Closed state of both binding domains of homodimeric mGlu receptors is required for full activity

Membrane receptors, key components in signal transduction, often function as dimers. These include some G protein-coupled receptors such as metabotropic glutamate (mGlu) receptors that have large extracellular domains (ECDs) where agonists bind. How agonist binding in dimeric ECDs activates the effector domains remains largely unknown. The structure of the dimeric ECDs of mGlu(1) solved in the presence of agonist revealed two specific conformations in which either one or both protomers are in an agonist-stabilized closed form. Here we examined whether both conformations correspond to an active form of the full-length receptor. Using a system that allows the formation of dimers made of a wild-type and a mutant subunit, we show that the closure of one ECD per dimer is sufficient to activate the receptor, but the closure of both ECDs is required for full activity.     

Prostaglandin E(2) metabolism is activated in Schwann cell lines derived from human NF1 malignant peripheral nerve sheath tumors

Malignant peripheral nerve sheath tumors (MPNSTs) are characteristic of Neurofibromatosis type 1 (NF1), a human genetic disorder affecting approximately 1 in 3000 individuals. The absence of neurofibromin in Schwann cells results in hyperactivation of Ras, which contributes to Schwann cell hyperplasia. However, additional intracellular abnormalities in Schwann cells might contribute to the malignancy. We now report that cell lines derived from MPNSTs secrete elevated levels of prostaglandin E(2) (PGE(2)), express higher levels of phosphorylated mitogen-activated protein kinase (MAPK), phosphorylated cytosolic phospholipaseA(2) (cPLA(2)) and cyclooxygenase 2 (COX-2) when compared to normal adult human Schwann cells (nhSCs). PCR analysis reveals that NF1 MPNST cell lines express mRNA for both EP2 and EP4 prostaglandin E2 receptors, whereas nhSCs express only the EP4 receptor. COX-2 inhibitors and PGE(2) receptor antagonists decrease the proliferation of MPNST cell lines. These results indicate that prostaglandin metabolism is activated in MPNSTs and might contribute to tumor growth in NF1.     

Oxidized mono-, di-, tri-, and polysaccharides as potential hemoglobin cross-linking reagents for the synthesis of high oxygen affinity artificial blood substitutes

Various oxidized mono/di/tri/poly saccharides were studied as potential hemoglobin (Hb) cross-linkers in order to produce oxygen carriers with high oxygen affinities (low P(50)'s) and high molecular weights (therefore lower macromolecular diffusivities compared to tetrameric Hb). Such physical properties were desired to produce polymerized hemoglobins (PolyHbs) with oxygen release profiles similar to that of human blood, as was demonstrated in work by Winslow (1). In this present study, bovine hemoglobin was cross-linked with a variety of oxidized (ring-opened) saccharides, which resulted in cross-linked Hb species ranging in size from 64 to 6400 kDa (depending on the particular oxidized saccharide used in the reaction) and P(50)'s ranging from 6 to 15 mmHg. A parallel synthetic approach was used to synthesize these carbohydrate-hemoglobin conjugates, and asymmetric flow field-flow fractionation (AFFF) coupled with multi-angle static light scattering (MASLS) was used to measure the absolute molecular weight distribution of these PolyHb dispersions. Cross-linking reactions were conducted at two pHs (6 and 8), with larger cross-linked Hb species produced at pH 8 (where hydrolysis was most likely to occur between glycosidic bonds linking adjacent saccharide rings) rather than at pH 6. The largest molecular weight species formed from these reactions consisted of Hb cross-linked with ring-opened lactose, maltose, methylglucopyranoside, sucrose, trehalose, and 15 kDa and 71 kDa dextran at high pH (pH 8). The most promising Hb cross-linker was methylglucopyranoside, which resulted in very large cross-linked Hb species, with low P(50)'s and lower methemoglobin (metHb) levels compared to the other Hb cross-linking reagents.     

Influence of form IA RubisCO and environmental dissolved inorganic carbon on the delta13C of the clam-chemoautotroph symbiosis Solemya velum

Many nutritive symbioses between chemoautotrophic bacteria and invertebrates, such as Solemya velum, have delta(13)C values of approximately -30 to -35%, considerably more depleted than phytoplankton. Most of the chemoautotrophic symbionts fix carbon with a form IA ribulose 1,5-bisphosphate carboxylase (RubisCO). We hypothesized that this form of RubisCO discriminates against (13)CO(2) to a greater extent than other forms. Solemya velum symbiont RubisCO was cloned and expressed in Escherichia coli, purified and characterized. Enzyme from this recombinant system fixed carbon most rapidly at pH 7.5 and 20-25 degrees C. Surprisingly, this RubisCO had an epsilon-value (proportional to the degree to which the enzyme discriminates against (13)CO(2)) of 24.4 per thousand, similar to form IB RubisCOs, and higher than form II RubisCOs. Samples of interstitial water from S. velum's habitat were collected to determine whether the dissolved inorganic carbon (DIC) could contribute to the negative delta(13)C values. Solemya velum habitat DIC was present at high concentrations (up to approximately 5 mM) and isotopically depleted, with delta(13)C values as low as approximately -6%. Thus environmental DIC, coupled with a high degree of isotopic fractionation by symbiont RubisCO likely contribute to the isotopically depleted delta(13)C values of S. velum biomass, highlighting the necessity of considering factors at all levels (from environmental to enzymatic) in interpreting stable isotope ratios.     

Adenylyl cyclase-associated protein Aca1 regulates virulence and differentiation of Cryptococcus neoformans via the cyclic AMP-protein kinase A cascade

The evolutionarily conserved cyclic AMP (cAMP) signaling pathway controls cell functions in response to environmental cues in organisms as diverse as yeast and mammals. In the basidiomycetous human pathogenic fungus Cryptococcus neoformans, the cAMP pathway governs virulence and morphological differentiation. Here we identified and characterized adenylyl cyclase-associated protein, Aca1, which functions in parallel with the Galpha subunit Gpa1 to control the adenylyl cyclase (Cac1). Aca1 interacted with the C terminus of Cac1 in the yeast two-hybrid system. By molecular and genetic approaches, Aca1 was shown to play a critical role in mating by regulating cell fusion and filamentous growth in a cAMP-dependent manner. Aca1 also regulates melanin and capsule production via the Cac1-cAMP-protein kinase A pathway. Genetic epistasis studies support models in which Aca1 and Gpa1 are necessary and sufficient components that cooperate to activate adenylyl cyclase. Taken together, these studies further define the cAMP signaling cascade controlling virulence of this ubiquitous human fungal pathogen.     

Promiscuous protein biotinylation by Escherichia coli biotin protein ligase

Biotin protein ligases (BPLs) are enzymes of extraordinary specificity. BirA, the BPL of Escherichia coli biotinylates only a single cellular protein. We report a mutant BirA that attaches biotin to a large number of cellular proteins in vivo and to bovine serum albumin, chloramphenicol acetyltransferase, immunoglobin heavy and light chains, and RNAse A in vitro. The mutant BirA also self biotinylates in vivo and in vitro. The wild type BirA protein is much less active in these reactions. The biotinylation reaction is proximity-dependent in that a greater extent of biotinylation was seen when the mutant ligase was coupled to the acceptor proteins than when the acceptors were free in solution. This approach may permit facile detection and recovery of interacting proteins by existing avidin/streptavidin technology.     

Listeria monocytogenes actin-based motility varies depending on subcellular location: a kinematic probe for cytoarchitecture

Intracellular Listeria monocytogenes actin-based motility is characterized by significant individual variability, which can be influenced by cytoarchitecture. L. monocytogenes was used as a probe to transmit information about structural variation among subcellular domains defined by mitochondrial density. By analyzing the movement of a large population of L. monocytogenes in PtK2 cells, we found that mean speed and trajectory curvature were significantly larger for bacteria moving in mitochondria-containing domains (generally perinuclear) than for bacteria moving in mitochondria-free domains (generally peripheral). Analysis of bacteria that traversed both mitochondria-containing and mitochondria-free domains revealed that these motile differences were not intrinsic to bacteria themselves. Disruption of mitochondrial respiration did not affect bacterial mean speed, speed persistence, or trajectory curvature. In contrast, microtubule depolymerization lead to decreased mean speed per bacterium and increased mean speed persistence of L. monocytogenes moving in mitochondria-free domains compared with untreated cells. L. monocytogenes were also observed to physically collide with mitochondria and push them away from the bacterial path of motion, causing bacteria to slow down before rapidly resuming their speed. Our results show that subcellular domains along with microtubule depolymerization may influence the actin cytoskeleton to affect L. monocytogenes speed, speed persistence, and trajectory curvature.