Additional file 2: of Mercury alters endogenous phosphorylation profiles of SYK in murine B cells

Consensus SYK phosphorylation sites. Singly, doubly or triply phosphorylated peptides were scored for peptide identification probability and modification site localization probability. Cutoff for accepted phosphosites was a localization probability of ≥95%. Data represent top scoring peptides. (PDF 219 kb

Additional file 1: of Mercury alters endogenous phosphorylation profiles of SYK in murine B cells

SYK protein sequencing coverage. These data represent results from a single anti-SYK immunoprecipitation experiment with WEHI cell extracts. The sample was digested with trypsin and analyzed by LC-MS/MS. MS2 spectral identifications were scored with Mascot and X!Tandem algorithms against forward and scrambled murine protein databases. Positive identifications were assigned below a 1% FDR threshold. (PDF 254 kb

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

Identification of an Intrinsic Determinant Critical for Maspin Subcellular Localization and Function

<div><p>Maspin, a multifaceted tumor suppressor, belongs to the serine protease inhibitor superfamily, but only inhibits serine protease-like enzymes such as histone deacetylase 1 (HDAC1). Maspin is specifically expressed in epithelial cells and it is differentially regulated during tumor progression. A new emerging consensus suggests that a shift in maspin subcellular localization from the nucleus to the cytoplasm stratifies with poor cancer prognosis. In the current study, we employed a rational mutagenesis approach and showed that maspin reactive center loop (RCL) and its neighboring sequence are critical for maspin stability. Further, when expressed in multiple tumor cell lines, single point mutation of Aspartate³⁴⁶ (D³⁴⁶) to Glutamate (E³⁴⁶), maspin^D346E, was predominantly nuclear, whereas wild type maspin (maspin^WT) was both cytoplasmic and nuclear. Evidence from cellular fractionation followed by immunological and proteomic protein identification, combined with the evidence from fluorescent imaging of endogenous proteins, fluorescent protein fusion constructs, as well as bimolecular fluorescence complementation (BiFC) showed that the increased nuclear enrichment of maspin^D346E was, at least in part, due to its increased affinity to HDAC1. Maspin^D346E was also more potent than maspin^WT as an HDAC inhibitor. Taken together, our evidence demonstrates that D³⁴⁶ is a critical <i>cis</i>-element in maspin sequence that determines the molecular context and subcellular localization of maspin. A mechanistic model derived from our evidence suggests a new window of opportunity for the development of maspin-based biologically competent HDAC inhibitors for cancer treatment.</p></div

A hypothetical model explaining disregulation of maspin in tumor progression.

<p>(<b>A</b>) Benign tumor or better differentiated cancer with better prognosis is associated with nuclear maspin. Nuclear maspin has a stronger affinity towards HDAC1 as compared to hypothetical HDAC1-associated nuclear factor X. (<b>B</b>) High grade carcinoma exhibiting a less differentiated phenotype and worse prognosis is associated with cytosolic maspin or loss of maspin. In cancer, hypothetical HDAC1- associated nuclear factor X is X′ and it has a stronger affinity towards HDAC1 as compared to maspin. (<b>C</b>) Therapeutic potential of bioengineered maspin mutant or its derivative in treating advanced disease. Maspin mutant restores maspin nuclear localization and HDAC1 inhibition in advanced disease.</p

Maspin nuclear localization correlates with increased histone acetylation and release of HDAC-repressed gene expression.

<p>(<b>A</b>) Western blot of recombinant maspin, HDAC1, and HDAC1 target protein (acetylated Histone 3 at Lysine 9 (H3 Acetyl K⁹)) in DU145 cells. GAPDH was used as a loading control. (<b>B</b>) Q-RT-PCR of HDAC1 targeted genes differentially regulated by maspin. The threshold cycle (ct) numbers obtained from qRT-PCR were normalized by the internal GAPDH controls and presented as the fold change. Maspin^WT: black bar; maspin^D346E: white bar.</p

Increased nuclear localization of maspin^D346E correlates with increased HDAC1 interaction.

<p>(<b>A</b>) Confocal imaging of endogenous maspin (green) and HDAC1 (red) immunofluorescence staining in MCF10A cells. Scale bar = 20 µm (<b>B</b>). Bimolecular fluorescence complementation (BIFC) of live PC3 and DU145 cells transiently transected with pBiFC maspin^WT YC or pBiFC maspin^D346E YC in combination with pBiFC HDAC1 YN. The BiFC of bJunYN and BiFC bFosYC and BiFC of bJunYN and BiFC bFosYC Δ179–193 were used as positive and negative controls, respectfully, in PC3 cells. Scale bar = 10 µm (<b>C</b>) Immunofluorescence imaging (x40) of live PC3 cells after co-transfection with either pEGFP N1-maspin^WT or pEGFP N1 maspin^D346E in combination with pcDNA3.1 HDAC1-RFP. → indicates cytosolic maspin/HDAC1 interaction and ▸ indicates nuclear maspin/HDAC1 interaction. Scale bar = 20 µm. (<b>D</b>) Western blot of recombinant maspin and HDAC1 after immunoprecipitation (IP) with maspin antibody in DU145 cells. The mouse IgG was used as a negative control. Total levels of maspin, HDAC1, and loading control GAPDH are shown in the input panel. Numbers below represent normalized HDAC1/maspin ratio.</p