Protein molecular function influences mutation rates in human genetic diseases with allelic heterogeneity

Molecular epidemiology studies have used the counts of different mutational types like transitions, transversions, etc. to identify putative mutagens, with little reference to gene organization and structure–function of the translated product. Moreover, geographical variation in the mutational spectrum is not limited to the mutational types at the nucleotide level but also have a bearing at the functional level. Here, we developed a novel measure to estimate the rate of spontaneous detrimental mutations called “mutation index” for comparing the mutational spectra consisting of all single base, missense, and non-missense changes. We have analyzed 1609 mutations occurring in 38 exons in 24 populations in three diseases viz. hemophilia B (F9 gene – 420 mutations in 9 populations across 8 exons), hemophilia A (F8 gene – 650, 8 and 26, respectively) and ovarian carcinoma (TP53 gene – 539, 7 and 4, respectively). We considered exons as units of evolution instead of the entire gene and observed feeble differences among populations implying lack of a mutagen-specific effect and the possibility of mutation causing endogenous factors. In all the three genes we observed elevated rates of detrimental mutations in exons encoding regions of significance for the molecular function of the protein. We propose that this can be extended to the entire exome with implications in exon-shuffling and complex human diseases.     

Anomalous spatial redistribution of competing bacteria under starvation conditions

Bacterial cells evolved under prolonged stress often have a growth advantage in stationary phase (GASP); we expect GASP cells to maintain a proliferative state and dominate wild-type cells during starvation, especially when nutrients are limited and the medium has been conditioned. However, when we compete GASP mutants against wild-type cells in a chain of microfluidic microhabitat patches (MHPs) with alternating nutrient-rich and nutrient-limited regions, we observe the reverse effect: wild-type cells achieve maximum relative density under nutrient-limited conditions, while GASP cells dominate nutrient-rich regions. We explain this surprising observation in terms of ideal free distributions, where we show that wild-type cells maximize their fitness at high cell density by redistributing themselves to sparsely populated MHPs. At the microscopic level, we describe how biofilm formation also contributes to the population redistribution. We conclude by discussing the implications of these results for social interactions of more complex organisms.     

Whole genome sequence of the rifamycin B-producing strain Amycolatopsis mediterranei S699

Amycolatopsis mediterranei S699 is an actinomycete that produces an important antibiotic, rifamycin B. Semisynthetic derivatives of rifamycin B are used for the treatment of tuberculosis, leprosy, and AIDS-related mycobacterial infections. Here, we report the complete genome sequence (10.2 Mb) of A. mediterranei S699, with 9,575 predicted coding sequences.     

A synthetic S6 segment derived from KvAP channel self-assembles, permeabilizes lipid vesicles, and exhibits ion channel activity in bilayer lipid membrane

KvAP is a voltage-gated tetrameric K(+) channel with six transmembrane (S1-S6) segments in each monomer from the archaeon Aeropyrum pernix. The objective of the present investigation was to understand the plausible role of the S6 segment, which has been proposed to form the inner lining of the pore, in the membrane assembly and functional properties of KvAP channel. For this purpose, a 22-residue peptide, corresponding to the S6 transmembrane segment of KvAP (amino acids 218-239), and a scrambled peptide (S6-SCR) with rearrangement of only hydrophobic amino acids but without changing its composition were synthesized and characterized structurally and functionally. Although both peptides bound to the negatively charged phosphatidylcholine/phosphatidylglycerol model membrane with comparable affinity, significant differences were observed between these peptides in their localization, self-assembly, and aggregation properties onto this membrane. S6-SCR also exhibited reduced helical structures in SDS micelles and phosphatidylcholine/phosphatidylglycerol lipid vesicles as compared with the S6 peptide. Furthermore, the S6 peptide showed significant membrane-permeabilizing capability as evidenced by the release of calcein from the calcein-entrapped lipid vesicles, whereas S6-SCR showed much weaker efficacy. Interestingly, although the S6 peptide showed ion channel activity in the bilayer lipid membrane, despite having the same amino acid composition, S6-SCR was significantly inactive. The results demonstrated sequence-specific structural and functional properties of the S6 wild type peptide. The selected S6 segment is probably an important structural element that could play an important role in the membrane interaction, membrane assembly, and functional property of the KvAP channel.     

Caspase-8-mediated cleavage inhibits IRF-3 protein by facilitating its proteasome-mediated degradation

Interferon regulatory factor 3 (IRF-3) plays a central role in inducing the expression of cellular antiviral genes, including the interferon-β gene, in response to Pattern Recognition Receptors. IRF-3 is targeted for proteasome-mediated degradation, which modulates the strength and duration of the innate immune responses that depend on it. We have found that caspase-8, which is activated by cytosolic RIG-I-dependent signaling, catalyzes an essential intermediate step in the ubiquitination and proteasome-mediated degradation of IRF-3. Mutation of a consensus cleavage site within IRF-3 generates a form that is not cleaved by caspase-8 and that is protected from ubiquitination and degradation. An in vitro assay confirms the direct action of caspase-8 cleavage on IRF-3. We also show that caspase-8-mediated cleavage of IRF-3 helps to modulate dsRNA-dependent gene induction.     

Clustering and internalization of toxic amylin oligomers in pancreatic cells require plasma membrane cholesterol

Self-assembly of the human pancreatic hormone amylin into toxic oligomers and aggregates is linked to dysfunction of islet β-cells and pathogenesis of type 2 diabetes mellitus. Recent evidence suggests that cholesterol, an essential component of eukaryotic cells membranes, controls amylin aggregation on model membranes. However, the pathophysiological consequence of cholesterol-regulated amylin polymerization on membranes and biochemical mechanisms that protect β-cells from amylin toxicity are poorly understood. Here, we report that plasma membrane (PM) cholesterol plays a key role in molecular recognition, sorting, and internalization of toxic amylin oligomers but not monomers in pancreatic rat insulinoma and human islet cells. Depletion of PM cholesterol or the disruption of the cytoskeleton network inhibits internalization of amylin oligomers, which in turn enhances extracellular oligomer accumulation and potentiates amylin toxicity. Confocal microscopy reveals an increased nucleation of amylin oligomers across the plasma membrane in cholesterol-depleted cells, with a 2-fold increase in cell surface coverage and a 3-fold increase in their number on the PM. Biochemical studies confirm accumulation of amylin oligomers in the medium after depletion of PM cholesterol. Replenishment of PM cholesterol from intracellular cholesterol stores or by the addition of water-soluble cholesterol restores amylin oligomer clustering at the PM and internalization, which consequently diminishes cell surface coverage and toxicity of amylin oligomers. In contrast to oligomers, amylin monomers followed clathrin-dependent endocytosis, which is not sensitive to cholesterol depletion. Our studies identify an actin-mediated and cholesterol-dependent mechanism for selective uptake and clearance of amylin oligomers, impairment of which greatly potentiates amylin toxicity.     

Optimization of protein production in mammalian cells with a coexpressed fluorescent marker

The expression of mammalian proteins in sufficient abundance and quality for structural studies often presents formidable challenges. Many express poorly in bacterial systems, whereas it can be time consuming and expensive to produce them from cells of higher organisms. Here we describe a procedure for the direct selection of stable mammalian cell lines that express proteins of interest in high yield. Coexpression of a marker protein, such as green fluorescent protein, is linked to that of the desired protein through an internal ribosome entry site in the vector that is transfected into cells in culture. The coexpressed marker is used to select for highly expressing clonal cell lines. Applications are described to a membrane protein, the 5HT2c serotonin receptor, and to a secreted cysteine-rich protein, resistin. Besides providing an expeditious means for producing mammalian proteins for structural work, the resulting cell lines also readily support tests of functional properties and structure-inspired hypotheses.     

Characterization of a novel endo-β-1,4-glucanase from Armillaria gemina and its application in biomass hydrolysis

A novel endo-β-1,4-glucanase (EG)-producing strain was isolated and identified as Armillaria gemina KJS114 based on its morphology and internal transcribed spacer rDNA gene sequence. A. gemina EG (AgEG) was purified using a single-step purification by gel filtration. The relative molecular mass of AgEG by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 65 kDa and by size exclusion chromatography was 66 kDa, indicating that the enzyme is a monomer in solution. The pH and temperature optima for hydrolysis were 5.0 and 60 °C, respectively. Purified AgEG had the highest catalytic efficiency with carboxymethylcellulose (k _cat/K _m?=?3,590 mg mL^?1 s^?1) unlike that reported for any fungal EG, highlighting the significance of the current study. The amino acid sequence of AgEG showed homology with hydrolases from the glycoside hydrolase family 61. The addition of AgEG to a Populus nigra hydrolysate reaction containing a commercial cellulase mixture (Celluclast 1.5L and Novozyme 188) showed a stimulatory effect on reducing sugar production. AgEG is a good candidate for applications that convert lignocellulosic biomass to biofuels and chemicals.     

C4′/H4′ selective,non-uniformly sampled 4D HC(P)CH experiment for sequential assignments of 13C-labeled RNAs

A through bond, C4′/H4′ selective, “out and stay” type 4D HC(P)CH experiment is introduced which provides sequential connectivity via H4′_(i)–C4′_(i)–C4′_(i?1)–H4′_(i?1) correlations. The ³¹P dimension (used in the conventional 3D HCP experiment) is replaced with evolution of better dispersed C4′ dimension. The experiment fully utilizes ¹³C-labeling of RNA by inclusion of two C4′ evolution periods. An additional evolution of H4′ is included to further enhance peak resolution. Band selective ¹³C inversion pulses are used to achieve selectivity and prevent signal dephasing due to the of C4′–C3′ and C4′–C5′ homonuclear couplings. For reasonable resolution, non-uniform sampling is employed in all indirect dimensions. To reduce sensitivity losses, multiple quantum coherences are preserved during shared-time evolution and coherence transfer delays. In the experiment the intra-nucleotide peaks are suppressed whereas inter-nucleotide peaks are enhanced to reduce the ambiguities. The performance of the experiment is verified on a fully ¹³C, ¹⁵N-labeled 34-nt hairpin RNA comprising typical structure elements.