The structure of human ubiquitin in 2-methyl-2,4-pentanediol: a new conformational switch

A new crystal structure of human ubiquitin is reported at 1.8 Å resolution. Compared with the other known crystal structure or the solution NMR structure of monomeric human ubiquitin, this new structure is similar in its overall fold but differs with respect to the conformation of the backbone in a surface‐exposed region. The conformation reported here resembles conformations previously seen in complex with deubiquinating enzymes, wherein the Asp52/Gly53 main chain and Glu24 side chain move. This movement exposes the backbone carbonyl of Asp52 to the exterior of the molecule, making it possible to engage in hydrogen‐bond contacts with neighboring molecules, rather than in an internal hydrogen bond with the backbone of Glu24. This particular crystal form of ubiquitin has been used in a large number of solid state NMR studies. The structure described here elucidates the origin of many of the chemical shift differences comparing solution and solid state studies.     

Triosephosphate Isomerase: N and C Chemical Shift Assignments and Conformational Change upon Ligand Binding by Magic-Angle Spinning Solid-State NMR Spectroscopy

Microcrystalline uniformly ¹³C,¹⁵N-enriched yeast triosephosphate isomerase (TIM) is sequentially assigned by high-resolution solid-state NMR (SSNMR). Assignments are based on intraresidue and interresidue correlations, using dipolar polarization transfer methods, and guided by solution NMR assignments of the same protein. We obtained information on most of the active-site residues involved in chemistry, including some that were not reported in a previous solution NMR study, such as the side-chain carbons of His95. Chemical shift differences comparing the microcrystalline environment to the aqueous environment appear to be mainly due to crystal packing interactions. Site-specific perturbations of the enzyme's chemical shifts upon ligand binding are studied by SSNMR for the first time. These changes monitor proteinwide conformational adjustment upon ligand binding, including many of the sites probed by solution NMR and X-ray studies. Changes in Gln119, Ala163, and Gly210 were observed in our SSNMR studies, but were not reported in solution NMR studies (chicken or yeast). These studies identify a number of new sites with particularly clear markers for ligand binding, paving the way for future studies of triosephosphate isomerase dynamics and mechanism.     

Good to the bone: microbial community thrives within bone cavities of a bison carcass at Yellowstone National Park

The discovery of unanticipated microbial diversity in remote, often hostile environments has led to a greater appreciation of the complexity and richness of the natural world. Yellowstone National Park (YNP) has long been a focus of work on taxa that inhabit extreme environments. Here we report the finding of microbial flora that inhabit an unexpected niche: the cavities of bone remnants from a bison carcass in Norris Geyser Basin in YNP. Although bleached white on the surface, the bone cavities are bright green due to the presence of Stichococcus-like trebouxiophyte green algae. The cavities also harbour different fungi and bacteria. Stichococcus species are common lichen photobionts and the Thelebolales fungi present in the bone cavities have previously been found in association with animal remains. Scanning electron microscope analysis suggests the fungi and algae do not form lichen-like associations in the bone. Rather these taxa and the bacteria appear to be opportunists that have colonized an isolated oasis that provides nutrients and protection from desiccation and UV radiation.     

Drosophila perlecan modulates FGF and hedgehog signals to activate neural stem cell division

Mutations in the Drosophila trol gene cause cell cycle arrest of neuroblasts in the larval brain. Here, we show that trol encodes the Drosophila homolog of Perlecan and regulates neuroblast division by modulating both FGF and Hh signaling. Addition of human FGF-2 to trol mutant brains in culture rescues the trol proliferation phenotype, while addition of a MAPK inhibitor causes cell cycle arrest of the regulated neuroblasts in wildtype brains. Like FGF, Hh activates stem cell division in the larval brain in a Trol-dependent fashion. Coimmunoprecipitation studies are consistent with interactions between Trol and Hh and between mammalian Perlecan and Shh that are not competed with heparin sulfate. Finally, analyses of mutations in trol, hh, and ttv suggest that Trol affects Hh movement. These results indicate that Trol can mediate signaling through both of the FGF and Hedgehog pathways to control the onset of stem cell proliferation in the developing nervous system.     

The contribution of tandem repeat number to the O-glycosylation of mucins

The serine- and threonine-rich tandem repeat (TR) units that make up the characteristic feature of mucin glycoproteins are often polymorphic with substantial genetic variation in TR number. The precise effect of TR number on O-glycosylation is not fully understood, although the TR number of several mucins may be associated with apparent susceptibility to certain human diseases. To evaluate the contribution of TR number to O-glycosylation, we generated a series of chimeric mucins carrying increasing numbers of TR units from the MUC5B mucin in the context of an epitope-tagged MUC1 mucin backbone. These mucins were expressed in Caco2 colon carcinoma cell clones and purified by immunoprecipitation. O-Glycosylation was investigated by western blotting with antibodies to known carbohydrate structures and by fast atom bombardment-mass spectrometry. Additional carbohydrate epitopes were detected with antibodies on chimeric mucins with a higher TR number in comparison to those with fewer TRs. Using mass spectrometry, higher-molecular-weight glycans were detected more frequently on the mucins with extended TRs compared to those with fewer TRs. However no novel carbohydrate structures were seen, suggesting that TR number does not affect the specificity of O-glycosylation.     

Caspase-9 activation in hypoxic human corneal epithelial cells

The purpose of this study was to determine the effect of hypoxia on caspase-8 and -9 gene and protein expression and activity in corneal epithelium. Non-transformed human corneal epithelial cells (HCEC) were cultured in 2% oxygen. A cDNA expression array coupled with densitometric analysis was used to compare relative mRNA expression levels of 96 apoptosis-related genes in hypoxic and normoxic HCEC. Caspase-8, caspase-9, FLIP, Fas, FasL, and TNF protein expression was assessed further using Western blot analysis and ELISA. Caspase-8 and -9 activities were measured using a fluorometric activity assay. Hypoxia did not affect caspase-8 or -9 gene or protein expression in HCEC, however caspase-9 activity was significantly increased. Hypoxia significantly suppressed the activity of caspase-8. FLIP and Fas gene and protein expression were not significantly altered in hypoxic cells compared to normoxic controls. mRNA and protein levels of TNF and TNFR-1 were significantly decreased, while FasL mRNA and proteins levels were significantly increased in hypoxic HCEC. In corneal epithelium stressed by hypoxia caspase-9 activity is upregulated, suggesting that apoptosis proceeds via the mitochondrial pathway. Caspase-8 activity may be suppressed because the loss of TNF and TNFR-1 gene and protein expression inhibits the initial formation of a death signaling complex.     

Assembly of retrovirus capsid-nucleocapsid proteins in the presence of membranes or RNA

Retrovirus Gag precursor (PrGag) proteins direct the assembly of roughly spherical immature virus particles, while after proteolytic processing events, the Gag capsid (CA) and nucleocapsid (NC) domains condense on viral RNAs to form mature retrovirus core structures. To investigate the process of retroviral morphogenesis, we examined the properties of histidine-tagged (His-tagged) Moloney murine leukemia (M-MuLV) capsid plus nucleocapsid (CANC) (His-MoCANC) proteins in vitro. The His-MoCANC proteins bound RNA, possessed nucleic acid-annealing activities, and assembled into strand, circle (or sphere), and tube forms in the presence of RNA. Image analysis of electron micrographs revealed that tubes were formed by cage-like lattices of CANC proteins surrounding at least two different types of protein-free cage holes. By virtue of a His tag association with nickel-chelating lipids, His-MoCANC proteins also assembled into planar sheets on lipid monolayers, mimicking the membrane-associated immature PrGag protein forms. Membrane-bound His-MoCANC proteins organized into two-dimensional (2D) cage-like lattices that were closely related to the tube forms, and in the presence of both nickel-chelating lipids and RNAs, 2D lattice forms appeared similar to lattices assembled in the absence of RNA. Our observations are consistent with a M-MuLV morphogenesis model in which proteolytic processing of membrane-bound Gag proteins permits CA and NC domains to rearrange from an immature spherical structure to a condensed mature form while maintaining local protein-protein contacts.