Differential binding of monomethylarsonous acid compared to arsenite and arsenic trioxide with zinc finger peptides and proteins.

Arsenic is an environmental toxin that enhances the carcinogenic effect of DNA-damaging agents, such as ultraviolet radiation and benzo[a]pyrene. Interaction with zinc finger proteins has been shown to be an important molecular mechanism for arsenic toxicity and cocarcinogenesis. Arsenicals such as arsenite, arsenic trioxide (ATO), and monomethylarsonous acid (MMA(III)) have been reported to interact with cysteine residues of zinc finger domains, but little is known about potential differences in their selectivity of interaction. Herein we analyzed the interaction of arsenite, MMA(III), and ATO with C2H2, C3H1, and C4 configurations of zinc fingers using UV-vis, cobalt, fluorescence, and mass spectrometry. We observed that arsenite and ATO both selectively bound to C3H1 and C4 zinc fingers, while MMA(III) interacted with all three configurations of zinc finger peptides. Structurally and functionally, arsenite and ATO caused conformational changes and zinc loss on C3H1 and C4 zinc finger peptide and protein, respectively, whereas MMA(III) changed conformation and displaced zinc on all three types of zinc fingers. The differential selectivity was also demonstrated in zinc finger proteins isolated from cells treated with these arsenicals. Our results show that trivalent inorganic arsenic compounds, arsenite and ATO, have the same selectivity and behavior when interacting with zinc finger proteins, while methylation removes the selectivity. These findings provide insights on the molecular mechanisms underlying the differential effects of inorganic versus methylated arsenicals, as well as the role of in vivo arsenic methylation in arsenic toxicity and carcinogenesis.

Identification of protein components of egg masses indicates parental investment in immunoprotection of offspring by Biomphalaria glabrata (gastropoda, mollusca).

The macromolecules contributed by the freshwater gastropod Biomphalaria glabrata, intermediate host of Schistosoma mansoni, to developing offspring inside egg masses are poorly known. SDS-PAGE fractionated egg mass fluids (EMF) of M line and BB02 B. glabrata were analyzed by MALDI-TOF (MS and tandem MS). A MASCOT database was assembled with EST data from B. glabrata and other molluscs to aid in sequence characterization. Of approximately 20 major EMF polypeptides, 16 were identified as defense-related, including protease inhibitors, a hemocyanin-like factor and tyrosinase (each with possible phenoloxidase activity), extracellular Cu-Zn SOD, two categories of C-type lectins, Gram-negative bacteria-binding protein (GNBP), aplysianin/achacin-like protein, as well as versions of lipopolysaccharide binding protein/bacterial permeability-increasing proteins (LBP/BPI) that differed from those previously described from hemocytes. Along with two sequences that were encoded by "unknown" ESTs, EMF also yielded a compound containing a vWF domain that is likely involved in defense and a polypeptide with homology to the Aplysia pheromone temptin. Further study of B. glabrata pheromones is warranted as these could be useful in efforts to control these schistosome-transmitting snails. Several of the EMF polypeptides were contained in the albumen gland, the organ that produces most EMF. Thus, parental investment of B. glabrata in immunoprotection of its offspring is indicated to be considerable.

The SET and transposase domain protein Metnase enhances chromosome decatenation: regulation by automethylation.

Metnase is a human SET and transposase domain protein that methylates histone H3 and promotes DNA double-strand break repair. We now show that Metnase physically interacts and co-localizes with Topoisomerase IIalpha (Topo IIalpha), the key chromosome decatenating enzyme. Metnase promotes progression through decatenation and increases resistance to the Topo IIalpha inhibitors ICRF-193 and VP-16. Purified Metnase greatly enhanced Topo IIalpha decatenation of kinetoplast DNA to relaxed circular forms. Nuclear extracts containing Metnase decatenated kDNA more rapidly than those without Metnase, and neutralizing anti-sera against Metnase reversed that enhancement of decatenation. Metnase automethylates at K485, and the presence of a methyl donor blocked the enhancement of Topo IIalpha decatenation by Metnase, implying an internal regulatory inhibition. Thus, Metnase enhances Topo IIalpha decatenation, and this activity is repressed by automethylation. These results suggest that cancer cells could subvert Metnase to mediate clinically relevant resistance to Topo IIalpha inhibitors.

Identification of proteins within the nuclear factor-kappa B transcriptional complex including estrogen receptor-alpha.

OBJECTIVE: The objective of the study was to determine whether cross-talk occurs between estrogen receptors (ERs) and nuclear factor-kappa-B (NF-kappaB), to assess the functional consequences of such an ER/NF-kappaB interaction, and to identify other unknown regulatory proteins that may participate in the NF-kappaB transcriptional complex. STUDY DESIGN: Electromobility gel shifts, reporter gene assays, and mass spectrometry were used to identify proteins interacting with the NF-kappaB deoxyribonucleic acid (DNA) response element. RESULTS: ER and the p65 subunit of NF-kappaB colocalized on DNA. This interaction was inhibitory for ER transcriptional activity. Sequencing of proteins bound to the NF-kappaB/DNA complex identified DNA-modifying enzymes, scaffolding proteins, chaperones, and elements of the nuclear matrix. CONCLUSION: These studies have identified an inhibitory interaction between estrogen receptors and the p65 subunit of NF-kappaB with implications for estrogen action in pregnancy and cancer. New accessory proteins have also been identified that bind to protein complexes on the NF-kappaB DNA response element.

Heat shock protein 70 and glycoprotein 96 are differentially expressed on the surface of malignant and nonmalignant breast cells.

Heat shock proteins (HSPs), which are important for a number of different intracellular functions, are occasionally found on the surface of cells. The function of heat shock protein on the cell surface is not understood, although it has been shown to be greater in some tumor cells and some virally infected cells. Surface expression of both glycoprotein 96 (gp96) and Hsp70 occurs on tumor cells, and this expression correlates with natural killer cell killing of the cells. We examined the surface expression of gp96 and Hsp70 on human breast cell lines MCF7, MCF10A, AU565, and HS578, and in primary human mammary epithelial cells by immunofluorescence microscopy and flow cytometry. The nonmalignant cell lines HS578, MCF10A, and HMEC showed no surface expression of gp96, whereas malignant cell lines MCF7 and AU565 were positive for gp96 surface expression. All of the breast cell lines examined showed Hsp70 surface expression. These results also confirm previous studies, demonstrating that Hsp70 is on the plasma membrane of tumor cell lines. Given the involvement of heat shock proteins, gp96 and Hsp70, in innate and adaptive immunity, these observations may be important in the immune response to tumor cells.

Determining the site of spin trapping of the equine myoglobin radical by combined use of EPR, electrophoretic purification, and mass spectrometry.

Although myoglobin protein radicals are thought important intermediates in peroxide-induced toxicity, the site of spin trapping of this radical in equine myoglobin using the trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) is unclear. We have combined EPR, electrophoretic adduct purification, and mass spectrometry approaches to unambiguously determine the site of trapping to be Tyr-103 and suggest that reports of trapping at Trp-7 or Trp-14 may be due to nonradical addition to proteolytically derived Trp-containing peptides with DBNBS. The technique developed here of combining electrophoretic separation of DBNBS adducts with MS of resultant peptides will also allow proteomic-like approaches to determining identities and sites of radical formation and translocation on complex mixtures of proteins.

Mass-spectrometric analysis of myelin proteolipids reveals new features of this family of palmitoylated membrane proteins.

In this study, we have investigated the structure of the native myelin proteolipid protein (PLP), DM-20 protein and several low molecular mass proteolipids by mass spectrometry. The various proteolipid species were isolated from bovine spinal cord by size-exclusion and ion-exchange chromatography in organic solvents. Matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) of PLP and DM-20 revealed molecular masses of 31.6 and 27.2 kDa, respectively, which is consistent with the presence of six and four molecules of thioester-bound fatty acids. Electrospray ionization-MS analysis of the deacylated proteins in organic solvents produced the predicted molecular masses of the apoproteins (29.9 and 26.1 kDa), demonstrating that palmitoylation is the major post-translational modification of PLP, and that the majority of PLP and DM-20 molecules in the CNS are fully acylated. A series of myelin-associated, palmitoylated proteolipids with molecular masses raging between 12 kDa and 18 kDa were also isolated and subjected to amino acid analysis, fatty acid analysis, N- and C-terminal sequencing, tryptic digestion and peptide mapping by MALDI-TOF-MS. The results clearly showed that these polypeptides correspond to the N-terminal region (residues 1-105/112) and C-terminal region (residues 113/131-276) of the major PLP, and they appear to be produced by natural proteolytic cleavage within the 60 amino acid-long cytoplasmic domain. These proteolipids are not postmortem artifacts of PLP and DM-20, and are differentially distributed across the CNS.