Long-residency hydration, cation binding, and dynamics of loop E/helix IV rRNA-L25 protein complex

Molecular dynamics simulations of RNA-protein complex between Escherichia coli loop E/helix IV (LE/HeIV) rRNA and L25 protein reveal a qualitative agreement between the experimental and simulated structures. The major groove of LE is a prominent rRNA cation-binding site. Divalent cations rigidify the LE major groove geometry whereas in the absence of divalent cations LE extensively interacts with monovalent cations via inner-shell binding. The HeIV region shows bistability of its major groove explaining the observed differences between x-ray and NMR structures. In agreement with the experiments, the simulations suggest that helix-alpha1 of L25 is the least stable part of the protein. Inclusion of Mg2+ cations into the simulations causes perturbation of basepairing at the LE/HeIV junction, which does not, however, affect the protein binding. The rRNA-protein complex is mediated by a number of highly specific hydration sites with long-residing water molecules and two of them are bound throughout the entire 24-ns simulation. Long-residing water molecules are seen also outside the RNA-protein contact areas with water-binding times substantially enhanced compared to simulations of free RNA. Long-residency hydration sites thus represent important elements of the three-dimensional structure of rRNA.     

Non-Watson-Crick basepairing and hydration in RNA motifs: molecular dynamics of 5S rRNA loop E

Explicit solvent and counterion molecular dynamics simulations have been carried out for a total of >80 ns on the bacterial and spinach chloroplast 5S rRNA Loop E motifs. The Loop E sequences form unique duplex architectures composed of seven consecutive non-Watson-Crick basepairs. The starting structure of spinach chloroplast Loop E was modeled using isostericity principles, and the simulations refined the geometries of the three non-Watson-Crick basepairs that differ from the consensus bacterial sequence. The deep groove of Loop E motifs provides unique sites for cation binding. Binding of Mg(2+) rigidifies Loop E and stabilizes its major groove at an intermediate width. In the absence of Mg(2+), the Loop E motifs show an unprecedented degree of inner-shell binding of monovalent cations that, in contrast to Mg(2+), penetrate into the most negative regions inside the deep groove. The spinach chloroplast Loop E shows a marked tendency to compress its deep groove compared with the bacterial consensus. Structures with a narrow deep groove essentially collapse around a string of Na(+) cations with long coordination times. The Loop E non-Watson-Crick basepairing is complemented by highly specific hydration sites ranging from water bridges to hydration pockets hosting 2 to 3 long-residing waters. The ordered hydration is intimately connected with RNA local conformational variations.     

Molecular dynamics simulations of RNA kissing-loop motifs reveal structural dynamics and formation of cation-binding pockets

Explicit solvent molecular dynamics (MD) simulations were carried out for three RNA kissing–loop complexes. The theoretical structure of two base pairs (2 bp) complex of H3 stem–loop of Moloney murine leukemia virus agrees with the NMR structure with modest violations of few NMR restraints comparable to violations present in the NMR structure. In contrast to the NMR structure, however, MD shows relaxed intermolecular G-C base pairs. The core region of the kissing complex forms a cation-binding pocket with highly negative electrostatic potential. The pocket shows nanosecond-scale breathing motions coupled with oscillations of the whole molecule. Additional simulations were carried out for 6 bp kissing complexes of the DIS HIV-1 subtypes A and B. The simulated structures agree well with the X-ray data. The subtype B forms a novel four-base stack of bulged-out adenines. Both 6 bp kissing complexes have extended cation-binding pockets in their central parts. While the pocket of subtype A interacts with two hexacoordinated Mg²⁺ ions and one sodium ion, pocket of subtype B is filled with a string of three delocalized Na⁺ ions with residency times of individual cations 1–2 ns. The 6 bp complexes show breathing motions of the cation-binding pockets and loop major grooves.     

Conservation of a Pumilio-Nanos complex from<Emphasis Type="Italic"> Drosophila</Emphasis> germ plasm to human germ cells

Germ cells are the cells which ultimately give rise to mature sperm and eggs. In model organisms such as flies and worms, several genes that are required for formation and maintenance of germ cells have been identified and their interactions are rapidly being delineated. By contrast, little is known of the genes required for development of human germ cells and it is not clear whether findings from model organisms will translate into knowledge of human germ cell development, especially given observations that reproductive pathways may evolve more rapidly than somatic pathways. The Pumilio and Nanos genes have been especially well-characterized in model organisms and encode proteins that interact and are required for development of germ stem cells in one or both sexes. Here we report the first characterization of a mammalian Nanos homolog, human NANOS1 ( NOS1). We show that human NOS1 protein interacts with the human PUMILIO-2 (PUM2) protein via highly conserved domains to form a stable complex. We also show that in men, the NOS1 and PUM2 proteins are particularly abundant in germline stem cells. These observations mirror those in distant species and document for the first time a conserved protein-protein interaction in germ cells from flies to humans. These results suggest the possibility that the interaction of PUM2 and NOS1 may play a conserved role in germ cell development and maintenance in humans as in model organisms.     

Purification of Drosophila melanogaster ultraspiracle protein and analysis of its A/B region-dependent dimerization behavior in vitro

Two members of the nuclear receptor superfamily, EcR (ecdysteroid receptor protein) and Usp (Ultraspiracle), heterodimerize to form a functional receptor for the steroid hormone 20-hydroxyecdysone and thus enable it to coordinate morphogenetic events during insect metamorphosis. N-terminally His-tagged Usp was overexpressed in E. coli cells as a non-truncated protein and purified to homogeneity in two chromatographic steps. It was demonstrated that the recombinant receptor specifically binds the ecdysone response element of the hsp27 gene promoter (hsp27EcRE). Moreover, a highly synergistically formed heterodimeric complex with the DNA-binding domain of EcR was observed on hsp27EcRE, but not on the native Usp response element from the chorion s15 gene promoter. Recombinant Usp forms homodimers and homotetramers in the absence of DNA, as judged from gel filtration and chemical crosslinking experiments. Truncation of its N-terminal A/B region changes molecular characteristics of Usp, considerably weakening its oligomerization potential under the same experimental conditions. This contrasts with the results obtained previously for the similarly truncated RXR--a vertebrate homolog of Usp.     

Topology determination and functional analysis of the Escherichia coli TatC protein

Misfolding of the prion protein yields amyloidogenic isoforms, and it shows exacerbating neuronal damage in neurodegenerative disorders including prion diseases. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) potently stimulate neuritogenesis and survival of neuronal cells in the central nervous system. Here, we tested these neuropeptides on neurotoxicity in PC12 cells induced by the prion protein fragment 106-126 [PrP (106-126)]. Concomitant application of neuropeptide with PrP(106-126) (5x10(-5) M) inhibited the delayed death of neuron-like PC12 cells. In particular, PACAP27 inhibited the neurotoxicity of PrP(106-126) at low concentrations (>10(-15) M), characterized by the deactivation of PrP(106-126)-stimulated caspase-3. The neuroprotective effect of PACAP27 was antagonized by the selective PKA inhibitor, H89, or the MAP kinase inhibitor, U0126. These results suggest that PACAP27 attenuates PrP(106-126)-induced delayed neurotoxicity in PC12 cells by activating both PKA and MAP kinases mediated by PAC1 receptor.     

Sucrose Is a Nonaccumulated Osmoprotectant in Sinorhizobium meliloti

Intracellular accumulation of sucrose in response to lowered water activity seems to occur only in photosynthetic organisms. Here we demonstrate, for the first time, the potent ability of this common sugar, supplied exogenously, to reduce growth inhibition of Sinorhizobium meliloti cells in media of inhibitory osmolarity. Independently of the nature of the growth substrates and the osmotic agent, sucrose appears particularly efficient in promoting the recovery of cytoplasmic volume after plasmolysis. Surprisingly, sucrose is not accumulated by the bacteria at an osmotically efficient level. Instead, it strongly stimulates the accumulation of the main endogenous osmolytes glutamate and N-acetylglutaminylglutamine amide (NAGGN). Examining cell volume changes during the hyperosmotic treatment, we found a close correlation between the enhancement of the osmotically active solute pool and the increase in cell volume. Sucrose shares several features with ectoine, another nonaccumulated osmoprotectant for S. meliloti. Overall, osmoregulation in S. meliloti appears to be strongly divergent from that in most bacteria.     

Glycogen phosphorylase inhibition improves cognitive function of aged mice

Inhibition of glycogen breakdown blocks memory formation in young animals, but it stimulates the maintenance of the long-term potentiation, a cellular mechanism of memory formation, in hippocampal slices of old animals. Here, we report that a 2-week treatment with glycogen phosphorylase inhibitor BAY U6751 alleviated memory deficits and stimulated neuroplasticity in old mice. Using the 2-Novel Object Recognition and Novel Object Location tests, we discovered that the prolonged intraperitoneal administration of BAY U6751 improved memory formation in old mice. This was accompanied by changes in morphology of dendritic spines in hippocampal neurons, and by “rejuvenation” of hippocampal proteome. In contrast, in young animals, inhibition of glycogen degradation impaired memory formation; however, as in old mice, it did not alter significantly the morphology and density of cortical dendritic spines. Our findings provide evidence that prolonged inhibition of glycogen phosphorolysis improves memory formation of old animals. This could lead to the development of new strategies for treatment of age-related memory deficits.     

Generation of dendritic cells from human peripheral blood monocytes--comparison of different culture media

Culture medium or medium supplement is one of the factors responsible for dendritic cell (DC) generation, but little is known about the influence of various media on DC culture. In our study we generated DC from adherent monocytes of human peripheral blood in the presence of GM-CSF, IL-4 and TNF-alpha. The following culture media were used: RPMI 1640 supplemented with 2% human serum albumin; RPMI 1640 supplemented with 2% TCH serum replacement; X-VIVO 15 and Panserin 501. Flow cytometry analysis revealed that in all media cells were CD83+ and lost CD14. Interestingly, the use of Panserin and RPMI with albumin preferentially gave rise to CD1a+ DC, whereas in X-VIVO and RPMI with TCH we observed both CD1a+ and CD1a-. Our results showed that RPMI with TCH yielded the highest percentage of cells expressing both CD80 and CD86 molecules and, in contrast to other media, the higher percentage of CD86+ cells in comparison to CD80+ cells.