Systematic in vivo RNAi analysis identifies IAPs as NEDD8-E3 ligases

The intimate relationship between mediators of the ubiquitin (Ub)-signaling system and human diseases has sparked profound interest in how Ub influences cell death and survival. While the consequence of Ub attachment is intensely studied, little is known with regards to the effects of other Ub-like proteins (UBLs), and deconjugating enzymes that remove the Ub or UBL adduct. Systematic in vivo RNAi analysis identified three NEDD8-specific isopeptidases that, when knocked down, suppress apoptosis. Consistent with the notion that attachment of NEDD8 prevents cell death, genetic ablation of deneddylase 1 (DEN1) suppresses apoptosis. Unexpectedly, we find that Drosophila and human inhibitor of apoptosis (IAP) proteins can function as E3 ligases of the NEDD8 conjugation pathway, targeting effector caspases for neddylation and inactivation. Finally, we demonstrate that DEN1 reverses this effect by removing the NEDD8 modification. Altogether, our findings indicate that IAPs not only modulate cellular processes via ubiquitylation but also through attachment of NEDD8, thereby extending the complexity of IAP-mediated signaling.     

Interaction of soluble CD163 with activated T lymphocytes involves its association with non-muscle myosin heavy chain type A

CD163 is a monocyte/macrophage-specific scavenger receptor that undergoes ectodomain shedding upon an inflammatory stimulus. Soluble CD163 (sCD163) actively inhibits lymphocyte proliferation, but to date exactly how it interacts with these cells has remained elusive. We screened T lymphocytes and endothelial cells for proteins binding to sCD163. In both cell types a high affinity binding protein was detected. Partial sequencing of the protein revealed sequence identity to a non-muscle myosin heavy chain type A. Employing labelled sCD163 we found little specific binding of sCD163 to the extracellular domains of T lymphocytes and human umbilical vein endothelial cells (HUVEC). In activated T lymphocytes we demonstrated specific binding of sCD163 to intracellular structures as well as the presence of the native protein within the cell after co-incubation with purified sCD163. Furthermore, we developed a novel ELISA for highly specific detection of sCD163-myosin complexes. These complexes were present in activated T lymphocytes after incubation with shed sCD163. Co-localization of sCD163 and cellular myosin in T lymphocytes was further confirmed by fluorescence microscopy. Our results suggest that sCD163 associates with cellular myosin, thereby possibly modulating the cells' response to an inflammatory stimulus.     

The Sla2p talin domain plays a role in endocytosis in Saccharomyces cerevisiae

Clathrin-binding adaptors play critical roles for endocytosis in multicellular organisms, but their roles in budding yeast have remained unclear. To address this question, we created a quadruple mutant yeast strain lacking the genes encoding the candidate clathrin adaptors Yap1801p, Yap1802p, and Ent2p and containing a truncated version of Ent1p, Ent1DeltaCBMp, missing its clathrin-binding motif. This strain was viable and competent for endocytosis, suggesting the existence of other redundant adaptor-like factors. To identify these factors, we mutagenized the quadruple clathrin adaptor mutant strain and selected cells that were viable in the presence of full-length Ent1p, but inviable with only Ent1DeltaCBMp; these strains were named Rcb (requires clathrin binding). One mutant strain, rcb432, contained a mutation in SLA2 that resulted in lower levels of a truncated protein lacking the F-actin binding talin homology domain. Analyses of this sla2 mutant showed that the talin homology domain is required for endocytosis at elevated temperature, that SLA2 exhibits genetic interactions with both ENT1 and ENT2, and that the clathrin adaptors and Sla2p together regulate the actin cytoskeleton and revealed conditions under which Yap1801p and Yap1802p contribute to viability. Together, our data support the view that Sla2p is an adaptor that links actin to clathrin and endocytosis.     

Clathrin function in yeast endocytosis

The process of endocytosis is a complex series of events involving the coordinated activity of many proteins. In animal cells, clathrin plays a vital role in the invagination of the plasma membrane leading to formation of vesicles during endocytosis. The study of endocytosis in yeast cells has been hindered by a debate about the role of clathrin in early internalization steps. This review summarizes the evidence for and against clathrin's involvement in internalization from the yeast plasma membrane.     

The development of a transformation system for the dimorphic plant pathogen Holleya sinecauda based on Ashbya gossypii DNA elements

We have developed a transformation system for the dimorphic plant pathogenic fungus Holleya sinecauda based on an electroporation protocol used for the closely related filamentous fungus Ashbya gossypii. DNA-mediated transformation of the dominant selection marker kanMX generated H. sinecauda transformants that were resistant to the antibiotic drug G418/geneticin. Freely replicating plasmids could be established in H. sinecauda using an A. gossypii autonomously replicating sequence (ARS) element, whereas Saccharomyces cerevisiae ARS elements, which are functional in A. gossypii, were not functional in H. sinecauda. In addition, centromeric DNA of A. gossypii stabilized the maintenance of plasmids in H. sinecauda under non-selective conditions. We isolated a fragment of the HsLEU2 gene and used this locus for targeted integration of kanMX3, consisting of the kanMX gene flanked by direct repeats. This allowed the construction of a Hsleu2 strain which became G418 sensitive after direct repeat-induced marker excision. The Hsleu2 strain can be complemented by the ScLEU2 gene. Finally, we constructed high- and low-copy shuttle vectors for H. sinecauda.     

Characterisation of mouse monoclonal antibodies against rhesus macaque killer immunoglobulin-like receptors KIR3D

Killer immunoglobulin-like receptors (KIRs) represent a highly polymorphic and diverse gene family in rhesus macaques. Analyses of the respective gene products have been hampered until now due to non-availability of specific monoclonal antibodies and failure of cross-reactivity of anti-human KIR antibodies. We utilised one activating (KIR3DSW08) and two inhibitory (KIR3DLW03 and KIR3DL05) rhesus macaque KIR-Fc fusion proteins for generation of monoclonal antibodies in mice. Besides broadly reacting ones, we obtained anti-rhesus macaque KIR antibodies with intermediate and with single specificity. These monoclonal antibodies were tested for binding to a panel of rhesus macaque KIR proteins after heterologous expression on transiently transfected cells. Epitope mapping identified two polymorphic regions that are located next to each other in the mature KIR proteins. The availability of monoclonal antibodies against rhesus macaque KIR proteins will enable future studies on KIR at the protein level in rhesus macaques as important animal models of human infectious diseases.     

Precursor Decomposition,Microstructure, and Porosity of Yttria Stabilized Zirconia Thin Films Prepared by Aerosol‐Assisted Chemical Vapor Deposition

Microstructures of yttria stabilized zirconia thin films deposited by aerosol assisted chemical vapor deposition (AA‐CVD) are correlated with the thermal decomposition behavior of the corresponding metal precursors, zirconium and yttrium 2,4‐pentanedionate. Process conditions of AA‐CVD are investigated with the aim of producing dense and compact YSZ thin films for applications as gas‐tight electrolyte. Based on systematic cross sectional scanning transmission electron microscopy (STEM) investigations and conductivity measurements, the development of percolating nanoporosity is observed in samples prepared at temperatures between 350 °C and 600 °C at standard solution throughput. Compact columnar thin films with bulk conductivity are obtained at 600 °C by reducing the metal content of the precursor solution and at 450 °C by reducing the solution throughput.     

EH proteins

Endocytosis is a protein and lipid-trafficking pathway that occurs in all eukaryotic cells. It involves the internalization of plasma membrane proteins and lipids into the cell and the subsequent degradation of proteins in the lysosome or the recycling of proteins and lipids back to the plasma membrane. Over the past decade, studies in yeast and mammalian cells have revealed endocytosis to be a very complex molecular process that depends on regulated interactions between a variety of proteins and lipids. The Eps15 homology (EH) domain is a conserved, modular protein-interaction domain found in several endocytosis proteins. EH proteins can function as key regulators of endocytosis through their ability to interact with many of the other proteins involved in this process.     

The Hfq-like protein NrfA of the phototrophic purple bacterium Rhodobacter capsulatus controls nitrogen fixation via regulation of nifA and anfA expression

A novel esterase catalyzing regioselective hydrolysis was purified from the membrane fraction of Microbacterium sp. 7-1W, and characterized. The enzyme was solubilized with Brij 58 and purified 13.8-fold to apparent homogeneity with 2.58% overall recovery. The relative molecular mass of the native enzyme as estimated by gel filtration was more than 600,000 Da, and the subunit molecular mass was 62,000 Da. The enzyme catalyzed cleavage of the terminal ester bonds of cetraxate esters and pantothenate esters. The K(m) and V(max) values for methyl cetraxate were 0.380 mM and 7.76 micromole min(-1) mg(-1) protein, respectively. The enzyme was inhibited by serine hydrolase inhibitors.