The Deubiquitinase Inhibitor PR-619 Sensitizes Normal Human Fibroblasts to Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-mediated Cell Death

TNF-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapy that selectively targets cancer cell death while non-malignant cells remain viable. Using a panel of normal human fibroblasts, we characterized molecular differences in human foreskin fibroblasts and WI-38 TRAIL-resistant cells and marginally sensitive MRC-5 cells compared with TRAIL-sensitive human lung and colon cancer cells. We identified decreased caspase-8 protein expression and protein stability in normal fibroblasts compared with cancer cells. Additionally, normal fibroblasts had incomplete TRAIL-induced caspase-8 activation compared with cancer cells. We found that normal fibroblasts lack the ubiquitin modification of caspase-8 required for complete caspase-8 activation. Treatment with the deubiquitinase inhibitor PR-619 increased caspase-8 ubiquitination and caspase-8 enzymatic activity and sensitized normal fibroblasts to TRAIL-mediated apoptosis. Therefore, posttranslational regulation of caspase-8 confers resistance to TRAIL-induced cell death in normal cells through blockade of initiation of the extrinsic cell death pathway.     

Structural basis for the role of Asp-120 in metallo-beta-lactamases

Metallo-beta-lactamases (mbetals) are zinc-dependent enzymes that hydrolyze a wide range of beta-lactam antibiotics. The mbetal active site features an invariant Asp-120 that ligates one of the two metal ions (Zn2) and a metal-bridging water/hydroxide (Wat1). Previous studies show that substitutions at Asp-120 dramatically affect mbetal activity, but no consensus exists as to its role in beta-lactam turnover. Here we present crystal structures of the Asn and Cys mutants of Asp-120 of the L1 mbetal from Stenotrophomonas maltophilia. Both mutants retain a dinuclear zinc center with Wat1 present. In the essentially inactive Cys enzyme Zn2 is displaced to a more buried position relative to that in the wild-type enzyme. In the catalytically impaired Asn enzyme the coordination of Zn2 is altered, neither it nor Wat1 is coordinated by Asn-120, and the N-terminal 19 amino acids, important to cooperative interactions between subunits in the wild-type enzyme, are disordered. Comparison with the structure of L1 complexed with the hydrolyzed oxacephem moxalactam suggests that in the Cys mutant Zn2 can no longer make stabilizing interactions with anionic nitrogen species formed in the hydrolytic reaction. The diminished activity of the Asn mutant arises from a combination of loss of intersubunit interactions and impaired proton transfer to, and reduced interaction of Zn2 with, the substrate amide nitrogen. We conclude that, while interactions of Asp-120 with active site water molecules are important to proton transfer and possibly nucleophilic attack by Wat1, its primary role is to optimally position Zn2 for catalytically important interactions with the charged amide nitrogen of substrate.     

Arabidopsis thaliana GLX2-1 contains a dinuclear metal binding site, but is not a glyoxalase 2

In an effort to probe the structure and function of a predicted mitochondrial glyoxalase 2, GLX2-1, from Arabidopsis thaliana, GLX2-1 was cloned, overexpressed, purified and characterized using metal analyses, kinetics, and UV-visible, EPR, and (1)H-NMR spectroscopies. The purified enzyme was purple and contained substoichiometric amounts of iron and zinc; however, metal-binding studies reveal that GLX2-1 can bind nearly two equivalents of either iron or zinc and that the most stable analogue of GLX2-1 is the iron-containing form. UV-visible spectra of the purified enzyme suggest the presence of Fe(II) in the protein, but the Fe(II) can be oxidized over time or by the addition of metal ions to the protein. EPR spectra revealed the presence of an anti-ferromagnetically-coupled Fe(III)Fe(II) centre and the presence of a protein-bound high-spin Fe(III) centre, perhaps as part of a FeZn centre. No paramagnetically shifted peaks were observed in (1)H-NMR spectra of the GLX2-1 analogues, suggesting low amounts of the paramagnetic, anti-ferromagnetically coupled centre. Steady-state kinetic studies with several thiolester substrates indicate that GLX2-1 is not a GLX2. In contrast with all of the other GLX2 proteins characterized, GLX2-1 contains an arginine in place of one of the metal-binding histidine residues at position 246. In order to evaluate further whether Arg(246) binds metal, the R246L mutant was prepared. The metal binding results are very similar to those of native GLX2-1, suggesting that a different amino acid is recruited as a metal-binding ligand. These results demonstrate that Arabidopsis GLX2-1 is a novel member of the metallo-beta-lactamase superfamily.     

Comparing the refuge strategy for managing the evolution of insect resistance under different reproductive strategies

Genetically modified (GM) crops are used extensively worldwide to control diploid agricultural insect pests that reproduce sexually. However, future GM crops will likely soon target haplodiploid and parthenogenetic insects. As rapid pest adaptation could compromise these novel crops, strategies to manage resistance in haplodiploid and parthenogenetic pests are urgently needed. Here, we developed models to characterize factors that could delay or prevent the evolution of resistance to GM crops in diploid, haplodiploid, and parthenogenetic insect pests. The standard strategy for managing resistance in diploid pests relies on refuges of non-GM host plants and GM crops that produce high toxin concentrations. Although the tenets of the standard refuge strategy apply to all pests, this strategy does not greatly delay the evolution of resistance in haplodiploid or parthenogenetic pests. Two additional factors are needed to effectively delay or prevent the evolution of resistance in such pests, large recessive or smaller non-recessive fitness costs must reduce the fitness of resistance individuals in refuges (and ideally also on GM crops), and resistant individuals must have lower fitness on GM compared to non-GM crops (incomplete resistance). Recent research indicates that the magnitude and dominance of fitness costs could be increased by using specific host–plants, natural enemies, or pathogens. Furthermore, incomplete resistance could be enhanced by engineering desirable traits into novel GM crops. Thus, the sustainability of GM crops that target haplodiploid or parthenogenetic pests will require careful consideration of the effects of reproductive mode, fitness costs, and incomplete resistance.     

Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

Background

Protein translocation across the membrane of the Endoplasmic Reticulum (ER) is the first step in the biogenesis of secretory and membrane proteins. Proteins enter the ER by the Sec61 translocon, a proteinaceous channel composed of three subunits, α, β and γ. While it is known that Sec61α forms the actual channel, the function of the other two subunits remains to be characterized.

Results

In the present study we have investigated the function of Sec61β in Drosophila melanogaster. We describe its role in the plasma membrane traffic of Gurken, the ligand for the Epidermal Growth Factor (EGF) receptor in the oocyte. Germline clones of the mutant allele of Sec61β show normal translocation of Gurken into the ER and transport to the Golgi complex, but further traffic to the plasma membrane is impeded. The defect in plasma membrane traffic due to absence of Sec61β is specific for Gurken and is not due to a general trafficking defect.

Conclusion

Based on our study we conclude that Sec61β, which is part of the ER protein translocation channel affects a post-ER step during Gurken trafficking to the plasma membrane. We propose an additional role of Sec61β beyond protein translocation into the ER.     

Eating their way to the top? Mechanisms underlying the success of invasive insect generalist predators

Insect generalist predators have been introduced outside of their native range intentionally to improve biological control, or accidentally during commerce, and can subsequently become invasive. Invasive insect generalist predators (IIGP) have widespread impacts on invaded communities because they consume both herbivores and other predators. Also, they often reach higher densities than and displace similar native species. Reflecting the complexity of their ecological roles, a wide variety of mechanisms might contribute to invasive success by IIGP. These species often drive resources to lower levels than do natives, leading to intense resource competition and sometimes competitive exclusion of other predators. The broader range of resources used by many IIGP can heighten their competitive advantage, particularly when IIGP exploit modified habitats. In either case, IIGP improve herbivore suppression by depressing prey densities below pre-invasion levels. Coexistence among native and invasive generalists is fostered when species differ substantially in their niche requirements. In this case, a larger proportion of the total prey population is subject to attack post- than pre-invasion, which strengthens prey suppression. On the other hand, some IIGP feed heavily on other predators, at times leading to a weakening of prey suppression. Future research should continue to explore the roles of competition and niche partitioning on larger spatial scales, and in both the native and invasive ranges of IIGP. Additionally, combining data from empirical studies with theory might be an effective way to predict the spread and community impacts of IIGP invasions.     

Avoiding a crisis of motivation for ocean management under global environmental change

Climate change and ocean acidification are altering marine ecosystems and, from a human perspective, creating both winners and losers. Human responses to these changes are complex, but may result in reduced government investments in regulation, resource management, monitoring and enforcement. Moreover, a lack of peoples’ experience of climate change may drive some towards attributing the symptoms of climate change to more familiar causes such as management failure. Taken together, we anticipate that management could become weaker and less effective as climate change continues. Using diverse case studies, including the decline of coral reefs, coastal defences from flooding, shifting fish stocks and the emergence of new shipping opportunities in the Arctic, we argue that human interests are better served by increased investments in resource management. But greater government investment in management does not simply mean more of “business‐as‐usual.” Management needs to become more flexible, better at anticipating and responding to surprise, and able to facilitate change where it is desirable. A range of technological, economic, communication and governance solutions exists to help transform management. While not all have been tested, judicious application of the most appropriate solutions should help humanity adapt to novel circumstances and seek opportunity where possible.     

The Hepatitis C virus NS5A protein activates a phosphoinositide 3-kinase-dependent survival signaling cascade

Hepatitis C virus (HCV) establishes a persistent infection, with up to 80% of infected individuals proceeding to chronic hepatitis, which in many cases may result in liver cirrhosis and hepatocellular carcinoma (HCC); indeed HCV infection is increasingly associated with the development of HCC. The long time period (up to 30 years) between primary infection and the onset of HCC implies that HCV is not directly oncogenic but in some way predisposes patients to develop tumors, though the mechanism for this is unclear as yet. We report here that NS5A binds directly to the Src homology 3 domain of the p85 regulatory subunit of phosphoinositide 3-kinase (PI3K), and this interaction is mediated by a novel (non-proline-rich) motif within NS5A. Coimmunoprecipitation analysis revealed that NS5A bound native heterodimeric PI3K and enhanced the phosphotransferase activity of the catalytic (p110) subunit both in vitro and in human cell lines harboring a subgenomic HCV replicon or expressing NS5A alone. NS5A-mediated activation of PI3K resulted in increased phosphorylation and activity of Akt/protein kinase B and concomitantly provided protection against the induction of apoptosis in both replicon-harboring cells and cells stably expressing NS5A alone. These data suggest that stimulation of PI3K by NS5A may represent an indirect mechanism for development of HCC in HCV-infected patients and further suggests potential therapeutic strategies to counteract the occurrence of HCV-related HCC.     

Combinatorial polymer electrospun matrices promote physiologically-relevant cardiomyogenic stem cell differentiation

Myocardial infarction results in extensive cardiomyocyte death which can lead to fatal arrhythmias or congestive heart failure. Delivery of stem cells to repopulate damaged cardiac tissue may be an attractive and innovative solution for repairing the damaged heart. Instructive polymer scaffolds with a wide range of properties have been used extensively to direct the differentiation of stem cells. In this study, we have optimized the chemical and mechanical properties of an electrospun polymer mesh for directed differentiation of embryonic stem cells (ESCs) towards a cardiomyogenic lineage. A combinatorial polymer library was prepared by copolymerizing three distinct subunits at varying molar ratios to tune the physicochemical properties of the resulting polymer: hydrophilic polyethylene glycol (PEG), hydrophobic poly(ε-caprolactone) (PCL), and negatively-charged, carboxylated PCL (CPCL). Murine ESCs were cultured on electrospun polymeric scaffolds and their differentiation to cardiomyocytes was assessed through measurements of viability, intracellular reactive oxygen species (ROS), α-myosin heavy chain expression (α-MHC), and intracellular Ca(2+) signaling dynamics. Interestingly, ESCs on the most compliant substrate, 4%PEG-86%PCL-10%CPCL, exhibited the highest α-MHC expression as well as the most mature Ca(2+) signaling dynamics. To investigate the role of scaffold modulus in ESC differentiation, the scaffold fiber density was reduced by altering the electrospinning parameters. The reduced modulus was found to enhance α-MHC gene expression, and promote maturation of myocyte Ca(2+) handling. These data indicate that ESC-derived cardiomyocyte differentiation and maturation can be promoted by tuning the mechanical and chemical properties of polymer scaffold via copolymerization and electrospinning techniques.