Strategy for membrane protein crystallization exemplified with OmpA and OmpX

The bacterial outer membrane proteins OmpA and OmpX were modified in such a manner that they yielded bulky crystals diffracting X-rays isotropically beyond 2 A resolution and permitting detailed structural analyses. The procedure involved semi-directed mutagenesis, mass production into inclusion bodies, and (re)naturation therefrom; it should be applicable for a broader range of membrane proteins.     

Life stages and reproductive components of the Marmorkrebs (marbled crayfish), the first parthenogenetic decapod crustacean

Recently, we briefly reported on the first case of parthenogenesis in the decapod Crustacea which was found in the Marmorkrebs or marbled crayfish, a cambarid species of unknown geographic origin and species identity. Curiously, this animal is known only from aquarium populations, where it explosively propagates. By means of light and electron microscopic techniques we have now investigated the reproductive components of this crayfish, using more than 100 specimens ranging from hatchling to repeatedly spawned adult. Additionally, we documented its principal life stages. Our results revealed that the external sexual characters and also the gonads of the marbled crayfish are purely female, making this fast-reproducing species a good model for investigating female reproductive features in crayfish. Testicular tissues, ovotestes, or male gonoducts, gonopores, or gonopods were never found, either in small juveniles or large adult specimens, confirming the parthenogenetic nature of this crayfish. Parthenogenesis may have arisen spontaneously or by interspecific hybridization since Wolbachia-like feminizing microorganisms were not found in the ovaries. The external sexual characters of the marbled crayfish are first recognized in Stage 4 juveniles and are structurally complete approximately 2 months after hatching in specimens of approximately 2 cm total length. In the same life stage the ovary is fully differentiated as well, although the oocytes are in previtellogenic and primary vitellogenic stages only. The architecture of the mature ovary and also the synchronous maturation of cohorts of primary vitellogenic oocytes by secondary vitellogenesis are in general agreement with data published on ovaries of bisexual crayfish. New results were obtained with respect to the muscular nature of the ovarian envelope and its extensive proliferation after the first spawning, the distribution of hemal sinuses in the ovarian envelope and in the interstitium around the oogenetic pouches, the high transport activity of the follicle cells, and the colonization of oogenetic pouches by previtellogenic oocytes that originate in the germaria. Investigation of the nuclei of oocytes in the germaria and oogenetic pouches revealed no signs of meiosis, as usually found in females of bisexual decapods, suggesting that parthenogenesis in the marbled crayfish might be an apomictic thelytoky. The detection of new rickettsial and coccidian infections in the ovary and further organs raises fears that the marbled crayfish might endanger native European species by transmission of pathogens once escaped into the wild.     

Role of zinc in regulating the levels of hepatic elements following nickel toxicity in rats

This study was designed to determine the protective effects of zinc on the hepatotoxicity induced by nickel in rats. Female Sprague-Dawley (SD) rats received either nickel sulfate alone in the dose of 800 mg/L nickel in drinking water, zinc sulfate alone in the dose of 227 mg/L zinc in drinking water, and nickel plus zinc or drinking water alone for a total duration of 8 wk. The effects of different treatments were studied on activities of rat liver marker enzymes like alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferases (AST) and on the status of essential elements in rat liver. The study revealed a significant increase in the activities of enzymes ALP and ALT in rats subjected to nickel treatment. Interestingly, zinc supplementation to rats treated with nickel brought back the raised activities of these enzymes to within normal limits. Further, the levels of elements in liver that include zinc, copper, selenium, and potassium were found to be significantly suppressed following nickel treatment, whereas the levels of iron and sulfur were elevated. However, zinc treatment alone did not cause any appreciable change in the concentration of these elements. To the contrary, when zinc was given to nickel-treated rats, the concentrations of zinc, copper, potassium, and phosphorus were not significantly different from that of normal controls, whereas the levels of iron, selenium, and sulfur were improved in comparison to nickel-treated rats but were not within the normal limits. The present study concludes that zinc has the ability to maintain the levels of hepatic elements and has bearing in regulating the liver functions by maintaining the activities of marker enzymes in conditions of nickel toxicity.     

Activation of heterotrimeric G proteins by a high energy phosphate transfer via nucleoside diphosphate kinase (NDPK) B and Gbeta subunits. Specific activation of Gsalpha by an NDPK B.Gbetagamma complex in H10 cells

Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to G protein activation in vitro. To study the effect of NDPK on G protein activity in living cells, the NDPK isoforms A and B were stably expressed in H10 cells, a cell line derived from neonatal rat cardiomyocytes. Overexpression of either NDPK isoform had no effect on cellular GTP and ATP levels, basal cAMP levels, basal adenylyl cyclase activity, and the expression of G(s)alpha and G(i)alpha proteins. However, co-expression of G(s)alpha led to an increase in cAMP synthesis that was largely enhanced by the expression of NDPK B, but not NDPK A, and that was confirmed by direct measurement of adenylyl cyclase activity. Cells expressing an inactive NDPK B mutant (H118N) exhibited a decreased cAMP formation in response to G(s)alpha. Co-immunoprecipitation studies demonstrated a complex formation of the NDPK with Gbetagamma dimers. The overexpression of NDPK B, but not its inactive mutant or NDPK A, increased the phosphorylation of Gbeta subunits. In summary, our data demonstrate a specific NDPK B-mediated activation of a G protein in intact cells, which is apparently caused by formation of NDPK B.Gbetagamma complexes and which appears to contribute to the receptor-independent activation of heterotrimeric G proteins.     

Functional studies on native and mutated forms of perilipins. A role in protein kinase A-mediated lipolysis of triacylglycerols

Perilipin A coats the lipid storage droplets in adipocytes and is polyphosphorylated by protein kinase A (PKA); the fact that PKA activates lipolysis in adipocytes suggests a role for perilipins in this process. To assess whether perilipins participate directly in PKA-mediated lipolysis, we have expressed constructs coding for native and mutated forms of the two major splice variants of the perilipin gene, perilipins A and B, in Chinese hamster ovary fibroblasts. Perilipins localize to lipid droplet surfaces and displace the adipose differentiation-related protein that normally coats the droplets in these cells. Perilipin A inhibits triacylglycerol hydrolysis by 87% when PKA is quiescent, but activation of PKA and phosphorylation of perilipin A engenders a 7-fold lipolytic activation. Mutation of PKA sites within the N-terminal region of perilipin abrogates the PKA-mediated lipolytic response. In contrast, perilipin B exerts only minimal protection against lipolysis and is unresponsive to PKA activation. Since Chinese hamster ovary cells contain no PKA-activated lipase, we conclude that the expression of perilipin A alone is sufficient to confer PKA-mediated lipolysis in these cells. Moreover, the data indicate that the unique C-terminal portion of perilipin A is responsible for its protection against lipolysis and that phosphorylation at the N-terminal PKA sites attenuates this protective effect.     

The promyelocytic leukemia protein protects p53 from Mdm2-mediated inhibition and degradation

The p53 protein is kept labile under normal conditions. This regulation is governed largely by its major negative regulator, Mdm2. In response to stress however, p53 accumulates and becomes activated. For this to occur, the inhibitory effects of Mdm2 have to be neutralized. Here we investigated the role of the promyelocytic leukemia protein (PML) in the activation of p53 in response to stress. We found that PML is critical for the accumulation of p53 in response to DNA damage under physiological conditions. PML protects p53 from Mdm2-mediated ubiquitination and degradation, and from inhibition of apoptosis. PML neutralizes the inhibitory effects of Mdm2 by prolonging the stress-induced phosphorylation of p53 on serine 20, a site of the checkpoint kinase 2 (Chk2). PML recruits Chk2 and p53 into the PML nuclear bodies and enhances p53/Chk2 interaction. Our results provide a novel mechanistic explanation for the cooperation between PML and p53 in response to DNA damage.     

The organization of mature Rous sarcoma virus as studied by cryoelectron microscopy

We have studied the organization of mature infectious Rous sarcoma virus (RSV), suspended in vitreous ice, using transmission electron microscopy. The enveloped virions are spherical in shape, have a mean diameter of 127 nm, and vary significantly in size. Image processing reveals the presence of the viral matrix protein underlying the lipid bilayer and the viral envelope proteins external to the lipid bilayer. In the interior of the virus, the characteristic mature retroviral core is clearly imaged. In contrast to lentiviruses, such as human immunodeficiency virus, the core of RSV is essentially isometric. The capsid, or external shell of the core, has a faceted, almost polygonal appearance in electron micrographs, but many capsids also exhibit continuous surface curvature. Cores are not uniform in size or shape. Serrations observed along the projected faces of the core suggest a repetitive molecular structure. Some isolated cores were observed in the sample, confirming that cores are at least transiently stable in the absence of the viral envelope. Using an approach grounded in geometric probability, we estimate the size of the viral core from the projection data. We show that the size of the core is not tightly controlled and that core size and virion size are positively correlated. From estimates of RNA packing density we conclude that either the RNA within the core is loosely packed or, more probably, that it does not fill the core.     

Regulation of inositol phospholipid binding and signaling through syndecan-4

Syndecan-4 is a transmembrane heparan sulfate proteoglycan that can regulate cell-matrix interactions and is enriched in focal adhesions. Its cytoplasmic domain contains a central region unlike that of any other vertebrate or invertebrate syndecan core protein with a cationic motif that binds inositol phospholipids. In turn, lipid binding stabilizes the syndecan in oligomeric form, with subsequent binding and activation of protein kinase C. The specificity of phospholipid binding and its potential regulation are investigated here. Highest affinity of the syndecan-4 cytoplasmic domain was seen with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5P)(2)) and phosphatidylinositol 4-phosphate, and both promoted syndecan-4 oligomerization. Affinity was much reduced for 3-phosphorylated inositides while no binding of diacylglycerol was detected. Syndecan-2 cytoplasmic domain had negligible affinity for any lipid examined. Inositol hexakisphosphate, but not inositol tetrakisphosphate, also had high affinity for the syndecan-4 cytoplasmic domain and could compete effectively with PtdIns(4,5)P(2). Since inositol hexaphosphate binding to syndecan-4 does not promote oligomer formation, it is a potential down-regulator of syndecan-4 signaling. Similarly, phosphorylation of serine 183 in syndecan-4 cytoplasmic domain reduced PtdIns(4,5)P(2) binding affinity by over 100-fold, although interaction could still be detected by nuclear magnetic resonance spectroscopy. Only protein kinase Calpha was up-regulated in activity by the combination of syndecan-4 and PtdIns(4,5)P(2), with all other isoforms tested showing minimal response. This is consistent with the codistribution of syndecan-4 with the alpha isoform of protein kinase C in focal adhesions.