A conserved element that stabilizes the group II intron active site

The internal loop at the base of domain 3 (D3) is one of the most conserved and catalytically important elements of a group II intron. However, the location and molecular nature of its tertiary interaction partners has remained unknown. By employing a combination of site-directed photo-cross-linking and nucleotide analog interference suppression (NAIS), we show that the domain 3 internal loop (D3IL) interacts with the epsilon-epsilon' duplex, which is an active-site element located near the 5'-splice site in D1. Our data also suggest that the D3IL may interact with the bulge of D5, which is a critical active site component. The results of this and other recent studies indicate that the D3IL participates in a complex network of tertiary interactions involving epsilon-epsilon', the bulge of D5 and J23, and that it helps to optimize active site architecture by supporting interactions among these catalytic motifs. Our results are consistent with the role of D3 as a catalytic effector that enhances intron reactivity through active site stabilization.     

Genetic Control of Mitosis: Features of Anaphase Separation of Sister Chromatid Sets in Mutant Strain aar V158 in Drosophila melanogaster

The effect of mutation aar ^V158 on anaphase separation of chromatids was studied on fixed cells of neural ganglia of Drosophila melanogaster larvae. It was shown that mutation aar ^V158 causes three types of defective chromosome segregation manifested as (1) monopolar anaphase, (2) separation of chromatids to an abnormally short distance in anaphase, and (3) bridging and lagging of some chromatids or prolonged asynchronous separation of sister chromatid sets to the poles in anaphase. We believe that the former two types of defective segregation are caused by disturbed centrosome separation at the beginning of mitosis and the third type, by defects in chromatid separation during anaphase. During the two-year maintenance of the mutation in a heterozygous state, partial correction (adaptive modification) of the defects of type 1 and type 2 (but not type 3) occurred. The correction of type 1 and type 2 defects during adaptogenesis depended on the genotype: in heterozygotes and homozygotes, respectively type 1 and type 2 were preferentially corrected. The frequency of type 3 defects remained constant during the two-year period of maintenance of the mutation in a heterozygous state. However, in all variants of the experiment, their frequency decreased with increasing distance between the sister chromatid sets. In the cells that completed the previous division with abnormalities, the checkpoint system is supposed to effectively arrest the cell cycle in the subsequent division.     

Reversible and irreversible modifications of skeletal muscle proteins in a rat model of acute oxidative stress

Oxidative stress caused by an imbalance of the production of “reactive oxygen species” (ROS) and cellular scavenging systems is known to a play a key role in the development of various diseases and aging processes. Such elevated ROS levels can damage all components of cells, including proteins, lipids and DNA. Here, we study the influence of highly reactive ROS species on skeletal muscle proteins in a rat model of acute oxidative stress caused by X-ray irradiation at different time points. Protein preparations depleted for functional actin by polymerization were separated by gel electrophoresis in two dimensions by applying first non-reductive and then reductive conditions in SDS-PAGE. This diagonal redox SDS-PAGE revealed significant alterations to intra- and inter-molecular disulfide bridges for several proteins, but especially actin, creatine kinase and different isoforms of the myosin light chain. Though the levels of these reversible modifications were increased by oxidative stress, all proteins followed different kinetics. Moreover, a significant degree of protein was irreversibly oxidized (carbonylated), as revealed by western blot analyses performed at different time points.     

Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches

Lethal factor (LF), a zinc-dependent protease of high specificity produced by Bacillus anthracis, is the effector component of the binary toxin that causes death in anthrax. New therapeutics targeting the toxin are required to reduce systemic anthrax-related fatalities. In particular, new insights into the LF catalytic mechanism will be useful for the development of LF inhibitors. We evaluated the minimal length required for formation of bona fide LF substrates using substrate phage display. Phage-based selection yielded a substrate that is cleaved seven times more efficiently by LF than the peptide targeted in the protein kinase MKK6. Site-directed mutagenesis within the metal-binding site in the LF active center and within phage-selected substrates revealed a complex pattern of LF-substrate interactions. The elementary steps of LF-mediated proteolysis were resolved by the stopped-flow technique. Pre-steady-state kinetics of LF proteolysis followed a four-step mechanism as follows: initial substrate binding, rearrangement of the enzyme-substrate complex, a rate-limiting cleavage step, and product release. Examination of LF interactions with metal ions revealed an unexpected activation of the protease by Ca²⁺ and Mn²⁺. Based on the available structural and kinetic data, we propose a model for LF-substrate interaction. Resolution of the kinetic and structural parameters governing LF activity may be exploited to design new LF inhibitors.Anthrax is an infectious disease caused by the encapsulated, spore-forming bacterium Bacillus anthracis. Systemic forms of the disease, such as inhalational anthrax, are characterized by nonspecific early symptoms, rapid progression, and lethality approaching 100% (1). The lethality of inhalational anthrax is high even with antibiotic treatment and is caused by accumulation of secreted anthrax toxin (2), which consists of three proteins as follows: protective antigen (PA),² lethal factor (LF), and edema factor. PA binds to membrane receptors, forms pore complexes, and translocates LF and edema factor into the host cell (3, 4). The PA·LF complex is known as the lethal toxin, a virulence factor with pleiotropic action that facilitates establishment of the B. anthracis infection. LF is a Zn²⁺-dependent metalloprotease related to the thermolysin family that cleaves mitogen-activated protein kinase kinases (5).Although the complete mechanism by which LF causes fatal intoxication is still unclear, inhibition of LF proteolytic activity may be an efficient means of preventing anthrax lethality. A better understanding of the LF catalytic mechanism will facilitate rational design and optimization of LF inhibitors with potential clinical applicability. Recent structural (6, 7), mechanistic (8), and in vivo studies (9, 10) of LF point to a sophisticated catalytic mechanism involving accurate recognition of multiple target substrates.Here we use substrate phage display and stopped-flow fluorimetry kinetics to examine both the substrate specificity and elementary steps of substrate processing by LF. Our data allow us to construct a working model of LF-substrate binding and cleavage.     

The pattern of major urinary proteins (MUPS) expression during postnatal ontogenesis of the laboratory mouse depends on genotype and sex

We investigated the specific pattern of major urinary proteins (MUPs) expression in 3-, 4-, and 12-week old mice of CBA/LacY and C57BL/6JY inbred strains using polyacrylamide gel electrophoresis. Quantitative evaluation of 8 protein fractions A-H with regard to sex, age, and genotype of the animals is presented for the first time. Actual problems of genetic control and neuroendocrine regulation of MUPs expression during ontogenesis are discussed. In the light of current views on MUPs as a key component in intrapopulation information exchange via pheromones, we put forward the idea that the genetically determined structure of the olfactory code of the definitive type is formed at an early ontogenetic stage on the basis of the MUPs combinatorial pattern.     

Low in vivo brain glucose consumption and high oxidative stress in accelerated aging

The validity of the free radical theory of aging has been recently questioned. Our aim was to test whether there is oxidative stress in tissues critically involved in accelerated aging (senescence-accelerated mice, SAM) and whether this correlates with lower glucose consumption in vivo and behavioural tests. Positron emission tomography shows that brains of old SAM-prone animals consume less glucose than young ones. Behavioural characteristics, mitochondrial peroxide production, and damage in both the central nervous system and bone marrow stem cells also indicate that SAM-prone animals age faster than SAM-resistant ones. Our results support the role of the free radical theory of aging in critical tissues involved in aging and that this correlates with glucose consumption.     

Cardiotonic steroids stimulate glycogen synthesis in human skeletal muscle cells via a Src- and ERK1/2-dependent mechanism

The cardiotonic steroid, ouabain, a specific inhibitor of Na(+),K(+)-ATPase, initiates protein-protein interactions that lead to an increase in growth and proliferation in different cell types. We explored the effects of ouabain on glucose metabolism in human skeletal muscle cells (HSMC) and clarified the mechanisms of ouabain signal transduction. In HSMC, ouabain increased glycogen synthesis in a concentration-dependent manner reaching the maximum at 100 nM. The effect of ouabain was additive to the effect of insulin and was independent of phosphatidylinositol 3-kinase inhibitor LY294002 but was abolished in the presence of a MEK1/2 inhibitor (PD98059) or a Src inhibitor (PP2). Ouabain increased Src-dependent tyrosine phosphorylation of alpha(1)- and alpha(2)-subunits of Na(+),K(+)-ATPase and promoted interaction of alpha(1)- and alpha(2)-subunits with Src, as assessed by co-immunoprecipitation with Src. Phosphorylation of ERK1/2 and GSK3alpha/beta, as well as p90rsk activity, was increased in response to ouabain in HSMC, and these responses were prevented in the presence of PD98059 and PP2. Incubation of HSMC with 100 nM ouabain increased phosphorylation of the alpha-subunits of the Na-pump at a MAPK-specific Thr-Pro motif. Ouabain treatment decreased the surface abundance of alpha(2)-subunit, whereas abundance of the alpha(1)-subunit was unchanged. Marinobufagenin, an endogenous vertebrate bufadienolide cardiotonic steroid, increased glycogen synthesis in HSMC at 10 nM concentration, similarly to 100 nM ouabain. In conclusion, ouabain and marinobufagenin stimulate glycogen synthesis in skeletal muscle. This effect is mediated by activation of a Src-, ERK1/2-, p90rsk-, and GSK3-dependent signaling pathway.     

Genetic and Biochemical Characterization of Human AP Endonuclease 1 Mutants Deficient in Nucleotide Incision Repair Activity

Background

Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair enzyme involved in both base excision repair (BER) and nucleotide incision repair (NIR) pathways. In the BER pathway, APE1 cleaves DNA at AP sites and 3′-blocking moieties generated by DNA glycosylases. In the NIR pathway, APE1 incises DNA 5′ to a number of oxidatively damaged bases. At present, physiological relevance of the NIR pathway is fairly well established in E. coli, but has yet to be elucidated in human cells.

Methodology/Principal Finding

We identified amino acid residues in the APE1 protein that affect its function in either the BER or NIR pathway. Biochemical characterization of APE1 carrying single K98A, R185A, D308A and double K98A/R185A amino acid substitutions revealed that all mutants exhibited greatly reduced NIR and 3′→5′ exonuclease activities, but were capable of performing BER functions to some extent. Expression of the APE1 mutants deficient in the NIR and exonuclease activities reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to an alkylating agent, methylmethanesulfonate, suggesting that our APE1 mutants are able to repair AP sites. Finally, the human NIR pathway was fully reconstituted in vitro using the purified APE1, human flap endonuclease 1, DNA polymerase β and DNA ligase I proteins, thus establishing the minimal set of proteins required for a functional NIR pathway in human cells.

Conclusion/Significance

Taken together, these data further substantiate the role of NIR as a distinct and separable function of APE1 that is essential for processing of potentially lethal oxidative DNA lesions.     

Cytokinins contribute to realization of nitric oxide growth-stimulating and protective effects on wheat plants

We investigated effects of sodium nitroprusside (SNP), the donor of nitric oxide (NO), on the growth and hormonal system of wheat plants (Triticum aestivum L.) in normal conditions and after salt stress (2% NaCl). During germination of seeds treated with SNP (50–500 μM), we obtained the SNP concentration (200 μM) optimal for stimulation of seedling growth estimated by increase in seed germination capacity and seedlings' linear sizes and their fresh and dry biomass. A comparative analysis of SNP (200 μM) effects, after seed germination in the medium with SNP or pretreatment of 3-day-old seedlings, showed SNP ability to increase the wheat plant resistance to subsequent effects of sodium chloride salinity at both treatment methods. Protective SNP effects appeared in the reduction of stress inhibitory action on seedling growth rates and significant reduction in the level of lipid peroxidation and exosmosis of electrolytes. An important contribution to realization of the growth-stimulating and protective effects of NO is associated with its ability to influence the state of the hormonal system of wheat plants due to an increase in the concentration of hormones of a cytokinin nature under normal conditions and the prevention of a decrease in their level under stress.     

Proteomic analysis of the 20S proteasome (PSMA3)-interacting proteins reveals a functional link between the proteasome and mRNA metabolism

The 26S proteasome is a large multi-subunit protein complex that exerts specific degradation of proteins in the cell. The 26S proteasome consists of the 20S proteolytic particle and the 19S regulator. In order to be targeted for proteasomal degradation most of the proteins must undergo the post-translational modification of poly-ubiquitination. However, a number of proteins can also be degraded by the proteasome via a ubiquitin-independent pathway. Such degradation is exercised largely through the binding of substrate proteins to the PSMA3 (alpha 7) subunit of the 20S complex. However, a systematic analysis of proteins interacting with PSMA3 has not yet been carried out. In this report, we describe the identification of proteins associated with PSMA3 both in the cytoplasm and nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and tandem mass-spectrometry revealed a large number of PSMA3-bound proteins that are involved in various aspects of mRNA metabolism, including splicing. In vitro biochemical studies confirmed the interactions between PSMA3 and splicing factors. Moreover, we show that 20S proteasome is involved in the regulation of splicing in vitro of SMN2 (survival motor neuron 2) gene, whose product controls apoptosis of neurons.