rapid assembly leading to stable insoluble aggregates of Aβ(25-35) only when incubated at pH 7.4, might be helpful for designing novel aggregation inhibitors and to shed light on the in vivo environment in which fi bril formation takes place