Constitutive and accelerated shedding of murine syndecan-1 is mediated by cleavage of its core protein at a specific juxtamembrane site

Syndecan-1 is a developmentally regulated cell surface heparan sulfate proteoglycan (HSPG). It functions as a coreceptor for a variety of soluble and insoluble ligands and is implicated in several biological processes, including differentiation, cell migration, morphogenesis, and recently feeding behavior. The extracellular domain of syndecan-1 is proteolytically cleaved at a juxtamembrane site by tissue inhibitor of metalloprotease-3 (TIMP-3)-sensitive metalloproteinases in response to a variety of physiological stimulators and stress in a process known as shedding. Shedding converts syndecan-1 from a membrane-bound coreceptor into a soluble effector capable of binding the same ligands. We found that replacing syndecan-1 juxtamembrane amino acid residues A243-S-Q-S-L247 with human CD4 amino acid residues can completely block PMA-induced syndecan-1 ectodomain shedding. Furthermore, using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS), we identified the proteolytic cleavage site of syndecan-1 as amino acids A243 and S244, generated by constitutive and PMA-induced shedding from murine NMuMG cells. Finally, we show that basal cleavage of syndecan-1 utilizes the same in vivo site as the in vitro site. Indeed, as predicted, transgenic mice expressing the syndecan-1/CD4 cDNA do not shed the syndecan-1 ectodomain in vivo. These results suggest that the same cleavage site is utilized for basal syndecan-1 ectodomain shedding both in vitro from NMuMG and CHO cells and in vivo.     

Selective impairment of TLR-mediated innate immunity in human newborns: neonatal blood plasma reduces monocyte TNF-alpha induction by bacterial lipopeptides, lipopolysaccharide, and imiquimod, but preserves the response to R-848

Newborns are at increased risk of overwhelming infection, yet the mechanisms underlying this susceptibility are incompletely defined. In this study we report a striking 1- to 3-log decrease in sensitivity of monocytes in human neonatal cord blood, compared with monocytes in adult peripheral blood, to the TNF-alpha-inducing effect of multiple TLR ligands, including bacterial lipopeptides (BLPs), LPS, and the imidazoquinoline compound, imiquimod. In marked contrast, TNF-alpha release in response to R-848, a TLR ligand that is a congener of imiquimod, was equivalent in newborn and adult blood. Differences in ligand-induced TNF-alpha release correlated with divergent ligand-induced changes in monocyte TNF-alpha mRNA levels. Newborn and adult monocytes did not differ in basal mRNA or protein expression of TLRs or mRNA expression of functionally related molecules. Newborn monocytes demonstrated diminished LPS-induced, but equivalent R-848-induced, phosphorylation of p38 mitogen-activated protein kinase and altered BLP- and LPS-induced acute modulation of cognate receptors, suggesting that the mechanism accounting for the observed differences may be localized proximal to ligand recognition by surface TLRs. Remarkably, newborn plasma conferred substantially reduced BLP-, LPS-, and imiquimod-induced TNF-alpha release on adult monocytes without any effect on R-848-induced TNF-alpha release, reflecting differences in a plasma factor(s) distinct from soluble CD14. Impaired response to multiple TLR ligands may significantly contribute to immature neonatal immunity. Conversely, relative preservation of responses to R-848 may present unique opportunities for augmenting innate and acquired immunity in the human newborn.     

Overlapping Messages and Survivability

The phenomenon of overlapping of various sequence messages in genomes is a puzzle for evolutionary theoreticians, geneticists, and sequence researchers. The overlapping is possible due to degeneracy of the messages, in particular, degeneracy of codons. It is often observed in organisms with a limited size of genome, possessing polymerases of low fidelity. The most accepted view considers the overlapping as a mechanism to increase the amount of information per unit length. Here we present a model that suggests direct evolutionary advantage of the message overlapping. Two opposing drives are considered: (a) reduction in the amount of vulnerable points when the overlapping of two messages involves common critical points and (b) cumulative compromising cost of coexistence of messages at the same site. Over a broad range of conditions the reduction of the target size prevails, thus making the overlapping of messages advantageous.[Reviewing Editor: Dr. Martin Kreitman]     

Regulation of Gap Junction Coupling Through the Neuronal Connexin Cx35 by Nitric Oxide and cGMP

Gap-junctional coupling among neurons is subject to regulation by a number of neurotransmitters including nitric oxide. We studied the mechanisms by which NO regulates coupling in cells expressing Cx35, a connexin expressed in neurons throughout the central nervous system. NO donors caused potent uncoupling of HeLa cells stably transfected with Cx35. This effect was mimicked by Bay 21-4272, an activator of guanylyl cyclase. A pharmacological analysis indicated that NO-induced uncoupling involved both PKG-dependent and PKG-independent pathways. PKA was involved in both pathways, suggesting that PKG-dependent uncoupling may be indirect. In vitro, PKG phosphorylated Cx35 at three sites: Ser110, Ser276, and Ser289. A mutational analysis indicated that phosphorylation on Ser110 and Ser276, sites previously shown also to be phosphorylated by PKA, had a significant influence on regulation. Ser289 phosphorylation had very limited effects. We conclude that NO can regulate coupling through Cx35 and that regulation is indirect in HeLa cells.     

Probing the Transition State of the Allosteric Pathway of the Shaker Kv Channel Pore by Linear Free-Energy Relations

Long-range coupling between distant functional elements of proteins may rely on allosteric communication trajectories lying along the protein structure, as described in the case of the Shaker voltage-activated potassium (Kv) channel model allosteric system. Communication between the distant Kv channel activation and slow inactivation pore gates was suggested to be mediated by a network of local pairwise and higher-order interactions among the functionally unique residues that constitute the allosteric trajectory. The mechanism by which conformational changes propagate along the Kv channel allosteric trajectory to achieve pore opening, however, remains unclear. Such conformational changes may propagate in either a concerted or a sequential manner during the reaction coordinate of channel opening. Residue-level structural information on the transition state of channel gating is required to discriminate between these possibilities. Here, we combine patch-clamp electrophysiology recordings of Kv channel gating and analysis using linear free-energy relations, focusing on a select set of residues spanning the allosteric trajectory of the Kv channel pore. We show that all allosteric trajectory residues tested exhibit an open-like conformation in the transition state of channel opening, implying that coupling interactions occur along the trajectory break in a concerted manner upon moving from the closed to the open state. Energetic coupling between the Kv channel gates thus occurs in a concerted fashion in both the spatial and the temporal dimensions, strengthening the notion that such trajectories correspond to pathways of mechanical deformation along which conformational changes propagate.     

Iron Reduction and Soil Phosphorus Solubilization in Humid Tropical Forests Soils: The Roles of Labile Carbon Pools and an Electron Shuttle Compound

The affinity of iron oxides and hydroxides for phosphorus is thought to contribute to phosphorus limitation to net primary productivity in humid tropical forests on acidic, highly weathered soils. Perennially warm, humid conditions and high biological activity in these soils can result in fluctuating redox potential that in turn leads to considerable iron reduction in the presence of labile carbon and humic substances. We investigated the effects of reducing conditions in combination with the addition of labile carbon substrates (glucose and acetate) and an electron shuttle compound on iron reduction and phosphorus release in a humid tropical forest soil. Glucose or acetate was added to soils as a single dose at the beginning of the experiment, and as pulsed inputs over time, which more closely mimics patterns in labile carbon availability. Iron reduction and phosphorus mobilization were weakly stimulated by a single low level addition of carbon, and the addition of the electron shuttle compound with or without added carbon. Pulsed labile carbon additions produced a significant increase in soil pH, soluble iron, and phosphorus concentrations. Pulsed labile carbon inputs also promoted the precipitation of ferrous hydroxide complexes which could increase the capacity for P sorption, although our results suggest that rates of P solubilization exceeded re-adsorption. Plant and microbial P demand are also likely to serve as an important sinks for released P, limiting the role of P re-adsorption. Our results suggest that reducing conditions coupled with periodic carbon inputs can stimulate iron reduction and a corresponding increase in soil phosphorus mobilization, which may provide a source of phosphorus to plants and microorganisms previously undocumented in these ecosystems.     

Insulin promotes shedding of syndecan ectodomains from 3T3-L1 adipocytes: a proposed mechanism for stabilization of extracellular lipoprotein lipase

Syndecans are a family of four transmembrane heparan sulfate proteoglycans that act as coreceptors for a variety of cell-surface ligands and receptors. Receptor activation in several cell types leads to shedding of syndecan-1 and syndecan-4 ectodomains into the extracellular space by metalloproteinase-mediated cleavage of the syndecan core protein. We have found that 3T3-L1 adipocytes express syndecan-1 and syndecan-4 and that their ectodomains are shed in response to insulin in a dose-, time-, and metalloproteinase-dependent manner. Insulin responsive shedding is not seen in 3T3-L1 fibroblasts. This shedding involves both Ras-MAP kinase and phosphatidylinositol 3-kinase pathways. In response to insulin, adipocytes are known to secrete active lipoprotein lipase, an enzyme that binds to heparan sulfate on the luminal surface of capillary endothelia. Lipoprotein lipase is transported as a stable enzyme from its site of synthesis to its site of action, but the transport mechanism is unknown. Our studies indicate that shed adipocyte syndecans associate with lipoprotein lipase. The shed syndecan ectodomain can stabilize active lipoprotein lipase. These data suggest that syndecan ectodomains, shed by adipocytes in response to insulin, are physiological extracellular chaperones for lipoprotein lipase as it translocates from its site of synthesis to its site of action.     

Independent axes of genetic variation and parallel evolutionary divergence of opercle bone shape in threespine stickleback

Evolution of similar phenotypes in independent populations is often taken as evidence of adaptation to the same fitness optimum. However, the genetic architecture of traits might cause evolution to proceed more often toward particular phenotypes, and less often toward others, independently of the adaptive value of the traits. Freshwater populations of Alaskan threespine stickleback have repeatedly evolved the same distinctive opercle shape after divergence from an oceanic ancestor. Here we demonstrate that this pattern of parallel evolution is widespread, distinguishing oceanic and freshwater populations across the Pacific Coast of North America and Iceland. We test whether this parallel evolution reflects genetic bias by estimating the additive genetic variance-covariance matrix (G) of opercle shape in an Alaskan oceanic (putative ancestral) population. We find significant additive genetic variance for opercle shape and that G has the potential to be biasing, because of the existence of regions of phenotypic space with low additive genetic variation. However, evolution did not occur along major eigenvectors of G, rather it occurred repeatedly in the same directions of high evolvability. We conclude that the parallel opercle evolution is most likely due to selection during adaptation to freshwater habitats, rather than due to biasing effects of opercle genetic architecture.