RAD51 foci formation in response to DNA damage is modulated by TIP49

Chromatin modification plays an important role in modulating the access of homologous recombination proteins to the sites of DNA damage. TIP49 is highly conserved component of chromatin modification/remodeling complexes, but its involvement in homologous recombination repair in mammalian cells has not been examined in details. In the present communication we studied the role of TIP49 in recruitment of the key homologous recombination protein RAD51 to sites of DNA damage. RAD51 redistribution to chromatin and nuclear foci formation induced by double-strand breaks and interstrand crosslinks were followed under conditions of TIP49 depletion by RNA interference. TIP49 silencing reduced RAD51 recruitment to chromatin and nuclear foci formation to about 50% of that of the control. Silencing of TIP48, which is closely related to TIP49, induced a similar reduction in RAD51 foci formation. RAD51 foci reduction in TIP49-silenced cells was not a result of defective DNA damage checkpoint signaling as judged by the normal histone H2AX phosphorylation and cell cycle distribution. Treatment with the histone deacetylase inhibitor sodium butyrate restored RAD51 foci formation in the TIP49-depleted cells. The results suggest that as a constituent of chromatin modification complexes TIP49 may facilitate the access of the repair machinery to the sites of DNA damage.     

Structure of the L1 protuberance in the ribosome

The L1 protuberance of the 50S ribosomal subunit is implicated in the release/disposal of deacylated tRNA from the E site. The apparent mobility of this ribosomal region has thus far prevented an accurate determination of its three-dimensional structure within either the 50S subunit or the 70S ribosome. Here we report the crystal structure at 2.65 A resolution of ribosomal protein L1 from Sulfolobus acidocaldarius in complex with a specific 55-nucleotide fragment of 23S rRNA from Thermus thermophilus. This structure fills a major gap in current models of the 50S ribosomal subunit. The conformations of L1 and of the rRNA fragment differ dramatically from those within the crystallographic model of the T. thermophilus 70S ribosome. Incorporation of the L1-rRNA complex into the structural models of the T. thermophilus 70S ribosome and the Deinococcus radiodurans 50S subunit gives a reliable representation of most of the L1 protuberance within the ribosome.     

Kinetics of actin unfolding induced by guanidine hydrochloride.

The kinetics of actin unfolding induced by guanidine hydrochloride has been studied. On the basis of obtained experimental data a new kinetic pathway of actin unfolding was proposed. We have shown that the transition from native to inactivated actin induced by guanidine hydrochloride (GdnHCl) passes through essential unfolding of the protein. This means that inactivated actin should be considered as the off-pathway species rather than an intermediate conformation between native and completely unfolded states of actin, as has been assumed earlier. The rate constants of the transitions that give rise to the inactivated actin were determined. At 1.0-2.0 M GdnHCl the value of the rate constant of the transition from native to essentially unfolded actin exceeds that of the following step of inactivated actin formation. It leads to the accumulation of essentially unfolded macromolecules early in the unfolding process, which in turn causes the minimum in the time dependencies of tryptophan fluorescence intensity, parameter A, characterizing the intrinsic fluorescence spectrum position, and tryptophan fluorescence anisotropy.     

The role of caldesmon in the regulation of endothelial cytoskeleton and migration

The actin- and myosin-binding protein, caldesmon (CaD) is an essential component of the cytoskeleton in smooth muscle and non-muscle cells and is involved in the regulation of cell contractility, division, and assembly of actin filaments. CaD is abundantly present in endothelial cells (EC); however, the contribution of CaD in endothelial cytoskeletal arrangement is unclear. To examine this contribution, we generated expression constructs of l-CaD cloned from bovine endothelium. Wild-type CaD (WT-CaD) and truncated mutants lacking either the N-terminal myosin-binding site or the C-terminal domain 4b (containing actin- and calmodulin-binding sites) were transfected into human pulmonary artery EC. Cell fractionation experiments and an actin overlay assay demonstrated that deleting domain 4b, but not the N-terminal myosin-binding site, resulted in decreased affinity to both the detergent-insoluble cytoskeleton and soluble actin. Recombinant WT-CaD co-localized with acto-myosin filaments in vivo, but neither of CaD mutants did. Thus both domain 4b and the myosin-binding site are essential for proper localization of CaD in EC. Overexpression of WT-CaD led to cell rounding and formation of a thick peripheral subcortical actin rim in quiescent EC, which correlated with decreased cellular migration. Pharmacological inhibition of p38 MAPK, but not ERK MAPK, caused disassembly of this peripheral actin rim in CaD-transfected cells and decreased CaD phosphorylation at Ser531 (Ser789 in human h-CaD). These results suggest that CaD is critically involved in the regulation of the actin cytoskeleton and migration in EC, and that p38 MAPK-mediated CaD phosphorylation may be involved in endothelial cytoskeletal remodeling.     

Does plasma membrane H+-ATPase activation by fusicoccin involve protein kinase?

Sugar beet ( Beta vulgaris L.) root suspension-cultured cells were converted to protoplasts which responded to fusicoccin (FC) by a rise in cytoplasmic pH (pH_cyt) averaging 0.25 units in the fluorimetric assay. This effect was blocked by erythrosin B, a specific inhibitor of the plasma membrane H₊-ATPase. A protein kinase inhibitor, staurosporine also caused cytosolic alkalinization that was sensitive to H⁺-ATPase inhibitors. Most strikingly, the effect of staurosporine was suppressed by fusicoccin and vice versa. Addition of okadaic acid, entailing overall protein phosphorylation, also led to H⁺-ATPase activation, whereupon fusicoccin lost its effect on proton transport. In parallel, kinetic and inhibitor analyses demonstrated that FC binding to the protoplast plasma membrane involved two sites with dissociation constants of 1 n M and 0.2 μ M and was indifferent to phosphorylation and dephosphorylation inhibitors. Thus, it could be concluded that (1) the effect of FC on cytoplasmic pH probably depends on the phosphorylation state of plasma membrane proteins and may have either sign; (2) the activation of H⁺-ATPase by FC most likely proceeds directly through conformational receptor-enzyme interaction.     

Sex-biased parasitism is not universal: evidence from rodent–flea associations from three biomes

The distribution of parasites among individual hosts is characterised by high variability that is believed to be a result of variations in host traits. To find general patterns of host traits affecting parasite abundance, we studied flea infestation of nine rodent species from three different biomes (temperate zone of central Europe, desert of Middle East and tropics of East Africa). We tested for independent and interactive effects of host sex and body mass on the number of fleas harboured by an individual host while accounting for spatial clustering of host and parasite sampling and temporal variation. We found no consistent patterns of the effect of host sex and body mass on flea abundance either among species within a biome or among biomes. We found evidence for sex-biased flea infestation in just five host species (Apodemus agrarius, Myodes glareolus, Microtus arvalis, Gerbillus andersoni, Mastomys natalensis). In six rodent species, we found an effect of body mass on flea abundance (all species mentioned above and Meriones crassus). This effect was positive in five species and negative in one species (Microtus arvalis). In M. glareolus, G. andersoni, M. natalensis, and M. arvalis, the relationship between body mass and flea abundance was mediated by host sex. This was manifested in steeper change in flea abundance with increasing body mass in male than female individuals (M. glareolus, G. andersoni, M. natalensis), whereas the opposite pattern was found in M. arvalis. Our findings suggest that sex and body mass are common determinants of parasite infestation in mammalian hosts, but neither of them follows universal rules. This implies that the effect of host individual characteristics on mechanisms responsible for flea acquisition may be manifested differently in different host species.     

HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia

Activation of glycolytic genes by HIF-1 is considered critical for metabolic adaptation to hypoxia through increased conversion of glucose to pyruvate and subsequently to lactate. We found that HIF-1 also actively suppresses metabolism through the tricarboxylic acid cycle (TCA) by directly trans-activating the gene encoding pyruvate dehydrogenase kinase 1 (PDK1). PDK1 inactivates the TCA cycle enzyme, pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA. Forced PDK1 expression in hypoxic HIF-1alpha null cells increases ATP levels, attenuates hypoxic ROS generation, and rescues these cells from hypoxia-induced apoptosis. These studies reveal a hypoxia-induced metabolic switch that shunts glucose metabolites from the mitochondria to glycolysis to maintain ATP production and to prevent toxic ROS production.     

Evolved distal tail carbohydrate binding modules of Lactobacillus phage J‐1: a novel type of anti‐receptor widespread among lactic acid bacteria phages

Bacteriophage replication requires specific host‐recognition. Some siphophages harbour a large complex, the baseplate, at the tip of their non‐contractile tail. This baseplate holds receptor binding proteins (RBPs) that can recognize the host cell‐wall polysaccharide (CWPS) and specifically attach the phage to its host. While most phages possess a dedicated RBP, the phage J‐1 that infects Lactobacillus casei seemed to lack one. It has been shown that the phage J‐1 distal tail protein (Dit) plays a role in host recognition and that its sequence comprises two inserted modules compared with ‘classical’ Dits. The first insertion is similar to carbohydrate‐binding modules (CBMs), whereas the second insertion remains undocumented. Here, we determined the structure of the second insertion and found it also similar to several CBMs. Expressed insertion CBM2, but not CBM1, binds to L. casei cells and neutralize phage attachment to the bacterial cell wall and the isolated and purified CWPS of L. casei BL23 prevents CBM2 attachment to the host. Electron microscopy single particle reconstruction of the J‐1 virion baseplate revealed that CBM2 is projected at the periphery of Dit to optimally bind the CWPS receptor. Taken together, these results identify J‐1 evolved Dit as the phage RBP.     

Ceramide synthase 4 and de novo production of ceramides with specific N-acyl chain lengths are involved in glucolipotoxicity-induced apoptosis of INS-1 β-cells

Pancreatic β-cell apoptosis induced by palmitate requires high glucose concentrations. Ceramides have been suggested to be important mediators of glucolipotoxicity-induced β-cell apoptosis. In INS-1 β-cells, 0.4 mM palmitate with 5 mM glucose increased the levels of dihydrosphingosine and dihydroceramides, two lipid intermediates in the de novo biosynthesis of ceramides, without inducing apoptosis. Increasing glucose concentrations to 30 mM amplified palmitate-induced accumulation of dihydrosphingosine and the formation of (dihydro)ceramides. Of note, glucolipotoxicity specifically induced the formation of C(18:0), C(22:0) and C(24:1) (dihydro)ceramide molecular species, which was associated with the up-regulation of CerS4 (ceramide synthase 4) levels. Fumonisin-B1, a ceramide synthase inhibitor, partially blocked apoptosis induced by glucolipotoxicity. In contrast, apoptosis was potentiated in the presence of D,L-threo-1-phenyl-2-palmitoylamino-3-morpholinopropan-1-ol, an inhibitor of glucosylceramide synthase. Moreover, overexpression of CerS4 amplified ceramide production and apoptosis induced by palmitate with 30 mM glucose, whereas down-regulation of CerS4 by siRNA (short interfering RNA) reduced apoptosis. CerS4 also potentiates ceramide accumulation and apoptosis induced by another saturated fatty acid: stearate. Collectively, our results suggest that glucolipotoxicity induces β-cell apoptosis through a dual mechanism involving de novo ceramide biosynthesis and the formation of ceramides with specific N-acyl chain lengths rather than an overall increase in ceramide content.     

An endo-(1→3)-β-d-glucanase from the scallop Chlamys albidus: catalytic properties, cDNA cloning and secondary-structure characterization

An endo-(1→3)-β-d-glucanase (L₀) with molecular mass of 37 kDa was purified to homogeneity from the crystalline style of the scallop Chlamys albidus. The endo-(1→3)-β-d-glucanase was extremely thermolabile with a half-life of 10 min at 37 °C. L₀ hydrolyzed laminaran with K_m ∼ 0.75 mg/mL, and catalyzed effectively transglycosylation reactions with laminaran as donor and p-nitrophenyl β d-glucoside as acceptor (K_m ∼ 2 mg/mL for laminaran) and laminaran as donor and as acceptor (K_m ∼ 5 mg/mL) yielding p-nitrophenyl β d-glucooligosaccharides (n = 2-6) and high-molecular branching (1→3),(1→6)-β-d-glucans, respectively. Efficiency of hydrolysis and transglycosylation processes depended on the substrate structure and decreased appreciably with the increase of the percentage of β-(1→6)-glycosidic bonds, and laminaran with 10% of β-(1→6)-glycosidic bonds was the optimal substrate for both reactions. The CD spectrum of L₀ was characteristic for a protein with prevailing β secondary-structural elements. Binding L₀ with d-glucose as the best acceptor for transglycosylation was investigated by the methods of intrinsic tryptophan fluorescence and CD. Glucose in concentration sufficient to saturate the enzyme binding sites resulted in a red shift in the maximum of fluorescence emission of 1-1.5 nm and quenching the Trp fluorescence up to 50%. An apparent association constant of L₀ with glucose (K_a = 7.4 × 10⁵ ± 1.1 × 10⁵ M⁻¹) and stoichiometry (n = 13.3 ± 0.7) was calculated. The cDNA encoding L₀ was sequenced, and the enzyme was classified in glycoside hydrolases family 16 on the basis of the amino acid sequence similarity.