Methylation and loss of Secreted Frizzled-Related Protein 3 enhances melanoma cell migration and invasion

Background

Wnt signaling is important in development and can also contribute to the initiation and progression of cancer. The Secreted Frizzled Related Proteins (SFRPs) constitute a family of Wnt modulators, crucial for controlling Wnt signaling. Here we investigate the expression and role of SFRP3 in melanoma.

Methodology/Principal Findings

We show that SFRP3 mRNA is down-regulated in malignant melanoma tumors as compared to normal/benign tissue. Furthermore, we found that SFRP3 expression was lost in the malignant melanoma cell lines, A2058, HTB63 and A375, but not in the non-transformed melanocyte cell line, Hermes 3A. Methylated CpG rich areas were detected in the SFRP3 gene in melanoma cell lines and their SFRP3 expression could be restored using the demethylating agent, 5′aza-deoxycytidine. Addition of recombinant SFRP3 to melanoma cells had no effect on viable cell numbers, but decreased cell migration and invasion. Wnt5a signaling has been shown to increase the migration and invasion of malignant melanoma cells, and high expression of Wnt5a in melanoma tumors has been connected to a poor prognosis. We found that recombinant SFRP3 could inhibit Wnt5a signaling, and that it inhibited melanoma cell migration and invasion in a Wnt5a-dependent manner.

Conclusion/Significance

We conclude that SFRP3 functions as a melanoma migration and invasion suppressor by interfering with Wnt5a signaling.     

On Polydispersity of Plant Biomass Recalcitrance and Its Effects on Pretreatment Optimization for Sugar Production

This paper discusses a property associated with plant biomass recalcitrance to enzyme and microbial deconstructions in sugar production from cellulose and hemicelluloses. The hemicelluloses are more readily hydrolyzed to sugars than is cellulose. As a result, optimization to maximize individual glucose and hemicellulose sugar recovery is not possible. This property is an inherent feature of plant biomass and is named polydispersity of plant biomass recalcitrance (PPBR) in this study. A set of pretreatment experiments using eucalyptus and sulfite pretreatment to overcome recalcitrance of lignocelluloses was conducted. The results were used to predict the conditions for individually maximizing enzymatic glucose and xylose yields. The predicted maximal yields were used to quantitatively illustrate the PPBR concept. The effect of PPBR on pretreatment optimization and strategies for maximal sugar recovery using two-stage pretreatment are discussed.     

Association of Influenza Virus Matrix Protein with Ribonucleoproteins

To characterize the sites and nature of binding of influenza A virus matrix protein (M1) to ribonucleoprotein (RNP), M1 of A/WSN/33 was altered by deletion or site-directed mutagenesis, expressed in vitro, and allowed to attach to RNP under a variety of conditions. Approximately 70% of the wild-type (Wt) M1 bound to RNP at pH 7.0, but less than 5% of M1 associated with RNP at pH 5.0. Increasing the concentration of NaCl reduced M1 binding, but even at a high salt concentration (0.6 M NaCl), approximately 20% of the input M1 was capable of binding to RNP. Mutations altering potential M1 RNA-binding regions (basic amino acids 101RKLKR105 and the zinc finger motif at amino acids 148 to 162) had varied effect: mutations of amino acids 101 to 105 reduced RNP binding compared to the Wt M1, but mutations of zinc finger motif did not. Treatment of RNP with RNase reduced M1 binding by approximately half, but even M1 mutants lacking RNA-binding regions had residual binding to RNase-treated RNP provided that the N-terminal 76 amino acids of M1 (containing two hydrophobic domains) were intact. Addition of detergent to the reaction mixture further reduced binding related to the N-terminal 76 amino acids and showed the greatest effect for mutations affecting the RNA-binding regions of basic amino acids. The data suggest that M1 interacts with both the RNA and protein components of RNP in assembly and disassembly of influenza A viruses.     

Structure-function relationships of the viral RNA-dependent RNA polymerase: fidelity, replication speed, and initiation mechanism determined by a residue in the ribose-binding pocket

Studies of the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV), 3Dpol, have shown that Asn-297 permits this enzyme to distinguish ribose from 2'-deoxyribose. All animal RNA viruses have Asn at the structurally homologous position of their polymerases, suggesting a conserved function for this residue. However, all prokaryotic RNA viruses have Glu at this position. In the presence of Mg2+, the apparent affinity of Glu-297 3Dpol for 2'-deoxyribonucleotides was decreased by 6-fold relative to wild type without a substantial difference in the fidelity of 2'-dNMP incorporation. The fidelity of ribonucleotide misincorporation for Glu-297 3Dpol was reduced by 14-fold relative to wild type. A 4- to 11-fold reduction in the rate of ribonucleotide incorporation was observed. Glu-297 PV was unable to grow in HeLa cells due to a replication defect equivalent to that observed for a mutant PV encoding an inactive polymerase. Evaluation of the protein-(VPg)-primed initiation reaction showed that only half of the Glu-297 3Dpol initiation complexes were capable of producing VPg-pUpU product and that the overall yield of uridylylated VPg products was reduced by 20-fold relative to wild-type enzyme, a circumstance attributable to a reduced affinity for UTP. These studies identify the first RdRp derivative with a mutator phenotype and provide a mechanistic basis for the elevated mutation frequency of RNA phage relative to animal RNA viruses observed in culture. Although protein-primed initiation and RNA-primed elongation complexes employ the same polymerase active site, the functional differences reported here imply significant structural differences between these complexes.     

A hierarchical multi‐scale analysis of the spatial relationship between parasitism and host density in urban habitats

Studies on spatial density dependence in parasitism have paid scarce attention to how changes in host density at different hierarchical scales could influence parasitism in an herbivore at a particular scale. Here, we evaluated if rates of parasitism per leaf (by the whole parasitic complex and by dominant species) of the specialist leaf miner Liriomyza commelinae (Diptera: Agromyzidae) respond to variations in host density at the leaf, plant patch and site levels in an urban setting. We used multi‐level Bayesian models that incorporate the spatial hierarchy occurring in this system, as well as habitat factors previously found to have an effect on the L. commelinae parasitoid community in an urban context (patch size, patch isolation and urbanization level). According to the fitted model, overall parasitism rates decreased with increasing number of mines per leaf, being independent of host‐density variations at patch and site level. Patch structure was found to have a strong effect on parasitism rates per leaf. The analysis of parasitism by parasitoid species separately showed consistent results with the response at community level. These results suggest that parasitism of the parasitoid community here studied would be sensitive to hierarchical cues related to the host at the leaf level and to the host habitat at the patch level.     

Consequences of injuries on survival and reproduction of common bottlenose dolphins (Tursiops truncatus) along the west coast of Florida

Accurate identification of human‐induced injuries that lead to death or interfere with reproduction is important for marine mammal management, as deaths exceeding established limits can lead to restrictions on fisheries or vessel operations. The fates of cetaceans last seen swimming with attached gear, particularly in pelagic fisheries, or with vessel strike lacerations, have been difficult to predict. Survival and reproduction data from long‐term research on resident common bottlenose dolphins near Sarasota, Florida were examined relative to consequences of fishing gear ingestion, line entanglements, vessel strikes, and amputations of unknown origins. Fishing hooks embedded in the throat, goosebeak, or esophagus, or line wrapped around the goosebeak, generally lead to death. Multiple, constrictive line wraps around fin insertions can lead to amputation, blood loss, impaired mobility, or infection. Dolphins with ingested gear or severe entanglements may swim away with the gear, but likely die later. Propeller injuries involving only soft tissue were often survivable. Some dolphins survived amputations of the distal ends of fins, and continued to reproduce. As a precautionary approach, dolphins with ingested gear or severe constrictive entanglements should be considered mortalities, but extrapolations of findings from coastal bottlenose dolphins to other cetaceans and different gear must be done with caution.     

A Laminin G-EGF-Laminin G module in Neurexin IV is essential for the apico-lateral localization of Contactin and organization of septate junctions

Septate junctions (SJs) display a unique ultrastructural morphology with ladder-like electron densities that are conserved through evolution. Genetic and molecular analyses have identified a highly conserved core complex of SJ proteins consisting of three cell adhesion molecules Neurexin IV, Contactin, and Neuroglian, which interact with the cytoskeletal FERM domain protein Coracle. How these individual proteins interact to form the septal arrays that create the paracellular barrier is poorly understood. Here, we show that point mutations that map to specific domains of neurexin IV lead to formation of fewer septae and disorganization of SJs. Consistent with these observations, our in vivo domain deletion analyses identified the first Laminin G-EGF-Laminin G module in the extracellular region of Neurexin IV as necessary for the localization of and association with Contactin. Neurexin IV protein that is devoid of its cytoplasmic region is able to create septae, but fails to form a full complement of SJs. These data provide the first in vivo evidence that specific domains in Neurexin IV are required for protein-protein interactions and organization of SJs. Given the molecular conservation of SJ proteins across species, our studies may provide insights into how vertebrate axo-glial SJs are organized in myelinated axons.     

Quaternary structure of a SPATE autotransporter protein

The temperature-sensitive hemagglutinin (Tsh) is a representative of the growing subfamily of secreted bacterial virulence factors, known as serine protease autotransporters of the Enterobacteriaceae (SPATEs). Expressed by avian and human pathogenic strains of Escherichia coli Tsh acts as a serine protease and an adhesin to erythrocytes, hemoglobin, and extracellular matrix proteins. Mature Tsh is comprised of a 106-kDa secreted domain (Tshs) and a 33-kDa outer membrane beta-domain (Tshbeta). Based on the size of beta-domains and functional properties of their passenger domains, all SPATEs are considered to be conventional autotransporters. However, it is unsettled if the conventional autotransporters exist as monomers, oligomers, or multimers (e.g., hexamers). To determine the quaternary structure of Tsh in vitro, we purified Tshbeta from the outer membranes and showed that it is natively folded because it is heat modifiable and resistant to protease digestion. Blue-native polyacrylamide gel electrophoresis of Tshbeta indicated that Tshbeta exists as a monomer or a dimer. The cross-linking analysis demonstrated that purified Tshbeta exists as a monomer. The size-exclusion chromatography and cross-linking analyses of purified Tshs also showed that the passenger domain of Tsh is a monomer. Overall, our data indicated that Tsh is a monomeric protein in vitro and support the concept that the SPATE autotransporters exist as monomers rather than as multimers. Implications of our findings on the mechanism of autotransporter secretion across the outer membrane are discussed.