4 research outputs found
Oxidation-Induced Conformational Changes in Calcineurin Determined by Covalent Labeling and Tandem Mass Spectrometry
The Ca<sup>2+</sup>/calmodulin activated
phosphatase, calcineurin,
is inactivated by H<sub>2</sub>O<sub>2</sub> 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
H<sub>2</sub>O<sub>2</sub>-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 Ca<sup>2+</sup>/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
Ca<sup>2+</sup> 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 Hg<sup>2+</sup>. These changes were compared
to changes in the phosphoproteome that were induced by pervanadate
or okadaic acid exposure. Both 250 μM HgCl<sub>2</sub> and pervanadate,
a known phosphotyrosine phosphatase inhibitor, caused an increase
in the number of proteins identified after TiO<sub>2</sub> affinity
selection and LC-MS/MS analysis. Pervanadate treatment had a larger
effect than Hg<sup>2+</sup> on the number of Scansite motifs that
were tyrosine-phosphorylated, 17, and Ingenuity canonical signaling
pathways activated, 4, with score >5.0. However, Hg<sup>2+</sup> 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
Hg<sup>2+</sup> overlap with those of pervanadate, indicates that
at high concentrations Hg<sup>2+</sup> 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 Hg<sup>2+</sup>. These changes were compared
to changes in the phosphoproteome that were induced by pervanadate
or okadaic acid exposure. Both 250 μM HgCl<sub>2</sub> and pervanadate,
a known phosphotyrosine phosphatase inhibitor, caused an increase
in the number of proteins identified after TiO<sub>2</sub> affinity
selection and LC-MS/MS analysis. Pervanadate treatment had a larger
effect than Hg<sup>2+</sup> on the number of Scansite motifs that
were tyrosine-phosphorylated, 17, and Ingenuity canonical signaling
pathways activated, 4, with score >5.0. However, Hg<sup>2+</sup> 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
Hg<sup>2+</sup> overlap with those of pervanadate, indicates that
at high concentrations Hg<sup>2+</sup> inhibits protein tyrosine phosphatases