Mitochondrial import receptors Tom20 and Tom22 have chaperone-like activity

Mitochondrial preproteins are synthesized in the cytosol with N-terminal signal sequences (presequences) or internal targeting signals. Generally, preproteins with presequences are initially recognized by Tom20 (translocase of the outer membrane) and, subsequently, by Tom22, whereas hydrophobic preproteins with internal targeting signals are first recognized by Tom70. Recent studies suggest that Tom70 associates with molecular chaperones, thereby maintaining their substrate preproteins in an import-competent state. However, such a function has not been reported for other Tom component(s). Here, we investigated a role for Tom20 in preventing substrate preproteins from aggregating. In vitro binding assays showed that Tom20 binds to guanidinium chloride unfolded substrate proteins regardless of the presence or absence of presequences. This suggests that Tom20 functions as a receptor not only for presequences but also for mature portions exposed in unfolded preproteins. Aggregation suppression assays on citrate synthase showed that the cytosolic domain of Tom20 has a chaperone-like activity to prevent this protein from aggregating. This activity was inhibited by a presequence peptide, suggesting that the binding site of Tom20 for presequence is identical or close to the active site for the chaperone-like activity. The cytosolic domain of Tom22 also showed a similar activity for citrate synthase, whereas Tom70 did not. These results suggest that the cytosolic domains of Tom20 and Tom22 function to maintain their substrate preproteins unfolded and prevent them from aggregating on the mitochondrial surface.     

General enzymatic screens identify three new nucleotidases in Escherichia coli. Biochemical characterization of SurE, YfbR, and YjjG

To find proteins with nucleotidase activity in Escherichia coli, purified unknown proteins were screened for the presence of phosphatase activity using the general phosphatase substrate p-nitrophenyl phosphate. Proteins exhibiting catalytic activity were then assayed for nucleotidase activity against various nucleotides. These screens identified the presence of nucleotidase activity in three uncharacterized E. coli proteins, SurE, YfbR, and YjjG, that belong to different enzyme superfamilies: SurE-like family, HD domain family (YfbR), and haloacid dehalogenase (HAD)-like superfamily (YjjG). The phosphatase activity of these proteins had a neutral pH optimum (pH 7.0-8.0) and was strictly dependent on the presence of divalent metal cations (SurE: Mn(2+) > Co(2+) > Ni(2+) > Mg(2+); YfbR: Co(2+) > Mn(2+) > Cu(2+); YjjG: Mg(2+) > Mn(2+) > Co(2+)). Further biochemical characterization of SurE revealed that it has a broad substrate specificity and can dephosphorylate various ribo- and deoxyribonucleoside 5'-monophosphates and ribonucleoside 3'-monophosphates with highest affinity to 3'-AMP. SurE also hydrolyzed polyphosphate (exopolyphosphatase activity) with the preference for short-chain-length substrates (P(20-25)). YfbR was strictly specific to deoxyribonucleoside 5'-monophosphates, whereas YjjG showed narrow specificity to 5'-dTMP, 5'-dUMP, and 5'-UMP. The three enzymes also exhibited different sensitivities to inhibition by various nucleoside di- and triphosphates: YfbR was equally sensitive to both di- and triphosphates, SurE was inhibited only by triphosphates, and YjjG was insensitive to these effectors. The differences in their sensitivities to nucleotides and their varied substrate specificities suggest that these enzymes play unique functions in the intracellular nucleotide metabolism in E. coli.     

A role for BiP as an adjustor for the endoplasmic reticulum stress-sensing protein Ire1

In the unfolded protein response, the type I transmembrane protein Ire1 transmits an endoplasmic reticulum (ER) stress signal to the cytoplasm. We previously reported that under nonstressed conditions, the ER chaperone BiP binds and represses Ire1. It is still unclear how this event contributes to the overall regulation of Ire1. The present Ire1 mutation study shows that the luminal domain possesses two subregions that seem indispensable for activity. The BiP-binding site was assigned not to these subregions, but to a region neighboring the transmembrane domain. Phenotypic comparison of several Ire1 mutants carrying deletions in the indispensable subregions suggests these subregions are responsible for multiple events that are prerequisites for activation of the overall Ire1 proteins. Unexpectedly, deletion of the BiP-binding site rendered Ire1 unaltered in ER stress inducibility, but hypersensitive to ethanol and high temperature. We conclude that in the ER stress-sensory system BiP is not the principal determinant of Ire1 activity, but an adjustor for sensitivity to various stresses.     

Degradation of NF-kappa B in T cells by gangliosides expressed on renal cell carcinomas

T cells from cancer patients are often functionally impaired, which imposes a barrier to effective immunotherapy. Most pronounced are the alterations characterizing tumor-infiltrating T cells, which in renal cell carcinomas includes defective NF-kappaB activation and a heightened sensitivity to apoptosis. Coculture experiments revealed that renal tumor cell lines induced a time-dependent decrease in RelA(p65) and p50 protein levels within both Jurkat T cells and peripheral blood T lymphocytes that coincided with the onset of apoptosis. The degradation of RelA/p50 is critical for SK-RC-45-induced apoptosis because overexpression of RelA in Jurkat cells protects against cell death. The loss of RelA/p50 coincided with a decrease in expression of the NF-kappaB regulated antiapoptotic protein Bcl-xL at both the protein and mRNA level. The disappearance of RelA/p50 protein was mediated by a caspase-dependent pathway because pretreatment of T lymphocytes with a pan caspase inhibitor before coculture with SK-RC-45 blocked RelA and p50 degradation. SK-RC-45 gangliosides appear to mediate this degradative pathway, as blocking ganglioside synthesis in SK-RC-45 cells with the glucosylceramide synthase inhibitor, PPPP, protected T cells from tumor cell-induced RelA degradation and apoptosis. The ability of the Bcl-2 transgene to protect Jurkat cells from RelA degradation, caspase activation, and apoptosis implicates the mitochondria in these SK-RC-45 ganglioside-mediated effects.     

Impaired receptor editing in the primary B cell repertoire of BASH-deficient mice

The editing of B cell Ag receptor (BCR) through successive rearrangements of Ig genes has been considered to be a major mechanism for the central B cell tolerance, which precludes appearance of self-reactive B cells, through studies using anti-self-Ig transgenic/knock-in mouse systems. However, contribution of the receptor editing in the development of the normal B cell repertoire remains unclear. In addition, the signaling pathway directing this event is unknown. In this study, we demonstrate that receptor editing in anti-DNA Ig knock-in mice is impaired in the absence of an adaptor protein BASH (BLNK/SLP-65) that is involved in BCR signaling. Remarkably, the supposed hallmarks of receptor editing such as Iglambda chain expression, recombination sequence rearrangements at Igkappa loci, and presence of in-frame VkappaJkappa joins in the Igkappa loci inactivated by the recombination sequence rearrangements, were all diminished in BASH-deficient mice with unmanipulated Ig loci. BCR ligation-induced Iglambda gene recombination in vitro was also impaired in BASH-deficient B cells. Furthermore, the BASH-deficient mice showed an excessive Ab response to a DNA carrier immunization, suggesting the presence of unedited DNA-reactive B cells in the periphery. These results not only define a signaling pathway required for receptor editing but indicate that the BCR-signaled receptor editing indeed operates in the development of normal B cell repertoire and contributes to establishing the B cell tolerance.     

Cholinergic modulation of microglial activation by alpha 7 nicotinic receptors

Almost all degenerative diseases of the CNS are associated with chronic inflammation. A central step in this process is the activation of brain mononuclear phagocyte cells, called microglia. While it is recognized that healthy neurons and astrocytes regulate the magnitude of microglia-mediated innate immune responses and limit excessive CNS inflammation, the endogenous signals governing this process are not fully understood. In the peripheral nervous system, recent studies suggest that an endogenous 'cholinergic anti-inflammatory pathway' regulates systemic inflammatory responses via alpha 7 nicotinic acetylcholinergic receptors (nAChR) found on blood-borne macrophages. These data led us to investigate whether a similar cholinergic pathway exists in the brain that could regulate microglial activation. Here we report for the first time that cultured microglial cells express alpha 7 nAChR subunit as determined by RT-PCR, western blot, immunofluorescent, and immunohistochemistry analyses. Acetylcholine and nicotine pre-treatment inhibit lipopolysaccharide (LPS)-induced TNF-alpha release in murine-derived microglial cells, an effect attenuated by alpha 7 selective nicotinic antagonist, alpha-bungarotoxin. Furthermore, this inhibition appears to be mediated by a reduction in phosphorylation of p44/42 and p38 mitogen-activated protein kinase (MAPK). Though preliminary, our findings suggest the existence of a brain cholinergic pathway that regulates microglial activation through alpha 7 nicotinic receptors. Negative regulation of microglia activation may also represent additional mechanism underlying nicotine's reported neuroprotective properties.     

Differential properties between TRK-820 and U-50,488H on the discriminative stimulus effects in rats

It has been demonstrated that the newly synthesized kappa-opioid receptor agonist TRK-820, which has a unique structure that is different from those of other prototypical kappa-opioid receptor agonists such as U-50,488H, exert some behavioral effects that differ from those induced by U-50,488H. Therefore, the present study was designed to examine the possible difference between the discriminative stimulus effects of TRK-820 and U-50,488H in rats. Substitution tests with several kappa-opioid receptor agonists were initiated in rats trained to discriminate between TRK-820 (40 microg/kg) or U-50,488H (3.0 mg/kg) and saline. In the cross-substitution tests, U-50,488H substituted for the discriminative stimulus effects of TRK-820, whereas TRK-820 did not substitute completely for those of U-50,488H, indicating that the discriminative stimulus effects of TRK-820 and U-50,488H were somewhat different. In the substitution tests, E-2078, but not R-84760, substituted for the discriminative stimulus effects of both TRK-820 and U-50,488H. KT-90, CI-977 and ICI-199441 each substituted for the discriminative stimulus effects of U-50,488H, but not to those of TRK-820. These results imply that these kappa-opioid receptor agonists possess U-50,488H-like discriminative stimulus effects. Furthermore, that U-50,488H and the other kappa-opioid receptor agonists substituted for the discriminative stimulus effects of U-50,488H, produced aversive effects in rats. These findings suggest the possibility that unlike those of TRK-820, the cue of the discriminative stimulus effects of U-50,488H may be, at least in part, associated with its aversive effects.     

Dehydroepiandrosterone decreases elevated hepatic glucose production in C57BL/KsJ-db/db mice

Dehydroepiandrosterone (DHEA) is known to improve hyperglycemia in diabetic db/db mice that are obese and insulin resistant. In a previous study, we reported that DHEA suppresses the elevated hepatic gluconeogenic glucose-6-phosphatase (G6Pase) activity and gene expression in C57BL/KsJ-db/db mice. In the present study, we evaluated the total amount of gluconeogenesis using NaH[(14)C]CO(3) and hepatic glucose production using fructose as a substrate in primary cultured hepatocytes. Despite hyperinsulinemia, the glucose production of db/db mice in the total body and hepatocytes was elevated as compared to their heterozygote littermate C57BL/KsJ-db/+m mice. Administration of DHEA significantly decreased the blood glucose level and increased the plasma insulin level in db/db mice. Administration of DHEA decreased the elevated total body and hepatic glucose production in db/db mice. In addition, the glucose production in the primary cultured hepatocytes of db/db mice was decreased significantly by the direct addition of DHEA or DHEA-S to the medium. These results suggest that administration of DHEA suppresses the elevated total body and hepatic glucose production in db/db mice, and this effect on the liver is considered to result from increased plasma insulin and DHEA or DHEA-S itself.     

The extracellular domain of p185(c-neu) induces density-dependent inhibition of cell growth in malignant mesothelioma cells and reduces growth of mesothelioma in vivo

EGFR is involved in the density-dependent inhibition of cell growth, while coexpression of EGFR with erbB2 can render normal cells transformed. In this study, we have examined the effect of a species of p185 that contains the transmembrane domain and the extracellular domain of p185(c-neu), on growth properties of a human malignant mesothelioma cell line that coexpresses EGFR and erbB2. The ectodomain form of p185(c-neu) enhanced density-dependent inhibition of cell growth and we found that p21 induction appeared to be responsible for this inhibitory effect. Previously, the extracellular domain species was shown to suppress the transforming abilities of EGFR and p185(c-neu/erbB2) in a dominant-negative manner. The ability of this subdomain to affect tumor growth is significant, as it reduced in vivo tumor growth. Unexpectedly, we found that the domain did not abrogate all of EGFR functions. We noted that EGFR-induced density-dependent inhibition of cell growth was retained. Tyrosine kinase inhibitors of EGFR did not cause density-dependent inhibition of cell growth of malignant mesothelioma cells. Therefore, simultaneously inhibiting the malignant phenotype and inducing density-dependent inhibition of cell growth in malignant mesothelioma cells by the extracellular domain of p185(c-neu) may represent an important therapeutic advance.     

Structure of the whole cytosolic region of ATP-dependent protease FtsH

An ATP-dependent protease, FtsH, digests misassembled membrane proteins in order to maintain membrane integrity and digests short-lived soluble proteins in order to control their cellular regulation. This enzyme has an N-terminal transmembrane segment and a C-terminal cytosolic region consisting of an AAA+ ATPase domain and a protease domain. Here we present two crystal structures: the protease domain and the whole cytosolic region. The cytosolic region fully retains an ATP-dependent protease activity and adopts a three-fold-symmetric hexameric structure. The protease domains displayed a six-fold symmetry, while the AAA+ domains, each containing ADP, alternate two orientations relative to the protease domain, making "open" and "closed" interdomain contacts. Apparently, ATPase is active only in the closed form, and protease operates in the open form. The protease catalytic sites are accessible only through a tunnel following from the AAA+ domain of the adjacent subunit, raising a possibility of translocation of polypeptide substrate to the protease sites through this tunnel.