Histone deacetylase 4 interacts with 53BP1 to mediate the DNA damage response

Anumber of proteins are recruited to nuclear foci upon exposure to double-strand DNA damage, including 53BP1 and Rad51, but the precise role of these DNA damage-induced foci remain unclear. Here we show in a variety of human cell lines that histone deacetylase (HDAC) 4 is recruited to foci with kinetics similar to, and colocalizes with, 53BP1 after exposure to agents causing double-stranded DNA breaks. HDAC4 foci gradually disappeared in repair-proficient cells but persisted in repair-deficient cell lines or cells irradiated with a lethal dose, suggesting that resolution of HDAC4 foci is linked to repair. Silencing of HDAC4 via RNA interference surprisingly also decreased levels of 53BP1 protein, abrogated the DNA damage-induced G2 delay, and radiosensitized HeLa cells. Our combined results suggest that HDAC4 is a critical component of the DNA damage response pathway that acts through 53BP1 and perhaps contributes in maintaining the G2 cell cycle checkpoint.     

Poly(ADP-ribose) polymerase-1: association with nuclear lamins in rodent liver cells

The distribution of poly(ADP-ribose) polymerase-1 (PARP-1) over different nuclear compartments was studied by nuclear fractionation procedures and Western analysis revealing a prominent role of the nuclear matrix. This structure is operationally defined by the solubility properties of the A- and B-type lamins under defined experimental conditions. We consistently observed that most of the nuclear matrix-associated PARP-1 partitioned, in an active form, with the insoluble, lamin-enriched protein fractions that were prepared by a variety of established biochemical procedures. These PARP-1-protein interactions resisted salt extraction, disulfide reduction, RNase and DNase digestion. An inherent ability of PARP-1 to reassemble with the lamins became evident after a cycle of solubilization/dialysis using either urea or Triton X-100 and disulfide reduction, indicating that these interactions were dominated by hydrophobic forces. Together with in vivo crosslinking and co-immunoprecipitation experiments our results show that the lamins are prominent PARP-1-binding partners which could contribute to the functional sequestration of the enzyme on the nuclear matrix.     

Naive and memory T lymphocytes migrate in comparable numbers through normal rat liver: activated T cells accumulate in the periportal field.

Although the liver is known to contain a significant number of lymphocytes, migration of these through the compartments of the liver, parenchyma and periportal field, has not been studied. The periportal field, in particular, is affected in several immunological disorders of the liver. Populations of labeled naive, activated, and memory T cells were injected into congenic rats. The recipient livers and draining lymph nodes were removed at various time points, and cryostat sections were analyzed for the presence of donor cells using quantitative immunohistology. Donor cell proliferation and apoptosis were examined in vivo by BrdU (5 microM 5-bromo-2-deoxyuridine) incorporation and the TUNEL technique, respectively. Early after injection (0.5-1 h), naive, activated, and memory T cells were localized to the parenchyma and periportal field in comparable numbers. With time, all T cell subsets left the parenchyma but remained or, in the case of activated T cells, significantly accumulated in the periportal field. Furthermore, 12% of activated donor T cells proliferated in vivo within the periportal field, and 0.5% showed evidence of apoptosis. Taken together, not only activated and memory, but also naive T cells continuously migrate through the liver, showing a preference for the periportal field, and activated T cells mainly proliferate there. This may explain why many immunological liver diseases predominantly affect the periportal field.     

CnOtx, a member of the Otx gene family, has a role in cell movement in hydra.

Otx genes have been identified in a variety of organisms and are commonly associated with the patterning of anterior structures. In some vertebrates, Otx genes are also expressed in the prechordal mesoderm, where they may have a role in cell movement. Here we report the characterization of CnOtx, an Otx gene in hydra, thereby providing evidence that Otx genes appeared early in metazoan evolution. CnOtx is expressed at high levels in developing buds and aggregates, where it appears to have a role in the cell movements that are involved in the formation of new axes. Further, the gene is expressed at a low level throughout the body column of hydra. This latter pattern may reflect a role for CnOtx in specifying tissue as competent to be anterior, although the gene does not have a direct role in the formation of the head.     

Identification of phosphatidylserine decarboxylases 1 and 2 from Pichia pastoris

Genetic manipulation of lipid biosynthetic enzymes allows modification of cellular membranes. We made use of this strategy and constructed mutants in phospholipid metabolism of Pichia pastoris , which is widely used in biotechnology for expression of heterologous proteins. Here we describe identification of two P. pastoris phosphatidylserine decarboxylases (PSDs) encoded by genes homologous to PSD1 and PSD2 from Saccharomyces cerevisiae . Using P. pastoris psd1 Δ and psd2 Δ mutants we investigated the contribution of the respective gene products to phosphatidylethanolamine synthesis, membrane composition and cell growth. Deletion of PSD1 caused loss of PSD activity in mitochondria, a severe growth defect on minimal media and depletion of cellular and mitochondrial phosphatidylethanolamine levels. This defect could not be compensated by Psd2p, but by supplementation with ethanolamine, which is the substrate for the cytidine diphosphate (CDP)–ethanolamine pathway, the third route of phosphatidylethanolamine synthesis in yeast. Fatty acid analysis showed selectivity of both Psd1p and Psd2p in vivo for the synthesis of unsaturated phosphatidylethanolamine species. Phosphatidylethanolamine species containing palmitic acid (16:0), however, were preferentially assembled into mitochondria. In summary, this study provides first insight into membrane manipulation of P. pastoris , which may serve as a useful method to modify cell biological properties of this microorganism for biotechnological purposes.     

Secretion and differential localization of the proteolytic cleavage products Abeta40 and Abeta42 of the Alzheimer amyloid precursor protein in human fetal myogenic cells

Abeta peptides are major components of the amyloid plaques that characterize Alzheimer's disease. These peptides are proteolytic cleavage products of the amyloid precursor protein (APP) and are generated by beta- and gamma-secretases. Here we show by multiparameter immunofluorescence imaging in muscle cells that localization of the Abeta40 and Abeta42 cleavage products reveals different myocyte types in a three-dimensional culture system. These myocyte types are heterogeneous by selective intracellular concentration of either Abeta40 or Abeta42 in vesicular structures, whilst only the Abeta40 peptide is secreted as indicated by Western blot analysis. This cellular pattern of APP proteolysis and Abeta peptide secretion correlates with lack of L-APP mRNA splice isoforms. Differential secretion and intracellular accumulation of Abeta peptides is characteristic for the early myocyte development and might be related to cell fusion.     

Refined 2 A X-ray crystal structure of porcine pancreatic kallikrein A, a specific trypsin-like serine proteinase. Crystallization, structure determination, crystallographic refinement, structure and its comparison with bovine trypsin

Porcine pancreas kallikrein A has been crystallized in the presence of the small inhibitor benzamidine, yielding tetragonal crystals of space group P4₁2₁2 containing two molecules per asymmetric unit. X-ray data up to 2·05 Å resolution have been collected using normal rotation anode as well as synchrotron radiation. The crystal structure of benzamidine-kallikrein has been determined using multiple isomorphous replacement techniques, and has subsequently been refined to a crystallographic R-value of 0·220 by applying a diagonal matrix least-squares energy constraint refinement procedure.Both crystallographically independent kallikrein molecules 1 and 2 are related by a non-integral screw axis and form open, heterologous “dimer” structures. The root-mean-square deviation of both molecules is 0·37 Å for all main-chain atoms. This value is above the estimated mean positional error of about 0·2 Å and reflects some significant conformational differences, especially at surface loops. The binding site of molecule 1 in the asymmetric unit is in contact with residues of molecule 2, whereas the binding site of the latter is free and accessible to the solvent. In both molecules the characteristic “kallikrein loop”, where the peptide chain of kallikrein A is cleaved, is only partially traceable. The carbohydrate attached to Asn95 in this loop, although detectable chemically, is not defined.A comparison of the refined structures of porcine kallikrein and bovine trypsin indicates spatial homology for these enzymes. The root-mean-square difference is 0·68 Å if we compare only main-chain atoms of internal segments. Remarkably large deviations are found in some external loops most of which surround the binding site and form a more compact rampart around it in kallikrein than in trypsin. This feature might explain the strongly reduced activity and accessibility of kallikrein towards large protein substrates and inhibitors (e.g. as shown by the model-building experiments on inhibitor complexes reported by Chen &; Bode. 1983).The conformation of the active site residues is very similar in both enzymes. Tyr99 of kallikrein, which is a leucyl residue in trypsin, protrudes into the binding site and interferes with the binding of peptide substrates (Chen &; Bode. 1983). The kallikrein specificity pocket is significantly enlarged compared with trypsin due to a longer peptide segment, 217 to 220, and to the unique outwards orientation of the carbonyl group of cis-Pro219. Further, the side-chain of Ser226 in porcine kallikrein, which is a glycyl residue in trypsin, partially covers Asp 189 at the bottom of the pocket. These features considerably affect the binding geometry and strength of binding of benzamidine.     

Surface microtopographies of tropical sea stars: lack of an efficient physical defence mechanism against fouling

Abstract

The role of surface topography as a defence against fouling in tropical sea stars was investigated. The sea stars Linckia laevigata, Fromia indica, Cryptasterina pentagona and Archaster typicus are not fouled and have paxillae (modified ossicles with a median vertical pillar) on their aboral surfaces, which varied in diameter, height and distance depending on species and position on the aboral surface, providing unique and complex surface microtopographies for each species. The surfaces of the sea stars L. laevigata, F. indica and A. typicus were moderately wettable, with their mean seawater contact angles, calculated from captive bubble measurements, being 60.1°, 70.3° and 57.3°, respectively. The seawater contact angle of C. pentagona could not be measured. To evaluate the effectiveness of the surface microtopographies in deterring the settlement of fouling organisms, field experiments with resin replicas of the four sea star species were conducted at three sites around Townsville, Australia, for 8 weeks during the dry and wet seasons. The fouling community and total fouling cover did not differ significantly between replicas of L. laevigata, F. indica, C. pentagona, A. typicus and control surfaces at any site during the dry season. Significant differences between fouling communities on the replicas of the sea stars and control surfaces were detected at two sites during the wet season. However, these differences were transitory, and the total fouling cover did not differ significantly between replicas of sea stars and control surfaces at two of the three sites. In contrast to recent literature on the effects of biofouling control by natural surfaces in the marine environment, the surface microtopographies of tropical sea stars alone were not effective in deterring the settlement and growth of fouling organisms.     

Novel fluorescent prothrombin analogs as probes of staphylocoagulase-prothrombin interactions

Staphylocoagulase (SC) is a potent nonproteolytic prothrombin (ProT) activator and the prototype of a newly established zymogen activator and adhesion protein family. The staphylocoagulase fragment containing residues 1-325 (SC-(1-325)) represents a new type of nonproteolytic activator with a unique fold consisting of two three-helix bundle domains. The N-terminal, domain 1 of SC (D1, residues 1-146) interacts with the 148 loop of thrombin and prethrombin 2 and the south rim of the catalytic site, whereas domain 2 of SC (D2, residues 147-325) occupies (pro)exosite I, the fibrinogen (Fbg) recognition exosite. Reversible conformational activation of ProT by SC-(1-325) was used to create novel analogs of ProT covalently labeled at the catalytic site with fluorescence probes. Analogs selected from screening 10 such derivatives were used to characterize quantitatively equilibrium binding of SC-(1-325) to ProT, competitive binding with native ProT, and SC domain interactions. The results support the conclusion that SC-(1-325) binds to a single site on fluorescein-labeled and native ProT with indistinguishable dissociation constants of 17-72 pM. The results obtained for isolated SC domains indicate that D2 binds ProT with approximately 130-fold greater affinity than D1, yet D1 binding accounts for the majority of the fluorescence enhancement that accompanies SC-(1-325) binding. The SC-(1-325).(pro)thrombin complexes and free thrombin showed little difference in substrate specificity for tripeptide substrates or with their natural substrate, Fbg. Lack of a significant effect of blockage of (pro)exosite I of (pro)thrombin by SC-(1-325) on Fbg cleavage indicates that a new Fbg substrate recognition exosite is expressed on the SC-(1-325).(pro)thrombin complexes. Our results provide new insight into the mechanism that mediates zymogen activation by this prototypical bacterial activator.