A Ruler Protein in a Complex for Antiviral Defense Determines the Length of Small Interfering CRISPR RNAs

Small RNAs undergo maturation events that precisely determine the length and structure required for their function. CRISPRs (clustered regularly interspaced short palindromic repeats) encode small RNAs (crRNAs) that together with CRISPR-associated (cas) genes constitute a sequence-specific prokaryotic immune system for anti-viral and anti-plasmid defense. crRNAs are subject to multiple processing events during their biogenesis, and little is known about the mechanism of the final maturation step. We show that in the Staphylococcus epidermidis type III CRISPR-Cas system, mature crRNAs are measured in a Cas10·Csm ribonucleoprotein complex to yield discrete lengths that differ by 6-nucleotide increments. We looked for mutants that impact this crRNA size pattern and found that an alanine substitution of a conserved aspartate residue of Csm3 eliminates the 6-nucleotide increments in the length of crRNAs. In vitro, recombinant Csm3 binds RNA molecules at multiple sites, producing gel-shift patterns that suggest that each protein binds 6 nucleotides of substrate. In vivo, changes in the levels of Csm3 modulate the crRNA size distribution without disrupting the 6-nucleotide periodicity. Our data support a model in which multiple Csm3 molecules within the Cas10·Csm complex bind the crRNA with a 6-nucleotide periodicity to function as a ruler that measures the extent of crRNA maturation.     

Crystal structure of head-to-head dimers of cholic and deoxycholic acid derivatives with different symmetric bridges

The crystal structure of three head-to-head dimers (having two cholic acid or deoxycholic acid units) linked at carbon atoms C3 by aromatic or alkyl bridges is studied. An internal coordinates system is necessary for describing the relative orientation in the space of the two bile acid residues. Five angles (three torsion and two common ones) are necessary for defining the relative position of both steroid residues in space. Carbon atoms C3 (which always carries a α-hydroxy group in natural bile acids), and C10 and C13 (which always carry β-methyl groups) of each steroid residue are suitable for this purpose. Furthermore, the distance between each C3 carbon atoms of both steroid residues will allow one to locate the steroids in space. The three dimers selected provide a large range of values for these angles. The packing, hydrogen bond network, and location of guest in the three crystals are discussed.     

Evaluating marine diets through radiocarbon dating and stable isotope analysis of victims of the AD79 eruption of vesuvius

The stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope values of bone collagen are frequently used in paleodietary studies to assess the marine contribution to an individual's diet. Surprisingly, the relationship between stable isotope these values characteristics and the percentage of marine foods in diet has never been effectively demonstrated. To clarify this relationship, the stable isotope values and radiocarbon dates of nine humans and one sheep from Herculaneum, all who perished simultaneously during the AD 79 eruption of Vesuvius, were determined. Significant differences were found in the radiocarbon dates which are attributable to the incorporation of “old” carbon from the marine reservoir. The magnitude of the observed differences was linearly correlated with both δ¹³C and δ¹⁵N values allowing the response of each isotope to increasing marine carbon in collagen to be independently verified. Regression analyses showed that for every 1‰ enrichment in δ¹³C and δ¹⁵N, 56 years and 34 years were added to the radiocarbon age, respectively. Predictions of the maximum marine reservoir age differed considerably depending on which stable isotope was considered. This discrepancy is attributed to some degree of macronutrient scrambling whereby nitrogen from marine protein is preferentially incorporated in collagen over marine carbon. It is suggested that the macronutrient scrambling explains the observed relationship between δ¹³C and δ¹⁵N from Roman coastal sites and should be considered when interpreting any diet which is not dominated by protein. Nevertheless, without knowing the degree of macronutrient scrambling in different dietary scenarios, the accuracy of dietary reconstructions is severely compromised. Am J Phys Anthropol 152:345–352, 2013. © 2013 Wiley Periodicals, Inc.     

Ascorbyl palmitate interaction with phospholipid monolayers: Electrostatic and rheological preponderancy

Ascorbyl palmitate (ASC₁₆) is an anionic amphiphilic molecule of pharmacological interest due to its antioxidant properties. We found that ASC₁₆ strongly interacted with model membranes. ASC₁₆ penetrated phospholipid monolayers, with a cutoff near the theoretical surface pressure limit. The presence of a lipid film at the interface favored ASC₁₆ insertion compared with a bare air/water surface. The adsorption and penetration time curves showed a biphasic behavior: the first rapid peak evidenced a fast adsorption of charged ASC₁₆ molecules to the interface that promoted a lowering of surface pH, thus partially neutralizing and compacting the film. The second rise represented an approach to the equilibrium between the ASC₁₆ molecules in the subphase and the surface monolayer, whose kinetics depended on the ionization state of the film. Based on the Langmuir dimiristoylphosphatidylcholine + ASC₁₆ monolayer data, we estimated an ASC₁₆ partition coefficient to dimiristoylphosphatidylcholine monolayers of 1.5 × 10⁵ and a ΔG_p = − 6.7 kcal·mol^− 1. The rheological properties of the host membrane were determinant for ASC₁₆ penetration kinetics: a fluid membrane, as provided by cholesterol, disrupted the liquid-condensed ASC₁₆-enriched domains and favored ASC₁₆ penetration. Subphase pH conditions affected ASC₁₆ aggregation in bulk: the smaller structures at acidic pHs showed a faster equilibrium with the surface film than large lamellar ones. Our results revealed that the ASC₁₆ interaction with model membranes has a highly complex regulation. The polymorphism in the ASC₁₆ bulk aggregation added complexity to the equilibrium between the surface and subphase form of ASC₁₆, whose understanding may shed light on the pharmacological function of this drug.     

Design,synthesis, functional and structural characterization of an inhibitor of N-acetylneuraminate-9-phosphate phosphatase: Observation of extensive dynamics in an enzyme/inhibitor complex

The design, synthesis and characterization of a phosphonate inhibitor of N-acetylneuraminate-9-phosphate phosphatase (HDHD4) is described. Compound 3, where the substrate C-9 oxygen was replaced with a nonlabile CH₂ group, inhibits HDHD4 with a binding affinity (IC₅₀ 11 μM) in the range of the native substrate Neu5Ac-9-P (compound 1, K_m 47 μM). Combined SAR, modeling and NMR studies are consistent with the phosphonate group in inhibitor 3 forming a stable complex with native Mg²⁺. In addition to this key interaction, the C-1 carboxylate of the sugar interacts with a cluster of basic residues, K141, R104 and R72. Comparative NMR studies of compounds 3 and 1 with Ca²⁺ and Mg²⁺ are indicative of a highly dynamic process in the active site for the HDHD4/Mg²⁺/3 complex. Possible explanations for this observation are discussed.     

A biophysical characterization of the folded domains of KCTD12: insights into interaction with the GABAB2 receptor

Recent investigations have shown that members of the KCTD family play important roles in fundamental biological processes. Despite their roles, very limited information is available on their structures and molecular organization. By combining different experimental and theoretical techniques, we have here characterized the two folded domains of KCTD12, an integral component and modulator of the GABA_B2 receptor. Secondary prediction methods and CD spectroscopy have shown that the N‐terminal domain KCTD12_BTB assumes an α/β structure, whereas the C‐terminal domain KCTD12_H1 is predominantly characterized by a β‐structure. Binding assays indicate that the two domains independently expressed show a good affinity for each other. This suggests that the overall protein is likely endowed with a rather compact structure with two interacting structured domains joint by a long disordered region. Notably, both KCTD12_BTB and KCTD12_H1 are tetrameric when individually expressed. This finding could modify the traditional view that ascribes only to POZ/BTB domain a specific oligomerization role. The first quantification of the affinity of KCTD12_POZ/BTB for the C‐terminal region of GABA_B2 shows that it falls in the low micromolar range. Interestingly, we also demonstrate that a GABA_B2‐related peptide is able to bind KCTD12_BTB with a very high affinity. This peptide may represent a useful tool for modulating KCTD12/GABA_B2 interaction in vitro and may also constitute the starting point for the development of peptidomimetic compounds with a potential for therapeutic applications. Copyright © 2013 John Wiley & Sons, Ltd.     

Negative modulation of mitochondrial oxidative phosphorylation by epigallocatechin-3 gallate leads to growth arrest and apoptosis in human malignant pleural mesothelioma cells

Increasing evidence reveals a large dependency of epithelial cancer cells on oxidative phosphorylation (OXPHOS) for energy production. In this study we tested the potential of epigallocatechin-3-gallate (EGCG), a natural polyphenol known to target mitochondria, in inducing OXPHOS impairment and cell energy deficit in human epitheliod (REN cells) and biphasic (MSTO-211H cells) malignant pleural mesothelioma (MMe), a rare but highly aggressive tumor with high unmet need for treatment. Due to EGCG instability that causes H₂O₂ formation in culture medium, the drug was added to MMe cells in the presence of exogenous superoxide dismutase and catalase, already proved to stabilize the EGCG molecule and prevent EGCG-dependent reactive oxygen species formation. We show that under these experimental conditions, EGCG causes the selective arrest of MMe cell growth with respect to normal mesothelial cells and the induction of mitochondria-mediated apoptosis, as revealed by early mitochondrial ultrastructure modification, swelling and cytochrome c release. We disclose a novel mechanism by which EGCG induces apoptosis through the impairment of mitochondrial respiratory chain complexes, particularly of complex I, II and ATP synthase. This induces a strong reduction in ATP production by OXPHOS, that is not adequately counterbalanced by glycolytic shift, resulting in cell energy deficit, cell cycle arrest and apoptosis. The EGCG-dependent negative modulation of mitochondrial energy metabolism, selective for cancer cells, gives an important input for the development of novel pharmacological strategies for MMe.     

Protection of Cells against Oxidative Stress by Nanomolar Levels of Hydroxyflavones Indicates a New Type of Intracellular Antioxidant Mechanism

Natural polyphenol compounds are often good antioxidants, but they also cause damage to cells through more or less specific interactions with proteins. To distinguish antioxidant activity from cytotoxic effects we have tested four structurally related hydroxyflavones (baicalein, mosloflavone, negletein, and 5,6-dihydroxyflavone) at very low and physiologically relevant levels, using two different cell lines, L-6 myoblasts and THP-1 monocytes. Measurements using intracellular fluorescent probes and electron paramagnetic resonance spectroscopy in combination with cytotoxicity assays showed strong antioxidant activities for baicalein and 5,6-dihydroxyflavone at picomolar concentrations, while 10 nM partially protected monocytes against the strong oxidative stress induced by 200 µM cumene hydroperoxide. Wide range dose-dependence curves were introduced to characterize and distinguish the mechanism and targets of different flavone antioxidants, and identify cytotoxic effects which only became detectable at micromolar concentrations. Analysis of these dose-dependence curves made it possible to exclude a protein-mediated antioxidant response, as well as a mechanism based on the simple stoichiometric scavenging of radicals. The results demonstrate that these flavones do not act on the same radicals as the flavonol quercetin. Considering the normal concentrations of all the endogenous antioxidants in cells, the addition of picomolar or nanomolar levels of these flavones should not be expected to produce any detectable increase in the total cellular antioxidant capacity. The significant intracellular antioxidant activity observed with 1 pM baicalein means that it must be scavenging radicals that for some reason are not eliminated by the endogenous antioxidants. The strong antioxidant effects found suggest these flavones, as well as quercetin and similar polyphenolic antioxidants, at physiologically relevant concentrations act as redox mediators to enable endogenous antioxidants to reach and scavenge different pools of otherwise inaccessible radicals.