Paracrine neuroprotective effect of nitric oxide in the developing retina

The retina of newborn rats consists of the ganglion cell layer (GCL), the inner plexiform layer (IPL), the inner nuclear layer (INL) containing amacrine cells and the neuroblastic layer (NBL). In retinal explants, the GCL enters cell death after sectioning of the optic nerve, whereas there is almost no cell death in the NBL. When protein synthesis is inhibited with anisomycin, cell death is blocked in the GCL and induced in the NBL. We tested the roles of nitric oxide (NO) on cell death in the retina in vitro. Either L-arginine, the substrate for NO synthase or the NO donor S:-nitroso-acetylpenicillamine (SNAP) blocked cell death induced by anisomycin in the NBL, but had no effect in the GCL. Sepiapterin, a precursor of the nitric oxide synthase (NOS)-cofactor tetrahydrobiopterin also had a protective effect against anisomycin. The use of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble form of guanylyl cyclase, showed that anti-apoptotic effect of SNAP is partially mediated by cGMP generated by activation of guanylyl cyclase. NADPH-diaphorase histochemistry stained cells only in the GCL and INL. Thus, the degenerative effect of anisomycin is observed within the NBL, whereas the localization of NOS is restricted to the GCL and INL. The protective effect of both the NO substrate and cofactor upon cell death induced by anisomycin in the NBL, indicates that NO produced by amacrine and ganglion cells is a paracrine modulator of cell death within the retinal tissue.     

Bacterial lipopolysaccharide induces expression of the stress response genes hop and H411.

CD14-transfected Chinese hamster ovary K1 fibroblasts (CHO/CD14) respond to lipopolysaccharide (LPS) by metabolizing arachidonic acid and with translocation of NF-kappaB to the nucleus. Although previous experiments failed to identify the production of tumor necrosis factor-alpha and interleukin (IL)-1beta by CHO/CD14 cells, LPS did induce the expression of IL-6 mRNA and the subsequent release of the IL-6 protein. To identify additional LPS-inducible genes, a cDNA library derived from LPS-stimulated CHO/CD14 cells was screened by subtractive hybridization. Fourteen genes were found to be expressed differentially, and two were analyzed in detail: hop (Hsp70/Hsp90-organizing protein), which is the hamster homologue of the stress-inducible yeast gene, STI1, and clone H411, which encodes a novel LPS-inducible growth factor. In response to LPS, the expression of Hop mRNA was also increased in both the murine macrophage cell line, RAW 264.7, as well as in primary hamster macrophages. This suggested that the up-regulation of Hop expression is part of the macrophage stress response to LPS. Clone H411 encodes a protein in the epidermal growth factor-like repeat protein family. Overexpression of H411 cDNA in the RAW 264.7 macrophage cell line promoted an increased growth rate, suggesting that expression of H411 is part of the proliferative cell response to LPS. Both Hop and H411 represent novel gene products not previously recognized as part of the complex biological response to endotoxin.     

Signaling induced by hop/STI-1 depends on endocytosis

The co-chaperone hop/STI-1 is a ligand of the cell surface prion protein (PrP(C)), and their interaction leads to signaling and biological effects. Among these, hop/STI-1 induces proliferation of A172 glioblastoma cells, dependent on both PrP(C) and activation of the Erk pathway. We tested whether clathrin-mediated endocytosis affects signaling induced by hop/STI-1. Both hyperosmolarity induced by sucrose and monodansyl-cadaverine blocked Erk activity induced by hop/STI-1, without affecting the high basal Akt activity typical of A172. The endocytosis inhibitors also affected the sub-cellular distribution of phosphorylated Erk, consistent with blockade of the latter's activity. The data indicate that signaling induced by hop/STI-1 depends on endocytosis. These findings are consistent with a role of sub-cellular trafficking in signal transduction following engagement by PrP(C) by ligands such as hop/STI-1, and may help help unravel both the functions of the prion protein, as well as possible loss-of-function components of prion diseases.     

Pituitary adenylyl cyclase-activating polypeptide prevents induced cell death in retinal tissue through activation of cyclic AMP-dependent protein kinase

Multiple neuroactive substances are secreted by neurons and/or glial cells and modulate the sensitivity to cell death. In the developing retina, it has been shown that increased intracellular levels of cAMP protect cells from degeneration. We tested the hypothesis that the neuroactive peptide pituitary adenylyl cyclase-activating polypeptide (PACAP) has neuroprotective effects upon the developing rat retina. PACAP38 prevented anisomycin-induced cell death in the neuroblastic layer (NBL) of retinal explants, and complete inhibition of induced cell death was obtained with 1 nm. A similar protective effect was observed with PACAP27 and with the specific PAC1 receptor agonist maxadilan but not with glucagon. Photoreceptor cell death induced by thapsigargin was also prevented by PACAP38. The neuroprotective effect of PACAP38 upon the NBL could be reverted by the competitive PACAP receptor antagonist PACAP6-38 and by the specific PAC1 receptor antagonist Maxd.4. Molecular and immunohistochemical analysis demonstrated PAC1 receptors, and treatment with PACAP38 induced phospho-cAMP-response element-binding protein immunoreactivity in the anisomycin-sensitive undifferentiated postmitotic cells within the NBL. PACAP38 produced an increase in cAMP but not inositol triphosphate, and treatment with the cAMP-dependent protein kinase inhibitor R(p)-cAMPS blocked the protective effect of PACAP38. The results indicate that activation of PAC1 receptors by PACAP38 modulates cell death in the developing retina through the intracellular cAMP/cAMP-dependent protein kinase pathway.     

PrP cooperates with STI1 to regulate SOD activity in PrP-deficient neuronal cell line

Cellular prion protein (PrP(C)) plays anti-apoptotic and anti-oxidative roles in apoptosis induced by serum deprivation in an immortalized prion protein gene (Prnp)-deficient neuronal cell line. The octapeptide repeat region (OR) and N-terminal half of the hydrophobic region (HR) of PrP(C) are indispensable for PrP(C) activity, but the mechanisms remain unclear. In the present study, elucidation of the mechanisms by which PrP(C) elicits the anti-oxidative activities was facilitated by evidence of stress-inducible protein 1 (STI1) mediating PrP(C)-dependent superoxide dismutase (SOD) activation. Immunoprecipitation revealed that PrP(C) was associated with STI1. The inhibitory peptides against PrP(C)-STI1 binding [STI1 pep.1 and PrP(113-132)] indicated toxic activity in PrP(C)-expressing cells by inhibiting SOD activity but not in Prnp(-/-) cells. Furthermore, OR and N-terminal half of the HR were required for the inhibitory effect of PrP(113-132) but not STI1 pep.1. These data are consistent with results established with a model where OR and N-terminal half of the HR mediate the action of STI1 upon cell survival and upregulation of SOD activity.     

1H, 15N and 13C backbone resonance assignments of the TPR1 and TPR2A domains of mouse STI1

Hop/STI1 (Hsp-organizing protein/stress-induced-phosphoprotein 1) is a molecular co-chaperone, which coordinates Hsp70 and Hsp90 activity during client protein folding through interactions with its TPR1 and TPR2A domains. Hsp90 substrates include a diverse set of proteins, many of which have been implicated in tumorigenesis. Over-expression of Hsp90 in cancer cells stabilizes mutant oncoproteins promoting cancer cell survival. Disruption of Hsp90 and its co-chaperone machinery has become a promising strategy for the treatment of cancer. STI1 has also been described as a neurotrophic signaling molecule through its interactions with the prion protein (PrP^C). Here, we report the ¹H, ¹³C and ¹⁵N backbone assignments of the TPR1 and TPR2A domains of mouse STI1, which interact with Hsp70 and Hsp90, respectively. ¹H-¹⁵N HSQC spectra of TPR2A domain in the presence of a peptide encoding the C-terminal Hsp90 binding site revealed significant chemical shift changes indicating complex formation. These results will facilitate the screening of potential molecules that inhibit STI1 complex formation with Hsp70 and/or Hsp90 for the treatment of cancer and detailed structural studies of the STI1-PrP^C complex.     

Hop cleavage and function in granzyme B-induced apoptosis

Granzyme B (GzmB) is a cytotoxic protease found in the granules of natural killer cells and cytotoxic T lymphocytes. GzmB cleaves multiple intracellular protein substrates, leading to caspase activation, DNA fragmentation, cytoskeletal instability, and rapid induction of target cell apoptosis. However, no known individual substrate is required for GzmB to induce apoptosis. GzmB is therefore thought to initiate multiple cell death pathways simultaneously to ensure the death of target cells. We previously identified Hop (Hsp70/Hsp90-organizing protein) as a GzmB substrate in a proteomic survey (Bredemeyer, A. J., Lewis, R. M., Malone, J. P., Davis, A. E., Gross, J., Townsend, R. R., and Ley, T. J. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 11785-11790). Hop is a co-chaperone for Hsp70 and Hsp90, which have been implicated in the negative regulation of apoptosis. We therefore hypothesized that Hop may have an anti-apoptotic function that is abolished upon cleavage, lowering the threshold for GzmB-induced apoptosis. Here, we show that Hop was cleaved directly by GzmB in vitro and in cells undergoing GzmB-induced apoptosis. Expression of the two cleavage fragments of Hop did not induce cell death. Although cleavage of Hop by GzmB destroyed Hop function in vitro, both cells overexpressing GzmB-resistant Hop and cells with a 90-95% reduction in Hop levels exhibited unaltered susceptibility to GzmB-induced death. We conclude that Hop per se does not set the threshold for susceptibility to GzmB-induced apoptosis. Although it is possible that Hop may be cleaved by GzmB as an "innocent bystander" during the induction of apoptosis, it may also act to facilitate apoptosis in concert with other GzmB substrates.     

Multiple domains of the co-chaperone Hop are important for Hsp70 binding

The Hop/Sti1 co-chaperone binds to both Hsp70 and Hsp90. Biochemical and co-crystallographic studies have suggested that the EEVD-containing C terminus of Hsp70 or Hsp90 binds specifically to one of the Hop tetratricopeptide repeat domains, TPR1 or TPR2a, respectively. Mutational analyses of Hsp70 and Hop were undertaken to better characterize interactions between the C terminus of Hsp70 and Hop domains. Surprisingly, truncation of EEVD plus as many as 34 additional amino acids from the Hsp70 C terminus did not reduce the ability of Hsp70 mutants to co-immunoprecipitate with Hop, although further truncation eliminated Hop binding. Hop point mutations targeting a carboxylate clamp position in TPR1 disrupted Hsp70 binding, as was expected; however, similar point mutations in TPR2a or TPR2b also inhibited Hsp70 binding in some settings. Using a yeast-based in vivo assay for Hop function, wild type Hop and TPR2b mutants could fully complement deletion of Sti1p; TPR1 and TPR2a point mutants could partially restore activity. Conformations of Hop and Hop mutants were probed by limited proteolysis. The TPR1 mutant digested in a similar manner to wild type; however, TPR2a and TPR2b mutants each displayed greater resistance to chymotryptic digestion. All point mutants retained an ability to dimerize, and none appeared to be grossly misfolded. These results raise questions about current models for Hop/Hsp70 interaction.     

Domain:domain interactions within Hop, the Hsp70/Hsp90 organizing protein, are required for protein stability and structure

The major heat shock protein (Hsp) chaperones Hsp70 and Hsp90 both bind the co-chaperone Hop (Hsp70/Hsp90 organizing protein), which coordinates Hsp actions in folding protein substrates. Hop contains three tetratricopeptide repeat (TPR) domains that have binding sites for the conserved EEVD C termini of Hsp70 and Hsp90. Crystallographic studies have shown that EEVD interacts with positively charged amino acids in Hop TPR-binding pockets (called carboxylate clamps), and point mutations of these carboxylate clamp positions can disrupt Hsp binding. In this report, we use circular dichroism to assess the effects of point mutations and Hsp70/Hsp90 peptide binding on Hop conformation. Our results show that Hop global conformation is destabilized by single point mutations in carboxylate clamp positions at pH 5, while the structure of individual TPR domains is unaffected. Binding of peptides corresponding to the C termini of Hsp70 and Hsp90 alters the global conformation of wild-type Hop, whereas peptide binding does not alter conformation of individual TPR domains. These results provide biophysical evidence that Hop-binding pockets are directly involved with domain:domain interactions, both influencing Hop global conformation and Hsp binding, and contributing to proper coordination of Hsp70 and Hsp90 interactions with protein substrates.     

Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection

Prions are composed of an isoform of a normal sialoglycoprotein called PrP(c), whose physiological role has been under investigation, with focus on the screening for ligands. Our group described a membrane 66 kDa PrP(c)-binding protein with the aid of antibodies against a peptide deduced by complementary hydropathy. Using these antibodies in western blots from two-dimensional protein gels followed by sequencing the specific spot, we have now identified the molecule as stress-inducible protein 1 (STI1). We show that this protein is also found at the cell membrane besides the cytoplasm. Both proteins interact in a specific and high affinity manner with a K(d) of 10(-7) M. The interaction sites were mapped to amino acids 113-128 from PrP(c) and 230-245 from STI1. Cell surface binding and pull-down experiments showed that recombinant PrP(c) binds to cellular STI1, and co-immunoprecipitation assays strongly suggest that both proteins are associated in vivo. Moreover, PrP(c) interaction with either STI1 or with the peptide we found that represents the binding domain in STI1 induce neuroprotective signals that rescue cells from apoptosis.     

Serum withdrawal-induced apoptosis in ZrchI prion protein (PrP) gene-deficient neuronal cell line is suppressed by PrP, independent of Doppel

Previous studies have shown that cellular prion protein (PrP(C)) plays anti-apoptotic and antioxidative role against cell death induced by serum-deprivation (SDP) in an immortalized prion protein gene-deficient neuronal cell line derived from Rikn prion protein (PrP) gene-deficient (Prnp(-/-)) mice, which ectopically produce excess Doppel (Dpl) (PrP-like glycoprotein). To investigate whether PrP(C) inhibits apoptotic neuronal cell death without Dpl, an immortalized cell line was established from the brain of ZrchI Prnp(-/-) mice, which do not show ectopic expression of Dpl. The results using a ZrchI neuronal Prnp(-/-) cell line (NpL2) showed that PrP(C) potently inhibited SDP-induced apoptotic cell death. Furthermore, PrP(C) expression enhanced the superoxide dismutase (SOD) activity in NpL2 cells. These results indicate that Dpl production did not affect anti-apoptotic and anti-oxidative functions of PrP, suggesting that PrP(C) may be directly correlated with protection against oxidative stress.     

The Non-canonical Hop Protein from Caenorhabditis elegans Exerts Essential Functions and Forms Binary Complexes with Either Hsc70 or Hsp90

Heat shock protein (Hsp) 70/Hsp90-organizing proteins (Hop/Sti1) are thought to function as adaptor proteins to link the two chaperone machineries Hsp70 and Hsp90 during the processing of substrate proteins in eukaryotes. Hop (Hsp70/Hsp90-organizing protein) is composed of three tetratricopeptide repeat (TPR) domains, of which the first (TPR1) binds to Hsp70, the second (TPR2A) binds to Hsp90, and the third (TPR2B) is of unknown function. Contrary to most other eukaryotes, the homologue closest to the Caenorhabditis elegans Hop homologue R09E12.3 (CeHop) lacks the TPR1 domain and the short linker region connecting it to TPR2A, questioning the reported function as an Hsp90/Hsp70 adaptor in vitro and in vivo. We observed high constitutive expression levels of CeHop and detected significant phenotypes upon knockdown, linking the protein to functions in gonad development. Interestingly, we observed physical interactions with both chaperones Hsp70 and Hsp90, albeit only the interaction with Hsp90 is strong and inhibition of the Hsp90 ATPase activity can be observed upon binding of CeHop. However, the formation of ternary complexes with both chaperone machineries is impaired, as Hsp70 and Hsp90 compete for CeHop interaction sites, in particular as Hsp90 binds to both TPR domains simultaneously and requires both TPR domains for ATPase regulation. These results imply that, at least in C. elegans, essential functions of Hop exist which apparently do not depend on the simultaneous binding of Hsp90 and Hsp70 to Hop.     

The cochaperone murine stress-inducible protein 1: overexpression, purification, and characterization

Murine stress-inducible protein 1 (mSTI1) is a cochaperone that is homologous with the human heat shock cognate protein 70 (Hsc70)/heat shock protein 90 (Hsp90)-organizing protein (Hop). To analyze the biochemical properties of mSTI1 and the stoichiometry of the Hsc70.mSTI1.Hsp90 association, recombinant mSTI1 was produced in untagged, histidine (His)-tagged, and glutathione S-transferase (GST)-tagged forms. His-mSTI1 was detected either as a dimer during size-exclusion-high-performance liquid chromatography (SE-HPLC) or as a monomer during Superdex 200 gel filtration chromatography. SE-HPLC on GST-mSTI1 and untagged mSTI1 suggested that mSTI1 existed as a monomer. Cross-linking of His-mSTI1 detected a compact monomeric species and a dimeric species. Gel filtration on the association of bovine STI1 or His-mSTI1 with Hsc70 detected species of molecular mass consistent with a dimeric STI1 species or a 1:1 complex of STI1 and Hsc70. Our data and that of others suggest that mSTI1 and its homologues exist as either a monomer or a dimer and that this facilitates its proposed function as an Hsc70/Hsp90 organizing protein.     

Partner choice during meiosis is regulated by Hop1-promoted dimerization of Mek1

Meiotic recombination differs from mitotic recombination in that DSBs are repaired using homologous chromosomes, rather than sister chromatids. This change in partner choice is due in part to a barrier to sister chromatid repair (BSCR) created by the meiosis-specific kinase, Mek1, in a complex with two other meiosis-specific proteins, Hop1 and Red1. HOP1 contains two functional domains, called the N and C domains. Analysis of a point mutation that specifically inactivates the C domain (hop1-K593A) reveals that the N domain is sufficient for Hop1 localization to chromosomes and for Red1 and Hop1 interactions. The C domain is needed for spore viability, for chromosome synapsis, and for preventing DMC1-independent DSB repair, indicating it plays a role in the BSCR. All of the hop1-K593A phenotypes can be bypassed by fusion of ectopic dimerization domains to Mek1, suggesting that the function of the C domain is to promote Mek1 dimerization. Hop1 is a DSB-dependent phosphoprotein, whose phosphorylation requires the presence of the C domain, but is independent of MEK1. These results suggest a model in which Hop1 phosphorylation in response to DSBs triggers dimerization of Mek1 via the Hop1 C domain, thereby enabling Mek1 to phosphorylate target proteins that prevent repair of DSBs by sister chromatids.     

Functional coevolutionary networks of the Hsp70-Hop-Hsp90 system revealed through computational analyses

Currently, the identification of groups of amino acid residues that are important in the function, structure, or interaction of a protein can be both costly and prohibitively complex, involving vast numbers of mutagenesis experiments. Here, we present the application of a novel computational method, which identifies the presence of coevolution in a data set, thereby enabling the a priori identification of amino acid residues that play an important role in protein function. We have applied this method to the heat shock protein (Hsp) protein-folding system, studying the network between Hsp70, Hsp90, and Hop (heat shock-organizing protein). Our analysis has identified functional residues within the tetratricopeptide repeat (TPR) 1 and 2A domains in Hop, previously shown to be interacting with Hsp70 and Hsp90, respectively. Further, we have identified significant residues elsewhere in Hop within domains that have been recently proposed as being important for Hop interaction with Hsp70 and/or Hsp90. In addition, several amino acid sites present in groups of coevolution were identified as 3-dimensionally or linearly proximal to functionally important sites or domains. Based on our results, we also investigate a further functional domain within Hop, between TPR1 and TPR2A, which we suggest as being functionally important in the interaction of Hop with both Hsp70 and Hsp90 whether directly or otherwise. Our method has identified all the previously characterized functionally important regions in this system, thereby indicating the power of this method in the a priori identification of important regions for site-directed mutagenesis studies.     

The Drosophila Jak kinase hopscotch is required for multiple developmental processes in the eye.

Jak kinases are critical signaling components in hematopoiesis. While a large number of studies have been conducted on the roles of Jak kinases in the hematopoietic cells, much less is known about the requirements for these tyrosine kinases in other tissues. We have used loss of function mutations in the Drosophila Jak kinase Hopscotch (Hop) to determine the role of Hop in eye development. We find that Hop is required for cell proliferation/survival in the eye imaginal disc, for the differentiation of photoreceptor cells, and for the establishment of the equator and of ommatidial polarity. These results indicate that hop activity is required for multiple developmental processes in the eye, both cell-autonomously and nonautonomously.