Increased expression of plasminogen activator inhibitor-1 (PAI-1) is associated with depression and depressive phenotype in C57Bl/6J mice.

Plasminogen activator inhibitor 1 (PAI-1), which is elevated in numerous disease states, has been implicated as a stress-related protein involved in the pathogenesis of depression. We measured PAI-1 in the plasma of healthy and depressed individuals and assessed plasminogen activator (PA) expression and regulation by PAI-1 in cultured normal human astrocytes (NHA). Elevated plasma PAI-1 levels were found in depressed patients. Brain tissues from depressed individuals also showed stronger expression of hippocampal PAI-1 by confocal imaging in comparison to healthy individuals. Using a lipopolysaccharide-induced inflammatory model of depression in mice, we measured PAI-1 in murine plasma and brain, by ELISA and immunohistochemistry, respectively. Similar elevations were seen in plasma but not in brain homogenates of mice exposed to LPS. We further correlated the findings with depressive behavior. Ex vivo experiments with NHA treated with proinflammatory cytokines implicated in the pathogenesis of depression showed increased PAI-1 expression. Furthermore, these studies suggest that urokinase-type plasminogen activator may serve as an astrocyte PA reservoir, able to promote cleavage of brain-derived neurotrophic factor (BDNF) during stress or inflammation. In summary, our findings confirm that derangements of PAI-1 variably occur in the brain in association with the depressive phenotype. These derangements may impede the availability of active, mature (m)BDNF and thereby promote a depressive phenotype.

Genetic evidence for a phosphorylation-independent signal transduction mechanism within the Bacillus subtilis stressosome.

The stressosome is a 1.8 MDa cytoplasmic complex that controls diverse bacterial signaling pathways. Its role is best understood in Bacillus subtilis, where it activates the σB transcription factor in response to a variety of sharp environmental challenges, including acid, ethanol, heat or salt stress. However, details of the signaling mechanism within the stressosome remain uncertain. The core of the complex comprises one or more members of the RsbR co-antagonist family together with the RsbS antagonist protein, which binds the RsbT kinase in the absence of stress. As part of the response, RsbT first phosphorylates the RsbRA co-antagonist on T171 and then RsbS on S59; this latter event correlates with the stress-induced release of RsbT to activate downstream signaling. Here we examine the in vivo consequence of S59 phosphorylation in a model strain whose stressosome core is formed solely with the RsbRA co-antagonist and RsbS. A phosphorylation-deficient S59A substitution in RsbS blocked response to mild stress but had declining impact as stress increased: with strong ethanol challenge response with S59A was 60% as robust as with wild type RsbS. Genetic analysis narrowed this S59-independent activation to the stressosome and established that significant signaling still occurred in a strain bearing both the T171A and S59A substitutions. We infer that S59 phosphorylation increases signaling efficiency but is not essential, and that a second (or underlying) mechanism of signal transduction prevails in its absence. This interpretation nullifies models in which stressosome signaling is solely mediated by control of RsbT kinase activity toward S59.

The structure of the NasR transcription antiterminator reveals a one-component system with a NIT nitrate receptor coupled to an ANTAR RNA-binding effector.

The nitrate- and nitrite-sensing NIT domain is present in diverse signal-transduction proteins across a wide range of bacterial species. NIT domain function was established through analysis of the Klebsiella oxytoca NasR protein, which controls expression of the nasF operon encoding enzymes for nitrite and nitrate assimilation. In the presence of nitrate or nitrite, the NasR protein inhibits transcription termination at the factor-independent terminator site in the nasF operon transcribed leader region. We present here the crystal structure of the intact NasR protein in the apo state. The dimeric all-helical protein contains a large amino-terminal NIT domain that associates two four-helix bundles, and a carboxyl-terminal ANTAR (AmiR and NasR transcription antitermination regulator) domain. The analysis reveals unexpectedly that the NIT domain is structurally similar to the periplasmic input domain of the NarX two-component sensor that regulates nitrate and nitrite respiration. This similarity suggests that the NIT domain binds nitrate and nitrite between two invariant arginyl residues located on adjacent alpha helices, and results from site-specific mutagenesis showed that these residues are critical for NasR function. The resulting structural movements in the NIT domain would provoke an active configuration of the ANTAR domains necessary for specific leader mRNA binding.

Two surfaces of a conserved interdomain linker differentially affect output from the RST sensing module of the Bacillus subtilis stressosome.

The stressosome is a 1.8-MDa cytoplasmic complex that conveys environmental signals to the σ(B) stress factor of Bacillus subtilis. A functionally irreducible complex contains multiple copies of three proteins: the RsbRA coantagonist, RsbS antagonist, and RsbT serine-threonine kinase. Homologues of these proteins are coencoded in different genome contexts in diverse bacteria, forming a versatile sensing and transmission module called RST after its common constituents. However, the signaling pathway within the stressosome itself is not well defined. The N-terminal, nonheme globin domains of RsbRA project from the stressosome and are presumed to channel sensory input to the C-terminal STAS domains that form the complex core. A conserved, 13-residue α-helical linker connects these domains. We probed the in vivo role of the linker using alanine scanning mutagenesis, assaying stressosome output in B. subtilis via a σ(B)-dependent reporter fusion. Substitutions at four conserved residues increased output 4- to 30-fold in unstressed cells, whereas substitutions at four nonconserved residues significantly decreased output. The periodicity of these effects supports a model in which RsbRA functions as a dimer in vivo, with the linkers forming parallel paired helices via a conserved interface. The periodicity further suggests that the opposite, nonconserved faces make additional contacts important for efficient stressosome operation. These results establish that the linker influences stressosome output under steady-state conditions. However, the stress response phenotypes of representative linker substitutions provide less support for the notion that the N-terminal globin domain senses acute environmental challenge and transmits this information via the linker helix.

Substitutions in the presumed sensing domain of the Bacillus subtilis stressosome affect its basal output but not response to environmental signals.

The stressosome is a multiprotein, 1.8-MDa icosahedral complex that transmits diverse environmental signals to activate the general stress response of Bacillus subtilis. The way in which it senses these cues and the pathway of signal propagation within the stressosome itself are poorly understood. The stressosome core consists of four members of the RsbR coantagonist family together with the RsbS antagonist; its cryo-electron microscopy (cryoEM) image suggests that the N-terminal domains of the RsbR proteins form homodimers positioned to act as sensors on the stressosome surface. Here we probe the role of the N-terminal domain of the prototype coantagonist RsbRA by making structure-based amino acid substitutions in potential interaction surfaces. To unmask the phenotypes caused by single-copy rsbRA mutations, we constructed strains lacking the other three members of the RsbR coantagonist family and assayed system output using a reporter fusion. Effects of five individual alanine substitutions in the prominent dimer groove did not match predictions from an earlier in vitro assay, indicating that the in vivo assay was necessary to assess their influence on signaling. Additional substitutions expected to negatively affect domain dimerization had substantial impact, whereas those that sampled other prominent surface features had no consequence. Notably, even mutations resulting in significantly altered phenotypes raised the basal level of system output only in unstressed cells and had little effect on the magnitude of subsequent stress signaling. Our results provide evidence that the N-terminal domain of the RsbRA coantagonist affects stressosome function but offer no direct support for the hypothesis that it is a signal sensor.

The blue-light receptor YtvA acts in the environmental stress signaling pathway of Bacillus subtilis.

The general stress response of the bacterium Bacillus subtilis is regulated by a partner-switching mechanism in which serine and threonine phosphorylation controls protein interactions in the stress-signaling pathway. The environmental branch of this pathway contains a family of five paralogous proteins that function as negative regulators. Here we present genetic evidence that a sixth paralog, YtvA, acts as a positive regulator in the same environmental signaling branch. We also present biochemical evidence that YtvA and at least three of the negative regulators can be isolated from cell extracts in a large environmental signaling complex. YtvA differs from these associated negative regulators by its flavin mononucleotide (FMN)-containing light-oxygen-voltage domain. Others have shown that this domain has the photochemistry expected for a blue-light sensor, with the covalent linkage of the FMN chromophore to cysteine 62 composing a critical part of the photocycle. Consistent with the view that light intensity modifies the output of the environmental signaling pathway, we found that cysteine 62 is required for YtvA to exert its positive regulatory role in the absence of other stress. Transcriptional analysis of the ytvA structural gene indicated that it provides the entry point for at least one additional environmental input, mediated by the Spx global regulator of disulfide stress. These results support a model in which the large signaling complex serves to integrate multiple environmental signals in order to modulate the general stress response.

In vivo phosphorylation of partner switching regulators correlates with stress transmission in the environmental signaling pathway of Bacillus subtilis.

Exposure of bacteria to diverse growth-limiting stresses induces the synthesis of a common set of proteins which provide broad protection against future, potentially lethal stresses. Among Bacillus subtilis and its relatives, this general stress response is controlled by the sigmaB transcription factor. Signals of environmental and energy stress activate sigmaB through a multicomponent network that functions via a partner switching mechanism, in which protein-protein interactions are governed by serine and threonine phosphorylation. Here, we tested a central prediction of the current model for the environmental signaling branch of this network. We used isoelectric focusing and immunoblotting experiments to determine the in vivo phosphorylation states of the RsbRA and RsbS regulators, which act in concert to negatively control the RsbU environmental signaling phosphatase. As predicted by the model, the ratio of the phosphorylated to unphosphorylated forms of both RsbRA and RsbS increased in response to salt or ethanol stress. However, these two regulators differed substantially with regard to the extent of their phosphorylation under both steady-state and stress conditions, with RsbRA always the more highly modified. Mutant analysis showed that the RsbT kinase, which is required for environmental signaling, was also required for the in vivo phosphorylation of RsbRA and RsbS. Moreover, the T171A alteration of RsbRA, which blocks environmental signaling, also blocked in vivo phosphorylation of RsbRA and impeded phosphorylation of RsbS. These in vivo results corroborate previous genetic analyses and link the phosphorylated forms of RsbRA and RsbS to the active transmission of environmental stress signals.

A multicomponent protein complex mediates environmental stress signaling in Bacillus subtilis.

Activity of the general stress transcription factor sigma(B) of Bacillus subtilis is regulated directly by a partner-switching mechanism in which key protein interactions are governed by serine phosphorylation. Signals of energy or environmental stress are conveyed to sigma(B) by independent pathways, each terminating with a differentially regulated serine phosphatase, whose activity is required to control the partner-switching regulators. We present genetic and biochemical evidence that activation of the RsbU environmental signaling phosphatase is modulated by a second, atypical partner switch that comprises redundant negative regulatory proteins in a large, multicomponent signaling complex. In the current model, negative regulation of the RsbU phosphatase depends solely on the RsbS antagonist protein. Here, we perform a critical genetic test that invalidates this model and demonstrates that the RsbS antagonist alone is insufficient to prevent environmental signaling. Also required is one of a family of four co-antagonist proteins, here renamed RsbRA, RsbRB, RsbRC, and RsbRD, each with a carboxyl-terminal domain closely resembling the entire RsbS protein. Because any single member of the RsbR family, together with RsbS, was sufficient for environmental signaling, we conclude that the RsbR proteins serve as redundant co-antagonists necessary for RsbS antagonist function. Moreover, purification of RsbRA from cell extracts by nickel affinity and gel-filtration chromatography found a multicomponent complex containing the RsbRA and RsbRB co-antagonists together with the RsbS antagonist. We propose that this complex serves as a machine to transmit stress signals to sigma(B), and that the properties of the complex may contribute to environmental stress sensing.

The PrpC serine-threonine phosphatase and PrkC kinase have opposing physiological roles in stationary-phase Bacillus subtilis cells.

Loss of the PrpC serine-threonine phosphatase and the associated PrkC kinase of Bacillus subtilis were shown to have opposite effects on stationary-phase physiology by differentially affecting cell density, cell viability, and accumulation of beta-galactosidase from a general stress reporter fusion. These pleiotropic effects suggest that PrpC and PrkC have important regulatory roles in stationary-phase cells. Elongation factor G (EF-G) was identified as one possible target of the PrpC and PrkC pair in vivo, and purified PrpC and PrkC manifested the predicted phosphatase and kinase activities against EF-G in vitro.