Extended JAK activation and delayed STAT1 dephosphorylation contribute to the distinct signaling profile of CNS neurons exposed to interferon-gamma.

Although interferon-gamma (IFN-γ) plays a critical role in the noncytolytic elimination of many neurotropic viral infections, the signaling response to this cytokine has not been extensively characterized in primary CNS neurons. We previously demonstrated that the IFN-γ response at the signaling and gene expression levels is temporally extended in primary mouse hippocampal neurons, as compared to the transient response of primary mouse embryonic fibroblasts (MEF). We hypothesize that the protracted kinetics of STAT1 phosphorylation in IFN-γ-treated neurons are due to extended receptor activation and/or delayed STAT1 dephosphorylation in the nucleus. Here, we show that in response to IFN-γ, the Janus kinases (JAK1/JAK2) associated with the neuronal IFN-γ receptor complex remain active for an extended period as compared to MEF. Experimental inactivation of JAK1/JAK2 in neurons after IFN-γ treatment did not reverse the extended STAT1 phosphorylation phenotype. These results suggest that the extended kinetics of neuronal IFN-γ signaling are a product of distinct negative feedback mechanisms operating at both the receptor and within the nucleus.

STAT1-independent control of a neurotropic measles virus challenge in primary neurons and infected mice.

Neurons are chiefly nonrenewable; thus, cytolytic immune strategies to clear or control neurotropic viral infections could have lasting neurologic consequences. IFN-γ is a potent antiviral cytokine that is critical for noncytolytic clearance of multiple neurotropic viral infections, including measles virus (MV); however, the downstream pathways through which IFN-γ functions in neurons have not been defined. Unlike most cell types studied to date in which IFN-γ affects gene expression via rapid and robust activation of STAT1, basal STAT1 levels in primary hippocampal neurons are constitutively low, resulting in attenuated STAT1 activation and consequently slower kinetics of IFN-γ-driven STAT1-dependent gene expression. Given this altered expression and activation of STAT1 in neurons, we sought to determine whether STAT1 was required for IFN-γ-mediated protection from infection in neurons. To do so, we evaluated the consequences of MV challenge of STAT1-deficient mice and primary hippocampal neurons explanted from these mice. Surprisingly, the absence of STAT1 did not restrict the ability of IFN-γ to control viral infection either in vivo or ex vivo. Moreover, the canonical IFN-γ-triggered STAT1 gene expression profile was not induced in STAT1-deficient neurons, suggesting that IFN-γ regulates neuronal STAT1-independent pathways to control viral replication.

Altered levels of STAT1 and STAT3 influence the neuronal response to interferon gamma.

As immune responses in the CNS are highly regulated, cell-specific differences in IFNgamma signaling may be integral in dictating the outcome of host cell responses. In comparing the response of IFNgamma-treated primary neurons to control MEF, we observed that neurons demonstrated lower basal expression of both STAT1 and STAT3, the primary signal transducers responsible for IFNgamma signaling. Following IFNgamma treatment of these cell populations, we noted muted and delayed STAT1 phosphorylation, no detectable STAT3 phosphorylation, and a 3-10-fold lower level of representative IFNgamma-responsive gene transcripts. Moreover, in response to a brief pulse of IFNgamma, a steady increase in STAT1 phosphorylation and IFNgamma gene expression over 48 h was observed in neurons, as compared to rapid attenuation in MEF. These distinct response kinetics in IFNgamma-stimulated neurons may reflect modifications in the IFNgamma negative feedback loop, which may provide a mechanism for the cell-specific heterogeneity of responses to IFNgamma.

The Haloferax volcanii FtsY homolog is critical for haloarchaeal growth but does not require the A domain.

The targeting of many Sec substrates to the membrane-associated translocation pore requires the cytoplasmic signal recognition particle (SRP). In Eukarya and Bacteria it has been shown that membrane docking of the SRP-substrate complex occurs via the universally conserved SRP receptor (Sralpha/beta and FtsY, respectively). While much has been learned about the archaeal SRP in recent years, few studies have examined archaeal Sralpha/FtsY homologs. In the present study the FtsY homolog of Haloferax volcanii was characterized in its native host. Disruption of the sole chromosomal copy of ftsY in H. volcanii was possible only under conditions where either the full-length haloarchaeal FtsY or an amino-terminally truncated version of this protein lacking the A domain, was expressed in trans. Subcellular fractionation analysis of H. volcanii ftsY deletion strains expressing either one of the complementing proteins revealed that in addition to a cytoplasmic pool, both proteins cofractionate with the haloarchaeal cytoplasmic membrane. Moreover, membrane localization of the universally conserved SRP subunit SRP54, the key binding partner of FtsY, was detected in both H. volcanii strains. These analyses suggest that the H. volcanii FtsY homolog plays a crucial role but does not require its A domain for haloarchaeal growth.

Prokaryotic utilization of the twin-arginine translocation pathway: a genomic survey.

The twin-arginine translocation (Tat) pathway, which has been identified in plant chloroplasts and prokaryotes, allows for the secretion of folded proteins. However, the extent to which this pathway is used among the prokaryotes is not known. By using a genomic approach, a comprehensive list of putative Tat substrates for 84 diverse prokaryotes was established. Strikingly, the results indicate that the Tat pathway is utilized to highly varying extents. Furthermore, while many prokaryotes use this pathway predominantly for the secretion of redox proteins, analyses of the predicted substrates suggest that certain bacteria and archaea secrete mainly nonredox proteins via the Tat pathway. While no correlation was observed between the number of Tat machinery components encoded by an organism and the number of predicted Tat substrates, it was noted that the composition of this machinery was specific to phylogenetic taxa.

Adaptation of protein secretion to extremely high-salt conditions by extensive use of the twin-arginine translocation pathway.

Halophilic archaea thrive in environments with salt concentrations approaching saturation. However, little is known about the way in which these organisms stabilize their secreted proteins in such 'hostile' conditions. Here, we present data suggesting that the utilization of protein translocation pathways for protein secretion by the Halobacteriaceae differs significantly from that of non-haloarchaea, and most probably represents an adaptation to the high-salt environment. Although most proteins are secreted via the general secretion (Sec) machinery, the twin-arginine translocation (Tat) pathway is mainly used for the secretion of redox proteins and is distinct from the Sec pathway, in that it allows cytoplasmic folding of secreted proteins. tatfind (developed in this study) was used for systematic whole-genome analysis of Halobacterium sp. NRC-1 and several other prokaryotes to identify putative Tat substrates. Our analyses revealed that the vast majority of haloarchaeal secreted proteins were predicted substrates of the Tat pathway. Strikingly, most of these putative Tat substrates were non-redox proteins, the homologues of which in non-haloarchaea were identified as putative Sec substrates. We confirmed experimentally that the secretion of one such putative Tat substrate depended on the twin-arginine motif in its signal sequence. This extensive utilization of the Tat pathway in haloarchaea suggests an evolutionary adaptation to high-salt conditions by allowing cytoplasmic folding of secreted proteins before their secretion.

Decorin suppresses tumor cell-mediated angiogenesis.

The progressive growth of most neoplasms is dependent upon the establishment of new blood vessels, a process regulated by tumor-secreted factors and matrix proteins. We examined the in vitro and in vivo angiogenic ability of conditioned media obtained from fibrosarcoma, carcinoma, and osteosarcoma cells and their decorin-transfected counterparts. Human endothelial cells were investigated in vitro by evaluating three essential steps of angiogenesis: migration, attachment, and differentiation. On the whole, wild-type tumor cell-secretions enhanced endothelial cell attachment, migration, and differentiation, whereas their decorin-expressing forms inhibited these processes. Similarly, decorin-containing media suppressed endothelial cell sprouting in an ex vivo aortic ring assay. Since angiogenesis is an important component of tumor expansion, the growth rate of these cells as tumor xenografts was examined by implantation in nude mice. In vivo, the decorin-expressing tumor xenografts grew at markedly lower rates and showed a significant suppression of neovascularization. Immunohistochemical, Northern and Western blot analyses indicated that the decorin-expressing cells produced vascular endothelial growth factor (VEGF) at markedly reduced rates vis-á-vis their wild-type counterparts. Specificity of this process was confirmed by experiments where addition of recombinant decorin to the wild-type tumor cells caused 80-95% suppression of VEGF mRNA and protein. These results provide a novel mechanism of action for decorin, and indicate that decorin could adversely affect in vivo tumor growth by suppressing the endogenous tumor cell production of a powerful angiogenic stimulus.

In vivo analysis of an essential archaeal signal recognition particle in its native host.

The evolutionarily conserved signal recognition particle (SRP) plays an integral role in Sec-mediated cotranslational protein translocation and membrane protein insertion, as it has been shown to target nascent secretory and membrane proteins to the bacterial and eukaryotic translocation pores. However, little is known about its function in archaea, since characterization of the SRP in this domain of life has thus far been limited to in vitro reconstitution studies of heterologously expressed archaeal SRP components identified by sequence comparisons. In the present study, the genes encoding the SRP54, SRP19, and 7S RNA homologs (hv54h, hv19h, and hv7Sh, respectively) of the genetically and biochemically tractable archaeon Haloferax volcanii were cloned, providing the tools to analyze the SRP in its native host. As part of this analysis, an hv54h knockout strain was created. In vivo characterization of this strain revealed that the archaeal SRP is required for viability, suggesting that cotranslational protein translocation is an essential process in archaea. Furthermore, a method for the purification of this SRP employing nickel chromatography was developed in H. volcanii, allowing the successful copurification of (i) Hv7Sh with a histidine-tagged Hv54h, as well as (ii) Hv54h and Hv7Sh with a histidine-tagged Hv19h. These results provide the first in vivo evidence that these components interact in archaea. Such copurification studies will provide insight into the significance of the similarities and differences of the protein-targeting systems of the three domains of life, thereby increasing knowledge about the recognition of translocated proteins in general.