Generation of reactive oxygen species (ROS) with the accumulation of oxidative damage has been implicated in neurodegenerative disease and in the degradation of nervous system function with age. Here we report that ROS inhibit the activity of ciliary neurotrophic factor (CNTF) in nerve cells. Treatment with hydrogen peroxide (H 2 O 2 ) as a generator of ROS inhibited CNTF-mediated Jak/STAT signaling in all cultured nerve cells tested, including chick ciliary ganglion neurons, chick neural retina, HMN-1 motor neuron hybrid cells, and SH-SY5Y and BE(2)-C human neuroblastoma cells. H 2 O 2 treatment of non-neuronal cells, chick skeletal muscle and HepG2 hepatoma cells, did not inhibit Jak/STAT signaling. The H 2 O 2 block of CNTF activity was seen at concentrations as low as 0.1 mM and within 15 min, and was reversible upon removal of H 2 O 2 from the medium. Also, two other mediators of oxidative stress, nitric oxide and rotenone, inhibited CNTF signaling. Treatment of neurons with H 2 O 2 and rotenone also inhibited interferon-c-mediated activation of Jak/STAT1. Depleting the intracellular stores of reduced glutathione by treatment of BE(2)-C cells with nitrofurantoin inhibited CNTF activity, whereas addition of reduced glutathione protected cells from the effects of H 2 O 2 . These results suggest that disruption of neurotrophic factor signaling by mediators of oxidative stress may contribute to the neuronal damage observed in neurodegenerative diseases and significantly affect the utility of CNTF-like factors as therapeutic agents in preventing nerve cell death. Keywords: ciliary neurotrophic factor, cytokine, gpl30, reactive oxygen species, signal transduction, tyrosine kinase. Signaling through the ciliary neurotrophic factor (CNTF) receptor has been implicated in the development, survival and maintenance of a broad range of neurons and glia in the PNS and CNS, as well as cardiac and skeletal muscle. CNTF receptors are essential for embryonic development and the receptor knockout phenotype in mice is embryonic lethal (DeChiara et al. 1995). Defects in CNTF expression result in an impaired neural injury response in mice (Masu et al. 1993;Yao et al. 1999;Linker et al. 2002) and decreased neuron numbers in embryonic chick (Ernsberger et al. 1989;Finn et al. 1998). In addition, a variety of neuronal, glial and muscle cell types show responses to CNTF in vivo or in vitro affecting neuronal phenotype, neurotransmitter receptor levels, neurotransmitter release, metabolism and survival (Adler et al.

SummaryMembrane receptors communicate between the external world and the cell interior. In bacteria, these receptors include the transmembrane sensor kinases, which control gene expression via their cognate response regulators, and chemoreceptors, which control the direction of flagellar rotation via the CheA kinase and CheY response regulator. Here, we show that a chimeric protein that joins the ligandbinding, transmembrane and linker domains of the NarX sensor kinase to the signalling and adaptation domains of the Tar chemoreceptor of Escherichia coli mediates repellent responses to nitrate and nitrite. Nitrate induces a stronger response than nitrite and is effective at lower concentrations, mirroring the relative sensitivity to these ligands exhibited by NarX itself. We conclude that the NarX-Tar hybrid functions as a bona fide chemoreceptor whose activity can be predicted from its component parts. This observation implies that ligand-dependent activation of a sensor kinase and repellent-initiated activation of receptorcoupled CheA kinase involve a similar transmembrane signal.

Myxococcus xanthus fibril exopolysaccharide (EPS), essential for the social gliding motility and development of this bacterium, is regulated by the Dif chemotaxis-like pathway. DifA, an MCP homolog, is proposed to mediate signal input to the Dif pathway. However, DifA lacks a prominent periplasmic domain, which in classical chemoreceptors is responsible for signal perception and for initiating transmembrane signaling. To investigate the signaling properties of DifA, we constructed a NarX-DifA (NafA) chimera from the sensory module of Escherichia coli NarX and the signaling module of M. xanthus DifA. We report here the first functional chimeric signal transducer constructed using genes from organisms in two different phylogenetic subdivisions. When expressed in M. xanthus, NafA restored fruiting body formation, EPS production, and S-motility to difA mutants in the presence of nitrate. Studies with various double mutants indicate that NafA requires the downstream Dif proteins to function. We propose that signal inputs to the Dif pathway and transmembrane signaling by DifA are essential for the regulation of EPS production in M. xanthus. Despite the apparent structural differences, DifA appears to share similar transmembrane signaling mechanisms with enteric sensor kinases and chemoreceptors.

Signal-transducing proteins that span the cytoplasmic membrane transmit information about the environment to the interior of the cell. In bacteria, these signal transducers include sensor kinases, which typically control gene expression via response regulators, and methyl-accepting chemoreceptor proteins, which control flagellar rotation via the CheA kinase and CheY response regulator. We previously reported that a chimeric protein (Nart) that joins the ligand-binding, transmembrane, and linker regions of the NarX sensor kinase to the signaling and adaptation domains of the Tar chemoreceptor elicits a repellent response to nitrate and nitrite. As with NarX, nitrate evokes a stronger response than nitrite. Here we show that mutations targeting a highly conserved sequence (the P box) in the periplasmic domain alter chemoreception by Nart and signaling by NarX similarly. In particular, the G51R substitution converts Nart from a repellent receptor into an attractant receptor for nitrate. Our results underscore the conclusion that the fundamental mechanism of transmembrane signaling is conserved between homodimeric sensor kinases and chemoreceptors. They also highlight the plasticity of the coupling between ligand binding and signal output in these systems.The Tar chemoreceptor of Escherichia coli mediates attractant responses to aspartate and maltose (39), the latter via maltose-binding protein (18), and repellent responses to Ni 2ϩ and Co 2ϩ (45). The other high-abundance chemoreceptor, Tsr, mediates attractant and repellent responses to serine (39) and leucine (45), respectively. Tar and Tsr form homodimers in the presence or absence of ligands (5, 31), and these dimers associate into both homogeneous (19,21,43,46) and mixed (3,25,36,43,46) trimers of dimers. A number of sensor kinases, including NarX and EnvZ, share predicted membrane topology with these chemoreceptors (10, 13).The Tar and Tsr proteins contain two membrane-spanning regions that connect an N-terminal periplasmic ligand recognition domain to a C-terminal cytoplasmic signaling and adaptation domain (23,24). Tar binds aspartate at the dimer interface near the apexes of the periplasmic domains (8,27,28,31), whereas ligand-bound maltose-binding protein binds asymmetrically at the apex of the Tar homodimer (6,16,53). Serine and leucine bind directly to the periplasmic domain of Tsr (23). The cytoplasmic regions of the high-abundance transducers are responsible for transmitting the signal received from the periplasmic region to the CheA kinase (17,30,44). When the chemical environment is homogeneous, CheA activity is at its baseline level, the flagellar motors alternate between counterclockwise (CCW) and clockwise (CW) rotation, and the cell exhibits normal run-tumble motility (37). The addition of attractants, or the removal of repellents, inhibits the activation of CheA by the cognate receptor, thereby decreasing the rate of phosphoryl group transfer from receptor-associated CheA to the response regulator CheY. Lowering the cytoplasmic level of phospho...