ATP-dependent chromatin-remodeling complexes are known to facilitate transcriptional activation by opening chromatin structures. We report a novel human complex, named NURD, which contains not only ATP-dependent nucleosome disruption activity, but also histone deacetylase activity, which usually associates with transcriptional repression. The deacetylation is stimulated by ATP on nucleosomal templates, suggesting that nucleosome disruption aids the deacetylase to access its substrates. One subunit of NURD was identified as MTA1, a metastasis-associated protein with a region similar to the nuclear receptor core-pressor, N-CoR; and antibodies against NURD partially relieve transcriptional repression by thyroid hormone receptor. These results suggest that ATP-dependent chromatin remodeling can participate in transcriptional repression by assisting repressors in gaining access to chromatin.

A scoring procedure is described for measuring the quality of the results for protein identifications obtained from spectral matching of MS/MS data using the Sequest database search program. The scoring system is essentially probabilistic and operates by estimating the probability that a protein identification has come about by chance. The probability is based on the number of identified peptides from the protein, the total number of identified peptides, and the fraction of distinct tryptic peptides from the database that are present in the identified protein. The score is not strictly a probability, as it also incorporates information about the quality of the individual peptide matches. The result of using Qscore on a large test set of data was similar to that achieved using approaches that validate individual spectral matches, with only a narrow overlap in scores between identified proteins and false positive matches. In direct comparison with a published method of evaluating Sequest results, Qscore was able to identify an equivalent number of proteins without any identifiable false positive assignments. Qscore greatly reduces the number of Sequest protein identifications that have to be validated manually. (J Am Soc Mass Spectrom 2002, 13, 378 -386) © 2002 American Society for Mass Spectrometry P roteolytic digestion followed by mass spectrometry and database searching has become the premier approach to sensitive identification of proteins. High throughput approaches to protein identification depend on minimizing human time investment in this analysis. A variety of techniques, including robotic gel band excision and digestion, automated matrix-assisted laser desorption/ionization (MALDI) spotting, autosampled nano-liquid chromatography tandem mass spectrometry (LC/MS/MS) analysis, and automated database searching, have been developed to further this aim. To make automated database searching possible, it is necessary to use a search program that can process spectra without need for human interpretation. In theory, it is also desirable to have an automated scheme to determine the significance and reliability of the database search results.

Bloom syndrome (BS) is a genetic disorder associated with dwarfism, immunodeficiency, reduced fertility, and an elevated risk of cancer. To investigate the mechanism of this disease, we isolated from human HeLa extracts three complexes containing the helicase defective in BS, BLM. Interestingly, one of the complexes, termed BRAFT, also contains five of the Fanconi anemia (FA) complementation group proteins (FA proteins). FA resembles BS in genomic instability and cancer predisposition, but most of its gene products have no known biochemical activity, and the molecular pathogenesis of the disease is poorly understood. BRAFT displays a DNA-unwinding activity, which requires the presence of BLM because complexes isolated from BLM-deficient cells lack such an activity. The complex also contains topoisomerase III␣ and replication protein A, proteins that are known to interact with BLM and could facilitate unwinding of DNA. We show that BLM complexes isolated from an FA cell line have a lower molecular mass. Our study provides the first biochemical characterization of a multiprotein FA complex and suggests a connection between the BLM and FA pathways of genomic maintenance. The findings that FA proteins are part of a DNA-unwinding complex imply that FA proteins may participate in DNA repair.

The SWI͞SNF family of chromatin-remodeling complexes facilitates gene expression by helping transcription factors gain access to their targets in chromatin. SWI͞SNF and Rsc are distinctive members of this family from yeast. They have similar protein components and catalytic activities but differ in biological function. Rsc is required for cell cycle progression through mitosis, whereas SWI͞ SNF is not. Human complexes of this family have also been identified, which have often been considered related to yeast SWI͞SNF. However, all human subunits identified to date are equally similar to components of both SWI͞SNF and Rsc, leaving open the possibility that some or all of the human complexes are rather related to Rsc. Here, we present evidence that the previously identified human SWI͞SNF-B complex is indeed of the Rsc type. It contains six components conserved in both Rsc and SWI͞SNF. Importantly, it has a unique subunit, BAF180, that harbors a distinctive set of structural motifs characteristic of three components of Rsc. Of the two mammalian ATPases known to be related to those in the yeast complexes, human SWI͞SNF-B contains only the homolog that functions like Rsc during cell growth. Immunofluorescence studies with a BAF180 antibody revealed that SWI͞ SNF-B localizes at the kinetochores of chromosomes during mitosis. Our data suggest that SWI͞SNF-B and Rsc represent a novel subfamily of chromatin-remodeling complexes conserved from yeast to human, and could participate in cell division at kinetochores of mitotic chromosomes.

Pre-mRNA introns are spliced in a macromolecular machine, the spliceosome. For each round of splicing, the spliceosome assembles de novo in a series of ATP-dependent steps involving numerous changes in RNA-RNA and RNA-protein interactions. As currently understood, spliceosome assembly proceeds by addition of discrete U1, U2, and U4/U6*U5 snRNPs to a pre-mRNA substrate to form functional splicing complexes. We characterized a 45S yeast penta-snRNP which contains all five spliceosomal snRNAs and over 60 pre-mRNA splicing factors. The particle is functional in extracts and, when supplied with soluble factors, is capable of splicing pre-mRNA. We propose that the spliceosomal snRNPs associate prior to binding of a pre-mRNA substrate rather than with pre-mRNA via stepwise addition of discrete snRNPs.

Previous compositional studies of pre-mRNA processing complexes have been performed in vitro on synthetic pre-mRNAs containing a single intron. To provide a more comprehensive list of polypeptides associated with the pre-mRNA splicing apparatus, we have determined the composition of the bulk pre-mRNA processing machinery in living cells. We purified endogenous nuclear pre-mRNA processing complexes from human and chicken cells comprising the massive (>200S) supraspliceosomes (a.k.a. polyspliceosomes). As expected, RNA components include a heterogeneous mixture of pre-mRNAs and the five spliceosomal snRNAs. In addition to known pre-mRNA splicing factors, 5′ end binding factors, 3′ end processing factors, mRNA export factors, hnRNPs and other RNA binding proteins, the protein components identified by mass spectrometry include RNA adenosine deaminases and several novel factors. Intriguingly, our purified supraspliceosomes also contain a number of structural proteins, nucleoporins, chromatin remodeling factors and several novel proteins that were absent from splicing complexes assembled in vitro. These in vivo analyses bring the total number of factors associated with pre-mRNA to well over 300, and represent the most comprehensive analysis of the pre-mRNA processing machinery to date.

Clostridium difficile ( C. difficile ) incidence has tripled over the past 15 years and is attributed to the emergence of hypervirulent strains. While it is clear that C. difficile toxins cause damaging colonic inflammation, the immune mechanisms protecting from tissue damage require further investigation. Through a transcriptome analysis, we identify IL-33 as an immune target upregulated in response to hypervirulent C. difficile . We demonstrate that IL-33 prevents C. difficile -associated mortality and epithelial disruption independently of bacterial burden or toxin expression. IL-33 drives colonic group 2 innate lymphoid cell (ILC2) activation during infection and IL-33 activated ILC2s are sufficient to prevent disease. Furthermore, intestinal IL-33 expression is regulated by the microbiota as fecal microbiota transplantation (FMT) rescues antibiotic-associated depletion of IL-33. Lastly, dysregulated IL-33 signaling via the decoy receptor, sST2, predicts C. difficile -associated mortality in human patients. Thus, IL-33 signaling to ILC2s is an important mechanism of defense from C. difficile colitis.

Immune dysregulation is characteristic of the more severe stages of SARS-CoV-2 infection. Understanding the mechanisms by which the immune system contributes to COVID-19 severity may open new avenues to treatment. Here we report that elevated interleukin-13 (IL-13) was associated with the need for mechanical ventilation in two independent patient cohorts.In addition, patients who acquired COVID-19 while prescribed Dupilumab, a mAb that blocks IL-13 and IL-4 signaling, had less severe disease. In SARS-CoV-2 infected mice, IL-13 neutralization reduced death and disease severity without affecting viral load, demonstrating an immunopathogenic role for this cytokine. Following anti-IL-13 treatment in infected mice, hyaluronan synthase 1 (Has1) was the most downregulated gene and accumulation of the hyaluronan polysaccharide was decreased in the lung. In patients with COVID-19, hyaluronan was increased in the lungs and plasma. Blockade of the hyaluronan receptor, CD44, reduced mortality in infected mice, supporting the importance of hyaluronan as a pathogenic mediator.Finally, hyaluronan was directly induced in the lungs of mice by administration of IL-13, indicating a new role for IL-13 in lung disease. Understanding the role of IL-13 and hyaluronan has important implications for therapy of COVID-19 and potentially other pulmonary diseases.Summary: IL-13 levels were elevated in patients with severe COVID-19. In a mouse model of disease, IL-13 neutralization reduced disease and decreased lung hyaluronan deposition.Administration of IL-13 induced hyaluronan in the lung. Blockade of the hyaluronan receptor CD44 prevented mortality, highlighting a novel mechanism for IL-13-mediated hyaluronan synthesis in pulmonary pathology.