Bacteria are thought to avoid using the essential metal ion copper in their cytosol due to its toxicity. Herein we characterize Csp3, the cytosolic member of a new family of bacterial copper storage proteins from Methylosinus trichosporium OB3b and Bacillus subtilis. These tetrameric proteins possess a large number of Cys residues that point into the cores of their four-helix bundle monomers. The Csp3 tetramers can bind a maximum of approximately 80 Cu(I) ions, mainly via thiolate groups, with average affinities in the (1–2) × 1017 M−1 range. Cu(I) removal from these Csp3s by higher affinity potential physiological partners and small-molecule ligands is very slow, which is unexpected for a metal-storage protein. In vivo data demonstrate that Csp3s prevent toxicity caused by the presence of excess copper. Furthermore, bacteria expressing Csp3 accumulate copper and are able to safely maintain large quantities of this metal ion in their cytosol. This suggests a requirement for storing copper in this compartment of Csp3-producing bacteria.
Global analysis of protein phosphorylation will provide insight into mechanisms by which this dynamic post-translational modification modulates diverse cellular processes. Mass spectrometry-based phosphoproteomics is a potentially powerful approach for global profiling and quantification of protein phosphorylation. Such studies usually involve selective isolation of phosphorylated peptides and their subsequent fragmentation in a mass spectrometer to assign the sequence and localize phosphorylation sites. Three strategies have been described for enriching phosphopeptides based on antibodies (1, 2), chemical derivatization (3, 4), or ionic interactions (e.g. IMAC and strong ion exchange chromatography) (5-9). These methods have achieved limited success in enriching phosphopeptides for proteomics studies. Among these approaches, IMAC is the most convenient and holds much potential for the efficient isolation of phosphopeptides (10 -13). The method has been used for several global analyses of protein phosphorylation in model organisms and cellular organelles (5-9). Nevertheless further refinement of extant IMAC protocols is required to achieve high reproducibility and efficiency (14).Poor fragmentation of phosphopeptides in the mass spectrometer represents the second major challenge for phosphoproteomics. The availability of a relatively low energy fragmentation pathway via -elimination of the phosphate moiety limits fragmentation at peptide bonds that would be informative for identifying the sequence and site(s) of phosphorylation of the peptide. Mass spectrometers under development, such as instruments using electron transfer dissociation (15), might address this problem. However, such mass spectrometers are not currently commercially available. In addition, fragmentation of doubly charged and singly charged peptides in electron transfer dissociation mass spectrometry is compromised. In summary, despite extensive effort in the past several years, efficient proteomics of protein phosphorylation remains a daunting challenge.Here we report analysis of the phosphoproteome of mitochondria using an improved method that integrates an optimized batchwise IMAC protocol for isolation of phosphopeptides and MS3 1 for fragmentation of phosphopeptides. The improved IMAC procedure allowed recovery of ϳ77% of phosphopeptides while retaining few unphosphorylated peptides. MS3 addressed the poor fragmentation of phosphopeptides by generating highly informative fragmentation patterns that allow peptide identification. Analysis of mitochondrial phosphorylation revealed 84 phosphorylation sites from 62 proteins. Most identified phosphorylation sites have not been reported before, providing novel information about mitochondrial regulatory mechanisms. The optimized batchwise IMAC protocol in combination with MS3 offers a relatively simple and more efficient approach for proteomics of protein phosphorylation.EXPERIMENTAL PROCEDURES
Although the etiology of Parkinson's disease (PD) remains elusive, recent studies suggest that oxidative stress contributes to the cascade leading to dopaminergic (DAergic) neurodegeneration. The Nrf2 signaling is the main pathway responsible for cellular defense system against oxidative stress. Nrf2 is a transcription factor that regulates environmental stress response by inducing expression of antioxidant enzyme genes. We have synthesized novel vinyl sulfone derivatives. They exhibited a broad range of activities in inducing HO-1, whose gene expression is under the control of Nrf2. Among them, compound 12g was confirmed to activate Nrf2 and induce expression of the Nrf2-dependent antioxidant enzymes NQO1, GCLC, GLCM, and HO-1, at both mRNA and protein levels in DAergic neuronal cells. This was accompanied by protection of DAergic neurons in both in vitro and MPTP-induced in vivo models of PD. In addition, compound 12g effectively resulted in attenuation of the PD-associated behavioral deficits in the mouse model.
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