Vasoactive agents which elevate either cGMP or cAMP inhibit platelet activation by pathways sharing at least one component, the 46/50 kDa vasodilator‐stimulated phosphoprotein (VASP). VASP is stoichiometrically phosphorylated by both cGMP‐dependent and cAMP‐dependent protein kinases in intact human platelets, and its phosphorylation correlates very well with platelet inhibition caused by cGMP‐ and cAMP‐elevating agents. Here we report that in human platelets spread on glass, VASP is associated predominantly with the distal parts of radial microfilament bundles and with microfilaments outlining the periphery, whereas less VASP is associated with a central microfilamentous ring. VASP is also detectable in a variety of different cell types including fibroblasts and epithelial cells. In fibroblasts, VASP is concentrated at focal contact areas, along microfilament bundles (stress fibres) in a punctate pattern, in the periphery of protruding lamellae, and is phosphorylated by cGMP‐ and cAMP‐dependent protein kinases in response to appropriate stimuli. Evidence for the direct binding of VASP to F‐actin is also presented. The data demonstrate that VASP is a novel phosphoprotein associated with actin filaments and focal contact areas, i.e. transmembrane junctions between microfilaments and the extracellular matrix.
Cytostatic metallo-drugs mostly bind to the nucleobases of DNA. A new family of dinuclear transition metal complexes was rationally designed to selectively target the phosphate diesters of the DNA backbone by covalent bonding. The synthesis and characterization of the first dinuclear NiII 2 complex of this family are presented, and its DNA binding and interference with DNA synthesis in polymerase chain reaction (PCR) are investigated and compared to those of the analogous CuII 2 complex. The NiII 2 complex also binds to DNA but forms fewer intermolecular DNA cross-links, while it interferes with DNA synthesis in PCR at lower concentrations than CuII 2. To simulate possible competing phosphate-based ligands in vivo, these effects have been studied for both complexes with 100–200-fold excesses of phosphate and ATP, which provided no disturbance. The cytotoxicity of both complexes has been studied for human cancer cells and human stem cells with similar rates of proliferation. CuII 2 shows the lowest IC50 values and a remarkable preference for killing the cancer cells. Three different assays show that the CuII 2 complex induces apoptosis in cancer cells. These results are discussed to gain insight into the mechanisms of action and demonstrate the potential of this family of dinuclear complexes as anticancer drugs acting by a new binding target.
We analyzed, with respect to heat shock proteins (HSPs), systemically reacting tobacco leaves inoculated with Tobacco mosaic virus (TMV), wild-type vulgare, and temperature-sensitive coat protein (CP) mutants Ni 118 (P20L) and flavum (D19A), kept at 23 or 30 degrees C. HSP18 and HSP70 mRNAs and proteins were induced with temperature-sensitive CP mutants after 1 to 2 days at 30 degrees C. After 4 to 6 days, HSP70 was also induced at 23 degrees C. The induction of HSPs paralleled the amount of insoluble TMV CP in leaf extracts, indicating that denatured TMV CP by itself induces a heat-shock response.
Myotonia is a symptom of various genetic and acquired skeletal muscular disorders and is characterized by hyperexcitability of the sarcolemma. Here, we have performed a comparative proteomic study of the genetic mouse models ADR, MTO and MTO*5J of human congenital myotonia in order to determine myotonia-specific changes in the global protein complement of gastrocnemius muscle. Proteomic analyses of myotonia in the mouse, which is caused by mutations in the gene encoding the muscular chloride channel Clc1, revealed a generally perturbed protein expression pattern in severely affected ADR and MTO muscle, but less pronounced alterations in mildly diseased MTO*5J mice. Alterations were found in major metabolic pathways, the contractile machinery, ion handling elements, the cellular stress response and cell signaling mechanisms, clearly confirming a glycolytic-to-oxidative transformation process in myotonic fast muscle. In the long-term, a detailed biomarker signature of myotonia will improve our understanding of the pathobiochemical processes underlying this disorder and be helpful in determining how a single mutation in a tissue-specific gene can trigger severe downstream effects on the expression levels of a very large number of genes in contractile tissues.
Mutant tobacco mosaic virus (TMV) coat proteins (CPs) with known amino acid replacements provide well defined examples of destabilized tertiary structures. Here we show that misfolded TMV CPs, but not functional wild-type CPs, induce massive ubiquitylation in tobacco cells and that denatured, insoluble CP subunits are the main substrates of ubiquitin conjugation. As TMV CPs can be easily manipulated they are unique tools to study the molecular basis of the plant cell's response to aberrant protein structures and the associated intracellular stress reactions.
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