Oxidation-Induced Conformational Changes in Calcineurin Determined by Covalent Labeling and Tandem Mass Spectrometry

The Ca²⁺/calmodulin activated phosphatase, calcineurin, is inactivated by H₂O₂ or superoxide-induced oxidation, both <i>in vivo</i> and <i>in vitro</i>. However, the potential for global and/or local conformation changes occurring within calcineurin as a function of oxidative modification, that may play a role in the inactivation process, has not been examined. Here, the susceptibility of calcineurin methionine residues toward H₂O₂-induced oxidation were determined using a multienzyme digestion strategy coupled with capillary HPLC–electrospray ionization mass spectrometry and tandem mass spectrometry analysis. Then, regions within the protein complex that underwent significant conformational perturbation upon oxidative modification were identified by monitoring changes in the modification rates of accessible lysine residues between native and oxidized forms of calcineurin, using an amine-specific covalent labeling reagent, <i>S</i>,<i>S</i>′-dimethylthiobutanoylhydroxysuccinimide ester (DMBNHS), and tandem mass spectrometry. Importantly, methionine residues found to be highly susceptible toward oxidation, and the lysine residues exhibiting large increases in accessibility upon oxidation, were all located in calcineurin functional domains involved in Ca²⁺/CaM binding regulated calcineurin stimulation. These findings therefore provide initial support for the novel mechanistic hypothesis that oxidation-induced global and/or local conformational changes within calcineurin contribute to inactivation via (i) impairing the interaction between calcineurin A and calcineurin B, (ii) altering the low-affinity Ca²⁺ binding site in calcineurin B, (iii) inhibiting calmodulin binding to calcineurin A, and/or (iv) by altering the affinity between the calcineurin A autoinhibitory domain and the catalytic center

Semi-Synthetic Proteins as Metal Ion Capture Agents: Catch and Release of Ni(II) and Cu(II) with Myoglobin Bioconjugates

Metals play a vital role in many industries, but their extraction through traditional mining methods poses significant environmental challenges. To address these issues, alternative sources such as coal fly ash (CFA) need to be explored for the recovery of transition metals. In this study, we describe a novel green strategy for the selective separation and recovery of valuable metal ions from complex solutions using semi-synthetic proteins. Myoglobin (Mb), a small protein available from renewable sources, was conjugated to the high-affinity metal chelator SG-20 to create the semi-synthetic protein Mb-SG-20. Mb-SG-20 was characterized by gel electrophoresis, mass spectrometry, and proteomic analysis and found to contain a distribution of 4–7 equiv of SG-20 conjugated to Mb surface lysine residues. Binding and recovery properties of Mb-SG-20 for nickel (Ni) and copper (Cu) ions were characterized. The results showed that Mb-SG-20 captures super-stoichiometric amounts of Ni or Cu while maintaining a strong affinity for Ni or Cu ions. Furthermore, we demonstrate the recyclability of Mb-SG-20 as a Ni and Cu ion capture agent and provide proof-of-concept experiments using CFA leachate solution. This study presents a promising approach for the sustainable recovery of valuable metals from non-traditional sources, such as CFA

Mercury Alters B‑Cell Protein Phosphorylation Profiles

Environmental exposure to mercury is suggested to contribute to human immune dysfunction. To shed light on the mechanism, we identified changes in the phosphoproteomic profile of the WEHI-231 B cell line after intoxication with Hg²⁺. These changes were compared to changes in the phosphoproteome that were induced by pervanadate or okadaic acid exposure. Both 250 μM HgCl₂ and pervanadate, a known phosphotyrosine phosphatase inhibitor, caused an increase in the number of proteins identified after TiO₂ affinity selection and LC-MS/MS analysis. Pervanadate treatment had a larger effect than Hg²⁺ on the number of Scansite motifs that were tyrosine-phosphorylated, 17, and Ingenuity canonical signaling pathways activated, 4, with score >5.0. However, Hg²⁺ had a more focused effect, primarily causing tyrosine-phosphorylation in src homology 2 domains in proteins that are in the B cell receptor signaling pathway. The finding that many of the changes induced by Hg²⁺ overlap with those of pervanadate, indicates that at high concentrations Hg²⁺ inhibits protein tyrosine phosphatases

Mercury Alters B‑Cell Protein Phosphorylation Profiles

Environmental exposure to mercury is suggested to contribute to human immune dysfunction. To shed light on the mechanism, we identified changes in the phosphoproteomic profile of the WEHI-231 B cell line after intoxication with Hg²⁺. These changes were compared to changes in the phosphoproteome that were induced by pervanadate or okadaic acid exposure. Both 250 μM HgCl₂ and pervanadate, a known phosphotyrosine phosphatase inhibitor, caused an increase in the number of proteins identified after TiO₂ affinity selection and LC-MS/MS analysis. Pervanadate treatment had a larger effect than Hg²⁺ on the number of Scansite motifs that were tyrosine-phosphorylated, 17, and Ingenuity canonical signaling pathways activated, 4, with score >5.0. However, Hg²⁺ had a more focused effect, primarily causing tyrosine-phosphorylation in src homology 2 domains in proteins that are in the B cell receptor signaling pathway. The finding that many of the changes induced by Hg²⁺ overlap with those of pervanadate, indicates that at high concentrations Hg²⁺ inhibits protein tyrosine phosphatases