Oxime esters as selective,covalent inhibitors of the serine hydrolase retinoblastoma-binding protein 9 (RBBP9)

We recently described a fluorescence polarization platform for competitive activity-based protein profiling (fluopol-ABPP) that enables high-throughput inhibitor screening for enzymes with poorly characterized biochemical activity. Here, we report the discovery of a class of oxime ester inhibitors for the unannotated serine hydrolase RBBP9 from a full-deck (200,000+ compound) fluopol-ABPP screen conducted in collaboration with the Molecular Libraries Screening Center Network (MLSCN). We show that these compounds covalently inhibit RBBP9 by modifying enzyme’s active site serine nucleophile and, based on competitive ABPP in cell and tissue proteomes, are selective for RBBP9 relative to other mammalian serine hydrolases.     

Altered DNA methylation in leukocytes with trisomy 21

The primary abnormality in Down syndrome (DS), trisomy 21, is well known; but how this chromosomal gain produces the complex DS phenotype, including immune system defects, is not well understood. We profiled DNA methylation in total peripheral blood leukocytes (PBL) and T-lymphocytes from adults with DS and normal controls and found gene-specific abnormalities of CpG methylation in DS, with many of the differentially methylated genes having known or predicted roles in lymphocyte development and function. Validation of the microarray data by bisulfite sequencing and methylation-sensitive Pyrosequencing (MS-Pyroseq) confirmed strong differences in methylation (p<0.0001) for each of 8 genes tested: TMEM131, TCF7, CD3Z/CD247, SH3BP2, EIF4E, PLD6, SUMO3, and CPT1B, in DS versus control PBL. In addition, we validated differential methylation of NOD2/CARD15 by bisulfite sequencing in DS versus control T-cells. The differentially methylated genes were found on various autosomes, with no enrichment on chromosome 21. Differences in methylation were generally stable in a given individual, remained significant after adjusting for age, and were not due to altered cell counts. Some but not all of the differentially methylated genes showed different mean mRNA expression in DS versus control PBL; and the altered expression of 5 of these genes, TMEM131, TCF7, CD3Z, NOD2, and NPDC1, was recapitulated by exposing normal lymphocytes to the demethylating drug 5-aza-2'deoxycytidine (5aza-dC) plus mitogens. We conclude that altered gene-specific DNA methylation is a recurrent and functionally relevant downstream response to trisomy 21 in human cells.     

Gene trees,species and species trees in the <Emphasis Type="Italic">Ctenosaura palearis</Emphasis> clade

The growing use of molecular systematics in conservation has increased the importance of accurate resolution of taxonomic units and relationships. DNA data relate most directly to genealogies, which need not have perfect relationships with species limits and phylogenies. We used a multilocus gene tree approach to elucidate the relationships between four endangered Central American iguanas. We found support for the proposition that the described species taxa correspond to distinct evolutionary lineages warranting individual protection. We combined gene trees to estimate a phylogeny using Bayesian Estimation of Species Trees (BEST), minimizing deep coalescence, Species Trees from Average Ranks (STAR), and traditional concatenation. The estimate from concatenation conflicted with the other methods, likely owing to the disproportionate effect of mtDNA on concatenated analyses. This illustrates the importance of appropriate treatment of multilocus sequence data in phylogenetics. Our results indicate that these species have gone through recent and rapid speciation, resulting in four closely related narrow-range endemics.     

Lack of DNA polymerase μ affects the kinetics of DNA double-strand break repair and impacts on cellular senescence

The specialised DNA polymerase μ (pol μ) affects a sub-class of immunoglobulin genes rearrangements and haematopoietic development in vivo. These effects appear linked to double-strand breaks (DSBs) repair, but it is still unclear how and to what extent pol μ intervenes in this process. Using high-resolution quantitative imaging of DNA damage in irradiated wild-type and pol μ^?/? mouse embryonic fibroblasts (MEFs) we show that lack of pol μ results in delayed DSB repair kinetics and in persistent DNA damage. DNA damage triggers cellular senescence, and this response is thought to suppress cancer. Independent investigations either report or not a proliferative decline for MEFs lacking pol μ. Here we show pronounced senescence in pol μ^?/? MEFs, associated with high levels of the tumor-suppressor p16^INK4A and the DNA damage response kinase CHK2. Importantly, cellular senescence is induced by culture stress and exacerbated by low doses of irradiation in pol μ^?/? MEFs. We also found that low doses of irradiation provoke delayed immortalisation in MEFs lacking pol μ. Pol μ^?/? MEFs thus exhibit a robust anti-proliferative defence in response to irreparable DNA damage. These findings indicate that sub-optimal DSB repair, due to the absence of an auxiliary DNA damage repair factor, can impact on cell fitness and thereby on cell fate.     

Labeling elastase digests with TMT: informational gain by identification of poorly detectable peptides with MALDI-TOF/TOF mass spectrometry

The applicability of the less specific protease elastase for the identification of membrane and cytosolic proteins has already been demonstrated. MALDI as ionization technique particularly favors the detection of basic and to a lesser extent of weakly acidic peptides, whereas neutral peptides often remain undetected. Moreover, peptides below 700 Da are routinely excluded. In the following study, the advantage of additional information gained from tandem mass tag zero labeled peptides and the resultant increase in sequence coverage was evaluated. Through derivatization with tandem mass tag reagents, peptide measurement within the standard mass range of the MALDI reflector mode is achievable due to the mass increase. Compared to the unlabeled sample, peptides exhibiting relatively low molecular masses, pI values or higher hydrophobicity could be identified.     

Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells

Intestinal stem cells, characterized by high Lgr5 expression, reside between Paneth cells at the small intestinal crypt base and divide every day. We have carried out fate mapping of individual stem cells by generating a multicolor Cre-reporter. As a population, Lgr5(hi) stem cells persist life-long, yet crypts drift toward clonality within a period of 1-6 months. We have collected short- and long-term clonal tracing data of individual Lgr5(hi) cells. These reveal that most Lgr5(hi) cell divisions occur symmetrically and do not support a model in which two daughter cells resulting from an Lgr5(hi) cell division adopt divergent fates (i.e., one Lgr5(hi) cell and one transit-amplifying [TA] cell per division). The cellular dynamics are consistent with a model in which the resident stem cells double their numbers each day and stochastically adopt stem or TA fates. Quantitative analysis shows that stem cell turnover follows a pattern of neutral drift dynamics.     

Cellulosilyticum ruminicola,a Newly Described Rumen Bacterium That Possesses Redundant Fibrolytic-Protein-Encoding Genes and Degrades Lignocellulose with Multiple Carbohydrate- Borne Fibrolytic Enzymes

Cellulosilyticum ruminicola H1 is a newly described bacterium isolated from yak (Bos grunniens) rumen and is characterized by its ability to grow on a variety of hemicelluloses and degrade cellulosic materials. In this study, we performed the whole-genome sequencing of C. ruminicola H1 and observed a comprehensive set of genes encoding the enzymes essential for hydrolyzing plant cell wall. The corresponding enzymatic activities were also determined in strain H1; these included endoglucanases, cellobiohydrolases, xylanases, mannanase, pectinases, and feruloyl esterases and acetyl esterases to break the interbridge cross-link, as well as the enzymes that degrade the glycosidic bonds. This bacterium appears to produce polymer hydrolases that act on both soluble and crystal celluloses. Approximately half of the cellulytic activities, including cellobiohydrolase (50%), feruloyl esterase (45%), and one third of xylanase (31%) and endoglucanase (36%) activities were bound to cellulosic fibers. However, only a minority of mannase (6.78%) and pectinase (1.76%) activities were fiber associated. Strain H1 seems to degrade the plant-derived polysaccharides by producing individual fibrolytic enzymes, whereas the majority of polysaccharide hydrolases contain carbohydrate-binding module. Cellulosome or cellulosomelike protein complex was never isolated from this bacterium. Thus, the fibrolytic enzyme production of strain H1 may represent a different strategy in cellulase organization used by most of other ruminal microbes, but it applies the fungal mode of cellulose production.The ruminant rumens are long believed to be the anaerobic environments efficiently degrading the plant-derived polysaccharides, which is attributed to the inhabited abundant rumen microorganisms. They implement the fibrolytic degradation by the combination of the enzymes comprising of cellulases, hemicellulases, and to a lesser extent pectinases and ligninases (12). The rumen bacteria are outnumbered of the other rumen microbes; however, only a few of cellulolytic bacteria have been isolated from rumens. Ruminococcus flavefaciens, Ruminococcus albus, and Fibrobacter succinogenes are considered to be the most important cellulose-degrading bacteria in the rumen (18), and they produce a set of cellulolytic enzymes, including endoglucanases, exoglucanases (generally cellobiohydrolase), and β-glucosidases, as well as hemicellulases. In addition, the predominant ruminal hemicellulose-digesting bacteria such as Butyrivibrio fibrisolvens and Prevotella ruminicola lack the ability to digest cellulose but degrade xylan and pectin and utilize the degraded soluble sugars as substrates (10, 14). Although the robust cellulolytic species F. succinogenes degrades xylan, it cannot use the pentose product as a carbon source (24). Culture-independent approaches indicate that the three cellulolytic bacterial species represent only ∼2% of the ruminal bacterial 16S rRNA (43). Therefore, many varieties of rumen microbes remain uncultured (2). In recent years, rumen metagenomics studies have revealed the vast diversity of fibrolytic enzymes, multiple domain proteins, and the complexity of microbial composition in the ecosystem (9, 17). Hence, it is likely that the entire microbial community is necessary for the implementation of an efficient fibrolytic process in the rumen, including the uncultured species.In the rumen and other fibrolytic ecosystems, cellulolytic bacteria have to cope with the structural complexity of lignocelluloses and the interspecies competition; thus, not only a variety of plant polymer-degrading enzymes but also a noncatalytic assistant strategy, such as including adhesion of cells to substrates by a variety of anchoring domains, is required (8, 33, 38, 39). The (hemi)cellulolytic enzyme systems have been intensively studied for nonrumen anaerobic bacteria, including Clostridium thermocellum (19, 40), Clostridium cellulolyticum (6), Clostridium cellulovorans (13), and Clostridium stercorarium (47), as well as the rumen species, Rumicoccocus albus (35), Ruminococcus flavefaciens (32), and Fibrobacter succinogenes (4). The results indicate that most of them, except for Fibrobacter succinogenes, produce multiple cellulolytic enzymes integrated in a complex, cellulosome, and free individual proteins.The yak (Bos grunniens) is a large ruminant (∼1,000 kg) in the bovine family that lives mainly on the Qinghai-Tibetan Plateau in China at an altitude of 3,000 m above sea level. It is a local species that lives mainly on the world''s highest plateau. Yaks live in a full-grazing style with grasses, straws, and lichens as their exclusive feed, so the yak rumen can harbor a microbial flora distinct from those of other ruminants due to their fiber-component diet, since diet can be a powerful factor in regulating mammalian gut microbiome (27). A very different prokaryote community structure was revealed for yak rumen in our previous work based on the 16S rRNA diversity, which showed fewer phyla than for cattle but that a higher ratio of sequences was related to uncultured bacteria (2).We previously isolated a novel anaerobic fibrolytic bacterium, Cellulosilyticum ruminicola H1, from the rumen of a domesticated yak (11). Strain H1 grew robustly on natural plant fibers such as corn cob, alfalfa, and ryegrass as the sole carbon and energy sources, as well as on a variety of polysaccharides, including cellulose, xylan, mannan, and pectin, but not monosaccharides such as glucose, which is preferred by most ruminal bacteria. In the present study, using a draft of its genome and enzymatic characterization, we analyzed the enzymatic activities and the structures of the polymer hydrolases of strain H1 that were involved in the hydrolysis of complex polysaccharides.