Differences in DNA double strand breaks repair in male germ cell types: lessons learned from a differential expression of Mdc1 and 53BP1

In male germ cells the repair of DNA double strand breaks (DSBs) differs from that described for somatic cell lines. Irradiation induced immunofluorescent foci (IRIF's) signifying a double strand DNA breaks, were followed in spermatogenic cells up to 16 h after the insult. Foci were characterised for Mdc1, 53BP1 and Rad51 that always were expressed in conjecture with gamma-H2AX. Subsequent spermatogenic cell types were found to have different repair proteins. In early germ cells up to the start of meiotic prophase, i.e. in spermatogonia and preleptotene spermatocytes, 53BP1 and Rad51 are available but no Mdc1 is expressed in these cells before and after irradiation. The latter might explain the radiosensitivity of spermatogonia. Spermatocytes from shortly after premeiotic S-phase till pachytene in epithelial stage IV/V express Mdc1 and Rad51 but no 53BP1 which has no role in recombination involved repair during the early meiotic prophase. Mdc1 is required during this period as in Mdc1 deficient mice all spermatocytes enter apoptosis in epithelial stage IV when they should start mid-pachytene phase of the meiotic prophase. From stage IV mid pachytene spermatocytes to round spermatids, Mdc1 and 53BP1 are expressed while Rad51 is no longer expressed in the haploid round spermatids. Quantifying foci numbers of gamma-H2AX, Mdc1 and 53BP1 at various time points after irradiation revealed a 70% reduction after 16 h in pachytene and diplotene spermatocytes and round spermatids. Although the DSB repair efficiency is higher then in spermatogonia where only a 40% reduction was found, it still does not compare to somatic cell lines where a 70% reduction occurs in 2 h. Taken together, DNA DSBs repair proteins differ for the various types of spermatogenic cells, no germ cell type possessing the complete set. This likely leads to a compromised efficiency relative to somatic cell lines. From the evolutionary point of view it may be an advantage when germ cells die from DNA damage rather than risk the acquisition of transmittable errors made during the repair process.     

S. cerevisiae Srs2 helicase ensures normal recombination intermediate metabolism during meiosis and prevents accumulation of Rad51 aggregates

We investigated the meiotic role of Srs2, a multi-functional DNA helicase/translocase that destabilises Rad51-DNA filaments and is thought to regulate strand invasion and prevent hyper-recombination during the mitotic cell cycle. We find that Srs2 activity is required for normal meiotic progression and spore viability. A significant fraction of srs2 mutant cells progress through both meiotic divisions without separating the bulk of their chromatin, although in such cells sister centromeres often separate. Undivided nuclei contain aggregates of Rad51 colocalised with the ssDNA-binding protein RPA, suggesting the presence of persistent single-strand DNA. Rad51 aggregate formation requires Spo11-induced DSBs, Rad51 strand-invasion activity and progression past the pachytene stage of meiosis, but not the DSB end-resection or the bias towards interhomologue strand invasion characteristic of normal meiosis. srs2 mutants also display altered meiotic recombination intermediate metabolism, revealed by defects in the formation of stable joint molecules. We suggest that Srs2, by limiting Rad51 accumulation on DNA, prevents the formation of aberrant recombination intermediates that otherwise would persist and interfere with normal chromosome segregation and nuclear division.

     

Identification of structural transitions in bacterial fatty acid binding proteins that permit ligand entry and exit at membranes

Fatty acid (FA) transfer proteins extract FA from membranes and sequester them to facilitate their movement through the cytosol. Detailed structural information is available for these soluble protein–FA complexes, but the structure of the protein conformation responsible for FA exchange at the membrane is unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we define the conformational change from a “closed” FakB1 state to an “open” state that associates with the membrane and provides a path for entry and egress of the FA. Using NMR spectroscopy, we identified a conformationally flexible dynamic region in FakB1, and X-ray crystallography of FakB1 mutants captured the conformation of the open state. In addition, molecular dynamics simulations show that the new amphipathic α-helix formed in the open state inserts below the phosphate plane of the bilayer to create a diffusion channel for the hydrophobic FA tail to access the hydrocarbon core and place the carboxyl group at the phosphate layer. The membrane binding and catalytic properties of site-directed mutants were consistent with the proposed membrane docked structure predicted by our molecular dynamics simulations. Finally, the structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a conceptual framework for how these proteins interact with the membrane to create a diffusion channel from the FA location in the bilayer to the protein interior.     

Inhibitor of apoptosis proteins are substrates for the mitochondrial serine protease Omi/HtrA2

The mature serine protease Omi/HtrA2 is released from the mitochondria into the cytosol during apoptosis. Suppression of Omi/HtrA2 by RNA interference in human cell lines reduces cell death in response to TRAIL and etoposide. In contrast, ectopic expression of mature wildtype Omi/HtrA2, but not an active site mutant, induces potent caspase activation and apoptosis. In vitro assays demonstrated that Omi/HtrA2 could degrade inhibitor of apoptosis proteins (IAPs). Consistent with this observation, increased expression of Omi/HtrA2 in cells increases degradation of XIAP, while suppression of Omi/HtrA2 by RNA interference has an opposite effect. Combined, our data demonstrate that IAPs are substrates for Omi/HtrA2, and their degradation could be a mechanism by which the mitochondrially released Omi/HtrA2 activates caspases during apoptosis.     

Unique conformer selection of human growth-regulatory lectin galectin-1 for ganglioside GM1 versus bacterial toxins

Endogenous lectins induce effects on cell growth by binding to antennae of natural glycoconjugates. These complex carbohydrates often present more than one potential lectin-binding site in a single chain. Using the growth-regulatory interaction of the pentasaccharide of ganglioside GM(1) with homodimeric galectin-1 on neuroblastoma cell surfaces as a model, we present a suitable strategy for addressing this issue. The approach combines NMR spectroscopic and computational methods and does not require isotope-labeled glycans. It involves conformational analysis of the two building blocks of the GM(1) glycan, i.e., the disaccharide Galbeta1-3GalNAc and the trisaccharide Neu5Acalpha2-3Galbeta1-4Glc. Their bound-state conformations were determined by transferred nuclear Overhauser enhancement spectroscopy. Next, measurements on the lectin-pentasaccharide complex revealed differential conformer selection regarding the sialylgalactose linkage in the tri- versus pentasaccharide (Phi and Psi value of -70 degrees and 15 degrees vs 70 degrees and 15 degrees, respectively). To proceed in the structural analysis, the characteristic experimentally detected spatial vicinity of a galactose unit and Trp68 in the galectin's binding site offered a means, exploiting saturation transfer from protein to carbohydrate protons. Indeed, we detected two signals unambiguously assigned to the terminal Gal and the GalNAc residues. Computational docking and interaction energy analyses of the entire set of ligands supported and added to experimental results. The finding that the ganglioside's carbohydrate chain is subject to differential conformer selection at the sialylgalactose linkage by galectin-1 and GM(1)-binding cholera toxin (Phi and Psi values of -172 degrees and -26 degrees, respectively) is relevant for toxin-directed drug design. In principle, our methodology can be applied in studies aimed at blocking galectin functionality in malignancy and beyond glycosciences.     

Characterization of a novel and specific inhibitor for the pro-apoptotic protease Omi/HtrA2

Omi/HtrA2 is a mammalian serine protease with high homology to bacterial HtrA chaperones. Omi/HtrA2 is localized in mitochondria and is released to the cytoplasm in response to apoptotic stimuli. Omi/HtrA2 induces cell death in a caspase-dependent manner by interacting with the inhibitor of apoptosis protein as well as in a caspase-independent manner that relies on its protease activity. We describe the identification and characterization of a novel compound as a specific inhibitor of the proteolytic activity of Omi/HtrA2. This compound (ucf-101) was isolated in a high throughput screening of a combinatorial library using bacterially made Omi-(134-458) protease and fluorescein-casein as a generic substrate. ucf-101 showed specific activity against Omi/HtrA2 and very little activity against various other serine proteases. This compound has a natural fluorescence that was used to monitor its ability to enter mammalian cells. ucf-101, when tested in caspase-9 (-/-) null fibroblasts, was found to inhibit Omi/HtrA2-induced cell death.     

Pressure-induced water transport in membrane channels studied by molecular dynamics

A method is proposed to measure the water permeability of membrane channels by means of molecular dynamics simulations. By applying a constant force to the bulk water molecules and a counter force on the complementary system, a hydrostatic pressure difference across the membrane can be established, producing a net directional water flow. The hydraulic or osmotic permeability can then be determined by the ratio of the water flux and the pressure difference. The method is applied and tested on an aquaglyceroporin channel through a series of simulations totaling 5 ns in duration.     

The relation of omentin gene expression and glucose homeostasis of visceral and subcutaneous adipose tissues in non-diabetic adults

Molecular Biology Reports - Adipose tissue (AT) is a passive reservoir for energy storage and an active endocrine organ responsible for synthesizing bioactive molecules called...     

Ocean resource use: building the coastal blue economy

Reviews in Fish Biology and Fisheries - Humans have relied on coastal resources for centuries. However, current growth in population and increased accessibility of coastal resources through...