The proinflammatory protein HMGB1 is a substrate of transglutaminase-2 and forms high-molecular weight complexes with autoantigens.

High-mobility group box 1 (HMGB1) is a chromatin-associated protein that, in response to stress or injury, translocates from the nucleus to the extracellular milieu, where it functions as an alarmin. HMGB1's function is in part determined by the complexes (HMGB1c) it forms with other molecules. However, structural modifications in the HMGB1 polypeptide that may regulate HMGB1c formation have not been previously described. In this report, we observed high-molecular weight, denaturing-resistant HMGB1c in the plasma and peripheral blood mononuclear cells of individuals with systemic lupus erythematosus (SLE) and, to a much lesser extent, in healthy subjects. Differential HMGB1c levels were also detected in mouse tissues and cultured cells, in which these complexes were induced by endotoxin or the immunological adjuvant alum. Of note, we found that HMGB1c formation is catalyzed by the protein-cross-linking enzyme transglutaminase-2 (TG2). Cross-link site mapping and MS analysis revealed that HMGB1 can be cross-linked to TG2 as well as a number of additional proteins, including human autoantigens. These findings have significant functional implications for studies of cellular stress responses and innate immunity in SLE and other autoimmune disease.

The novel deacetylase inhibitor AR-42 demonstrates pre-clinical activity in B-cell malignancies in vitro and in vivo.

BACKGROUND: While deacetylase (DAC) inhibitors show promise for the treatment of B-cell malignancies, those introduced to date are weak inhibitors of class I and II DACs or potent inhibitors of class I DAC only, and have shown suboptimal activity or unacceptable toxicities. We therefore investigated the novel DAC inhibitor AR-42 to determine its efficacy in B-cell malignancies. PRINCIPAL FINDINGS: In mantle cell lymphoma (JeKo-1), Burkitt's lymphoma (Raji), and acute lymphoblastic leukemia (697) cell lines, the 48-hr IC(50) (50% growth inhibitory concentration) of AR-42 is 0.61 microM or less. In chronic lymphocytic leukemia (CLL) patient cells, the 48-hr LC(50) (concentration lethal to 50%) of AR-42 is 0.76 microM. AR-42 produces dose- and time-dependent acetylation both of histones and tubulin, and induces caspase-dependent apoptosis that is not reduced in the presence of stromal cells. AR-42 also sensitizes CLL cells to TNF-Related Apoptosis Inducing Ligand (TRAIL), potentially through reduction of c-FLIP. AR-42 significantly reduced leukocyte counts and/or prolonged survival in three separate mouse models of B-cell malignancy without evidence of toxicity. CONCLUSIONS/SIGNIFICANCE: Together, these data demonstrate that AR-42 has in vitro and in vivo efficacy at tolerable doses. These results strongly support upcoming phase I testing of AR-42 in B-cell malignancies.

Monte carlo simulation-based algorithms for analysis of shotgun proteomic data.

Two new statistical models based on Monte Carlo Simulation (MCS) have been developed to score peptide matches in shotgun proteomic data and incorporated in a database search program, MassMatrix (www.massmatrix.net). The first model evaluates peptide matches based on the total abundance of matched peaks in the experimental spectra. The second model evaluates amino acid residue tags within MS/MS spectra. The two models provide complementary scores for peptide matches that result in higher confidence in peptide identification when significant scores are returned from both models. The MCS-based models use a variance reduction technique that improves estimation precision. Due to the high computational expense of MCS-based models, peptide matches were prefiltered by other statistical models before further evaluation by the MCS-based models. Receiver operating characteristic analysis of the data sets confirmed that MCS-based models improved the overall performance of the MassMatrix search software, especially for low-mass accuracy data sets.

Identification of histone demethylases in Saccharomyces cerevisiae.

Based on the prediction that histone lysine demethylases may contain the JmjC domain, we examined the methylation patterns of five knock-out strains (ecm5Delta, gis1Delta, rph1Delta, jhd1Delta, and jhd2Delta (yjr119cDelta)) of Saccharomyces cerevisiae. Mass spectrometry (MS) analyses of histone H3 showed increased modifications in all mutants except ecm5Delta. High-resolution MS was used to unequivocally differentiate trimethylation from acetylation in various tryptic fragments. The relative abundance of specific fragments indicated that histones K36me3 and K4me3 accumulate in rph1Delta and jhd2Delta strains, respectively, whereas both histone K36me2 and K36me accumulate in gis1Delta and jhd1Delta strains. Analyses performed with strains overexpressing the JmjC proteins yielded changes in methylation patterns that were the reverse of those obtained in the complementary knock-out strains. In vitro enzymatic assays confirmed that the JmjC domain of Rph1 specifically demethylates K36me3 primarily and K36me2 secondarily. Overexpression of RPH1 generated a growth defect in response to UV irradiation. The demethylase activity of Rph1 is responsible for the phenotype. Collectively, in addition to Jhd1, our results identified three novel JmjC domain-containing histone demethylases and their sites of action in budding yeast S. cerevisiae. Furthermore, the methodology described here will be useful for identifying histone demethylases and their target sites in other organisms.