Diarylcarbonates are a new class of deubiquitinating enzyme inhibitor

Promiscuous inhibitors of tyrosine protein kinases, proteases and phosphatases are useful reagents for probing regulatory pathways and stabilizing lysates as well as starting points for the design of more selective agents. Ubiquitination regulates many critical cellular processes, and promiscuous inhibitors of deubiquitinases (DUBs) would be similarly valuable. The currently available promiscuous DUB inhibitors are highly reactive electrophilic compounds that can crosslink proteins. Herein we introduce diarylcarbonate esters as a novel class of promiscuous DUB inhibitors that do not have the liabilities associated with the previously reported compounds. Diarylcarbonates stabilize the high molecular weight ubiquitin pools in cells and lysates. They also elicit cellular phenotypes associated with DUB inhibition, demonstrating their utility in ubiquitin discovery. Diarylcarbonates may also be a useful scaffold for the development of specific DUB inhibitors.     

Bibliography

Maytenus ilicifolia is one of frequently used medicinal plants in Brazil. Its leaves are used in homemade and industrial medicines for effective treatment of stomach ulcers. A polygalacturonic acid (PGA) was obtained from its leaves by aqueous extraction, followed by fractionation via a freezing–thawing process and Fehling precipitation. Methylation analysis and ¹³C NMR spectroscopy showed PGA to consist of (1 → 4)-linked α-d-GalpA repeating units. It significantly inhibited ethanol-induced gastric lesions in rats, with an ED₅₀ of 103 mg/kg, suggesting that it has a protective anti-ulcer effect. This polysaccharide may thus play an important role in the anti-ulcer effect of M. ilicifolia.     

Monoclonal antibodies specific for the empty conformation of HLA-DR1 reveal aspects of the conformational change associated with peptide binding

Class II major histocompatibility complex (MHC) proteins bind peptides and present them at the cell surface for interaction with CD4+ T cells as part of the system by which the immune system surveys the body for signs of infection. Peptide binding is known to induce conformational changes in class II MHC proteins on the basis of a variety of hydrodynamic and spectroscopic approaches, but the changes have not been clearly localized within the overall class II MHC structure. To map the peptide-induced conformational change for HLA-DR1, a common human class II MHC variant, we generated a series of monoclonal antibodies recognizing the beta subunit that are specific for the empty conformation. Each antibody reacted with the empty but not the peptide-loaded form, for both soluble recombinant protein and native protein expressed at the cell surface. Antibody binding epitopes were characterized using overlapping peptides and alanine scanning substitutions and were localized to two distinct regions of the protein. The pattern of key residues within the epitopes suggested that the two epitope regions undergo substantial conformational alteration during peptide binding. These results illuminate aspects of the structure of the empty forms and the nature of the peptide-induced conformational change.     

Genetic damage induced by in vitro irradiation of human G0 lymphocytes with low-energy protons (28 keV/microm): HPRT mutations and chromosome aberrations

Cell survival, mutations and chromosomal effects were studied in primary human lymphocytes exposed in G0 phase to a proton beam with an incident energy of 0.88 MeV (incident LET of 28 keV/microm) in the dose range 0.125-2 Gy. The curves for survival and mutations at the hypoxanthine-guanine phosphoribosyl transferase locus were obtained by fitting the experimental data to linear and linear-quadratic equations, respectively. In the dose interval 0-1.5 Gy, the alpha parameters of the curves were 0.42/Gy and 3.6 x 10(-6) mutants/Gy, respectively. The mutation types at the HPRT locus were analyzed by multiplex-PCR in 94 irradiated and 41 nonirradiated clones derived from T lymphocytes from five healthy donors. All clones showed a normal multiplex-PCR pattern and were classified as point mutations. Chromosome aberration data were fitted as a linear function of dose (alpha = 0.62 aberrations per cell Gy(-1)). By irradiating G0 lymphocytes from a single subject with 28 keV/microm protons and gamma rays, an RBE of 6.07 was obtained for chromosome aberrations. An overinvolvement of chromosome 9 relative to chromosome 7 was found in chromosome breaks after chromosome painting analysis.     

Azorellane diterpenes from Azorella cryptantha

Azorella cryptantha yielded the diterpenes, azorellolide and the dihydroderivative, dihydroazorellolide, together with the known yaretol and 1alpha,10beta,4beta,5alpha-diepoxy-7alpha-germacran-6beta-ol. Both possess a carbon skeleton type that may originate from rearrangement of the mulinane skeleton.     

Heterocyclization of 3-deoxy-D-erythro-hexos-2-ulose-1,2-bis(thiosemicarbazone). Crystal structure of the major diastereomer

Both thiosemicarbazone groups of the derivative 1 of 3-deoxy-D-erythro-hexos-2-ulose underwent, on acetylation, a heterocyclization process to give (5R,5'R)-2,2'-diacetamido-4,4'-di-N-acetyl-5'-(1-deoxy-2,3,4-tri-O-acetyl-D-erythritol-1-yl)-5,5'-bis(1,3,4-thiadiazoline) (2) as a major product. The X-ray diffraction data of a single crystal of 2 indicated the R,R configuration for the stereocenters of the thiadiazoline rings (C-5 and C-5'). In the solid state, 2 adopts a sickle conformation (by clockwise rotation of the C-2-C-3 axis of the sugar chain) which has a S//O 1,3-parallel interaction. In solution, as determined by (1)H NMR spectroscopy which included NOE experiments, a similar sickle conformation was observed. From the reaction mixture of acetylation of 1 was isolated the bis(thiadiazoline) 3 as a by-product. The configuration of the C-5 and C-5' stereocenters of 3 were respectively assigned as S,R by comparison of the physical and spectroscopic data of this compound with those of 2.     

Multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited environment

When microbes evolve in a continuous, nutrient-limited environment, natural selection can be predicted to favor genetic changes that give cells greater access to limiting substrate. We analyzed a population of baker's yeast that underwent 450 generations of glucose-limited growth. Relative to the strain used as the inoculum, the predominant cell type at the end of this experiment sustains growth at significantly lower steady-state glucose concentrations and demonstrates markedly enhanced cell yield per mole glucose, significantly enhanced high-affinity glucose transport, and greater relative fitness in pairwise competition. These changes are correlated with increased levels of mRNA hybridizing to probe generated from the hexose transport locus HXT6. Further analysis of the evolved strain reveals the existence of multiple tandem duplications involving two highly similar, high- affinity hexose transport loci, HXT6 and HXT7. Selection appears to have favored changes that result in the formation of more than three chimeric genes derived from the upstream promoter of the HXT7 gene and the coding sequence of HXT6. We propose a genetic mechanism to account for these changes and speculate as to their adaptive significance in the context of gene duplication as a common response of microorganisms to nutrient limitation.      

Crystal structure and magnetic properties of lanthanide-trans-2-butenoate polymers (Ln = La, Pr, Dy, Ho)

Four lanthanide coordination polymers formulated as {[La₃L₉(H₂O)₃]EtOH · H₂O}_n1, {[Pr₂L₆(H₂O)₂]HL · H₂O}_n2, {[Dy₂L₆(H₂O)]0.5HL · H₂O}_n3 and {[Ho₂L₆(H₂O)]0.5HL · H₂O}_n4 and HL = trans-2-butenoic acid have been synthesized from the corresponding pure lanthanide oxide and HL acid in water at pH 3. The compounds were characterized by chemical analysis, IR spectroscopy, thermogravimetry, variable-temperature magnetic susceptibility and single crystal X-ray diffraction studies. A common feature in these materials is the presence of two differentiated lanthanide nodes linked by carboxylates into extended chains. These are further connected by inter- and intra-molecular hydrogen-bonds involving ligand and solvate molecules. Weak ferromagnetic interactions appear to be operative in the Dy material.     

The Role of Diverse Strategies in Sustainable Knowledge Production

Online communities are becoming increasingly important as platforms for large-scale human cooperation. These communities allow users seeking and sharing professional skills to solve problems collaboratively. To investigate how users cooperate to complete a large number of knowledge-producing tasks, we analyze Stack Exchange, one of the largest question and answer systems in the world. We construct attention networks to model the growth of 110 communities in the Stack Exchange system and quantify individual answering strategies using the linking dynamics on attention networks. We identify two answering strategies. Strategy A aims at performing maintenance by doing simple tasks, whereas strategy B aims at investing time in doing challenging tasks. Both strategies are important: empirical evidence shows that strategy A decreases the median waiting time for answers and strategy B increases the acceptance rate of answers. In investigating the strategic persistence of users, we find that users tends to stick on the same strategy over time in a community, but switch from one strategy to the other across communities. This finding reveals the different sets of knowledge and skills between users. A balance between the population of users taking A and B strategies that approximates 2:1, is found to be optimal to the sustainable growth of communities.