In vivo studies fail to reveal a role for IL-4 or STAT6 signaling in Th2 lymphocyte differentiation.

The expression of interleukin-4 (IL-4) is viewed as the hallmark of a Th2 lymphocyte, whereas the subsequent action of IL-4 and IL-13, mediated through the STAT6 signaling pathway, is seen as a prerequisite for the full development of Th2 immune responses to parasites and allergens. G4 mice, whose IL-4 gene locus contains the fluorescent reporter eGFP, were used to quantify the number of Th2 cells that develop during Nippostrongylus brasiliensis- or allergen-induced immune responses under conditions where IL-4 or STAT6 was absent. Here, we show that deletion of IL-4 or STAT6 had little impact on the number or timing of appearance of IL-4-producing Th2 cells. These data indicate that in vivo differentiation of naïve CD4 T cells to Th2 status often occurs independently of IL-4 and STAT6 and that recently described pathways of Th2 cell differentiation may explain how allergens and parasites selectively induce Th2-mediated immunity.

Multiple proteins with single activities or a single protein with multiple activities: the conundrum of cell surface NADH oxidoreductases.

Reduction of the cell-impermeable tetrazolium salt WST-1 has been used to characterise two plasma membrane NADH oxidoreductase activities in human cells. The trans activity, measured with WST-1 and the intermediate electron acceptor mPMS, utilises reducing equivalents from intracellular sources, while the surface activity, measured with WST-1 and extracellular NADH, is independent of intracellular metabolism. Whether these two activities involve distinct proteins or are inherent to a single protein is unclear. In this work, we have attempted to address this question by examining the relationship between the trans and surface WST-1-reducing activities and a third well-characterised family of cell surface oxidases, the ECTO-NOX proteins. Using blue native-polyacrylamide gel electrophoresis, we have identified a complex in the plasma membranes of human 143B osteosarcoma cells responsible for the NADH-dependent reduction of WST-1. The dye-reducing activity of the 300 kDa complex was attributed to a 70 kDa NADH oxidoreductase activity that cross-reacted with antisera against the ECTO-NOX protein CNOX. Differences in enzyme activities and inhibitor profiles between the WST-1-reducing NADH oxidoreductase enzyme in the presence of NADH or mPMS and the ECTO-NOX family are reconciled in terms of the different purification methods and assay systems used to study these proteins.

Molecular mimicry in the decoding of translational stop signals.

Molecular mimicry was a concept that was revived as we understood more about the ligands that bound to the active center of the ribosome, and the characteristics of the active center itself. It has been particularly useful for the termination phase of protein synthesis, because for many years this major process seemed not only to be out of step) with the initiation and elongation phases but also there were no common features of the process between eubacteria and eukaryotes. As the facts that supported molecular mimicry emerged, it was seen that the protein factors that facilitated polypeptide chain release when the decoding of an mRNA was complete had common features with the ligands involved in the other phases. Moreover, now common features and mechanisms began to emerge between the eubacterial and eukaryotic RFs and suddenly there seemed to be remarkable synergy between the external ligands and commonality in at least some features of the mechanistic prnciples. Almost 10 years after molecular mimicry took hold as a framework concept, we can now see that this idea is probably too simple. For example, structural mimicry can be apparent if there are extensive conformational changes either in the ribosome active center or in the ligand itself or, most likely, both. Early indications are that the bacterial RF may indeed undergo extensive conformational changes from its solution structure to achieve this accommodation. Thus, as important if not more important than structural and functional mimicry among the ligands, might be their accomodation of a common single active center made up of at least three parts to carry out a complex series of reactions. One part of the ribosomal active center is committed to decoding, a second is committed to the chemistry of putting the protein together and releasing it, and a third part, perhaps residing in the subdomains, is committed to binding ligands so that they can perform their respective single or multiple functions. It might be more accurate to regard the decoding RF as the cuckoo taking over the nest that was crafted and honed through evolution by another, the tRNA. A somewhat ungainly RF, perhaps bigger in dimensions than the tRNA, is able, nevertheless, like the cuckoo, to maneuvre into the nest. Perhaps it pushes the nest a little out of shape, but is still able to use the site for its own functions of stop signal decoding and for facilitating the release of the polypeptide. The term molecular mimicry has been dominant in the literature for a period of important advances in the understanding of protein synthesis. When the first structures of the ribosome appeared, the concept survived and was seen to be valid still. Now, we are at the stage of understanding the more detailed molecular interactions between ligands and the rRNA in particular, and how subtle changes in localized spatial orientations of atoms occur within these interactions. The simplicity of the original concept of mimicry will inevitably be blurred by this more detailed analysis. Nevertheless, it has provided a significant set of principles that allowed development of experimental programs to enhance our understanding of the dynamic events at this remarkable active site at the interface between the two subunits of this fascinating cell organelle, the ribosome.

Cell surface oxygen consumption by mitochondrial gene knockout cells.

Mitochondrial gene knockout (rho(0)) cells that depend on glycolysis for their energy requirements show an increased ability to reduce cell-impermeable tetrazolium dyes by electron transport across the plasma membrane. In this report, we show for the first time, that oxygen functions as a terminal electron acceptor for trans-plasma membrane electron transport (tPMET) in HL60rho(0) cells, and that this cell surface oxygen consumption is associated with oxygen-dependent cell growth in the absence of mitochondrial electron transport function. Non-mitochondrial oxygen consumption by HL60rho(0) cells was extensively inhibited by extracellular NADH and NADPH, but not by NAD(+), localizing this process at the cell surface. Mitochondrial electron transport inhibitors and the uncoupler, FCCP, did not affect oxygen consumption by HL60rho(0) cells. Inhibitors of glucose uptake and glycolysis, the ubiquinone redox cycle inhibitors, capsaicin and resiniferatoxin, the flavin centre inhibitor, diphenyleneiodonium, and the NQO1 inhibitor, dicoumarol, all inhibited oxygen consumption by HL60rho(0) cells. Similarities in inhibition profiles between non-mitochondrial oxygen consumption and reduction of the cell-impermeable tetrazolium dye, WST-1, suggest that both systems may share a common tPMET pathway. This is supported by the finding that terminal electron acceptors from both pathways compete for electrons from intracellular NADH.

Mitochondrial gene-knockout (rho0) cells: a versatile model for exploring the secrets of trans-plasma membrane electron transport.

Plasma membrane electron transport (tPMET) pathways have been identified in all living cells, and a wide variety of tools have been used to study these processes. In our laboratory we have used the cell-impermeable tetrazolium dye WST-1, together with the mitochondrial gene knockout (rho0) cell model, to investigate one of these pathways. We have shown that growth of HL60rho0 cells is dependent on oxygen, and that these cells consume oxygen at the cell surface. Similarities in inhibition profiles between non-mitochondrial oxygen consumption and WST-1 reduction suggest that both systems share a common tPMET pathway. In support of this, oxygen was shown to compete with the intermediate electron acceptor that mediates WST-1 reduction, for reducing electrons. The observation that tPMET activity is higher in rho0 cells compared to their mitochondrially-competent counterparts was shown to be the result of competition between the mitochondrial and plasma membrane electron transport systems for intracellular reducing equivalents. Elevated rates of dye reduction appear to be mediated through increased expression of the key components of tPMET, which include the cell surface NADH oxidase, CNOX. These findings have played a critical role in shaping our current understanding of the mechanisms of this particular pathway of tPMET.

Accommodating the bacterial decoding release factor as an alien protein among the RNAs at the active site of the ribosome.

The decoding release factor (RF) triggers termination of protein synthesis by functionally mimicking a tRNA to span the decoding centre and the peptidyl transferase centre (PTC) of the ribosome. Structurally, it must fit into a site crafted for a tRNA and surrounded by five other RNAs, namely the adjacent peptidyl tRNA carrying the completed polypeptide, the mRNA and the three rRNAs. This is achieved by extending a structural domain from the body of the protein that results in a critical conformational change allowing it to contact the PTC. A structural model of the bacterial termination complex with the accommodated RF shows that it makes close contact with the first, second and third bases of the stop codon in the mRNA with two separate loops of structure: the anticodon loop and the loop at the tip of helix alpha5. The anticodon loop also makes contact with the base following the stop codon that is known to strongly influence termination efficiency. It confirms the close contact of domain 3 of the protein with the key RNA structures of the PTC. The mRNA signal for termination includes sequences upstream as well as downstream of the stop codon, and this may reflect structural restrictions for specific combinations of tRNA and RF to be bound onto the ribosome together. An unbiased SELEX approach has been investigated as a tool to identify potential rRNA-binding contacts of the bacterial RF in its different binding conformations within the active centre of the ribosome.

Mapping functionally important motifs SPF and GGQ of the decoding release factor RF2 to the Escherichia coli ribosome by hydroxyl radical footprinting. Implications for macromolecular mimicry and structural changes in RF2.

The function of the decoding release factor (RF) in translation termination is to couple cognate recognition of the stop codon in the mRNA with hydrolysis of the completed polypeptide from its covalently linked tRNA. For this to occur, the RF must interact with specific A-site components of the active centers within both the small and large ribosomal subunits. In this work, we have used directed hydroxyl radical footprinting to map the ribosomal binding site of the Escherichia coli class I release factor RF2, during translation termination. In the presence of the cognate UGA stop codon, residues flanking the universally conserved (250)GGQ(252) motif of RF2 were each shown to footprint to the large ribosomal subunit, specifically to conserved elements of the peptidyltransferase and GTPase-associated centers. In contrast, residues that flank the putative "peptide anticodon" of RF2, (205)SPF(207), were shown to make a footprint in the small ribosomal subunit at positions within well characterized 16 S rRNA motifs in the vicinity of the decoding center. Within the recently solved crystal structure of E. coli RF2, the GGQ and SPF motifs are separated by 23 A only, a distance that is incompatible with the observed cleavage sites that are up to 100 A apart. Our data suggest that RF2 may undergo gross conformational changes upon ribosome binding, the implications of which are discussed in terms of the mechanism of RF-mediated termination.