Anti-DNA antibodies are a major component of the intrathecal B cell response in multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of unknown cause that afflicts the central nervous system. MS is typified by a highly clonally restricted antigen-driven antibody response that is confined largely to the central nervous system. The major antigenic targets of this response and the role of antibody in disease pathogenesis remain unclear. To help resolve these issues, we cloned the IgG repertoire directly from active plaque and periplaque regions in MS brain and from B cells recovered from the cerebrospinal fluid of a patient with MS with subacute disease. We found that high-affinity anti-DNA antibodies are a major component of the intrathecal IgG response in the patients with MS that we studied. Furthermore, we show DNA-specific monoclonal antibodies rescued from two subjects with MS as well as a DNA-specific antibody rescued from an individual suffering from systemic lupus erythematosus bound efficiently to the surface of neuronal cells and oligodendrocytes. For two of these antibodies, cell-surface recognition was DNA dependent. Our findings indicate that anti-DNA antibodies may promote important neuropathologic mechanisms in chronic inflammatory disorders, such as MS and systemic lupus erythematosus.

DNA binding sites for the Mlc and NagC proteins: regulation of nagE, encoding the N-acetylglucosamine-specific transporter in Escherichia coli

The NagC and Mlc proteins are homologous transcriptional regulators that control the expression of several phosphotransferase system (PTS) genes in Escherichia coli. NagC represses nagE, encoding the N-acetylglucosamine-specific transporter, while Mlc represses three PTS operons, ptsG, manXYZ and ptsHIcrr, involved in the uptake of glucose. NagC and Mlc can bind to each others operator, at least in vitro. A binding site selection procedure was used to try to distinguish NagC and Mlc sites. The major difference was that all selected NagC binding sites had a G or a C at positions +11/–11 from the centre of symmetry. This is also the case for most native NagC sites, but not the nagE operator, which thus looks like a potential Mlc target. The nagE operator does exhibit a higher affinity for Mlc than NagC, but no regulation of nagE by physiological concentrations of Mlc was detected in vivo. Regulation of wild-type nagE by NagC is achieved because of the chelation effect due to a second high affinity NagC operator covering the nagB promoter. Replacing the A/T at +11/–11 with C/G allows repression by NagC in the absence of the nagB operator.

Mechanism of stimulation of the DNA glycosylase activity of hOGG1 by the major human AP endonuclease: bypass of the AP lyase activity step

The generation of reactive oxygen species in the cell provokes, among other lesions, the formation of 8-oxo-7,8-dihydroguanine (8-oxoG) in DNA. Due to mispairing with adenine during replication, 8-oxoG is highly mutagenic. To minimise the mutagenic potential of this oxidised purine, human cells have a specific 8-oxoG DNA glycosylase/AP lyase (hOGG1) that initiates the base excision repair (BER) of 8-oxoG. We show here that in vitro this first enzyme of the BER pathway is relatively inefficient because of a high affinity for the product of the reaction it catalyses (half-life of the complex is >2 h), leading to a lack of hOGG1 turnover. However, the glycosylase activity of hOGG1 is stimulated by the major human AP endonuclease, HAP1 (APE1), the enzyme that performs the subsequent step in BER, as well as by a catalytically inactive mutant (HAP1-D210N). In the presence of HAP1, the AP sites generated by the hOGG1 DNA glycosylase can be occupied by the endonuclease, avoiding the re-association of hOGG1. Moreover, the glycosylase has a higher affinity for a non-cleaved AP site than for the cleaved DNA product generated by HAP1. This would shift the equilibrium towards the free glycosylase, making it available to initiate new catalytic cycles. In contrast, HAP1 does not affect the AP lyase activity of hOGG1. This stimulation of only the hOGG1 glycosylase reaction accentuates the uncoupling of its glycosylase and AP lyase activities. These data indicate that, in the presence of HAP1, the BER of 8-oxoG residues can be highly efficient by bypassing the AP lyase activity of hOGG1 and thus excluding a potentially rate limiting step.

Evaluation of the role of heterogeneous nuclear ribonucleoprotein A1 as a host factor in murine coronavirus discontinuous transcription and genome replication

Viruses with RNA genomes often capture and redirect host cell components to assist in mechanisms particular to RNA-dependent RNA synthesis. The nidoviruses are an order of positive-stranded RNA viruses, comprising coronaviruses and arteriviruses, that employ a unique strategy of discontinuous transcription, producing a series of subgenomic mRNAs linking a 5′ leader to distal portions of the genome. For the prototype coronavirus mouse hepatitis virus (MHV), heterogeneous nuclear ribonucleoprotein (hnRNP) A1 has been shown to be able to bind in vitro to the negative strand of the intergenic sequence, a cis-acting element found in the leader RNA and preceding each downstream ORF in the genome. hnRNP A1 thus has been proposed as a host factor in MHV transcription. To test this hypothesis genetically, we initially constructed MHV mutants with a very high-affinity hnRNP A1 binding site inserted in place of, or adjacent to, an intergenic sequence in the MHV genome. This inserted hnRNP A1 binding site was not able to functionally replace, or enhance transcription from, the intergenic sequence. This finding led us to test more directly the role of hnRNP A1 by analysis of MHV replication and RNA synthesis in a murine cell line that does not express this protein. The cellular absence of hnRNP A1 had no detectable effect on the production of infectious virus, the synthesis of genomic RNA, or the quantity or quality of subgenomic mRNAs. These results strongly suggest that hnRNP A1 is not a required host factor for MHV discontinuous transcription or genome replication.

Brain 5α-dihydroprogesterone and allopregnanolone synthesis in a mouse model of protracted social isolation

Allopregnanolone (ALLO), is a brain endogenous neurosteroid that binds with high affinity to γ-aminobutyric acid type A (GABAA) receptors and positively modulates the action of GABA at these receptors. Unlike ALLO, 5α-dihydroprogesterone (5α-DHP) binds with high affinity to intracellular progesterone receptors that regulate DNA transcription. To investigate the physiological roles of ALLO and 5α-DHP synthesized in brain, we have adopted a mouse model involving protracted social isolation. In the frontal cortex of mice, socially isolated for 6 weeks, both neurosteroids were decreased by approximately 50%. After administration of (17β)-17-(bis-1-methyl amino carbonyl) androstane-3,5-diene-3-carboxylic acid (SKF105,111), an inhibitor of the enzyme (5α-reductase Type I and II) that converts progesterone into 5α-DHP, the ALLO and 5α-DHP content of frontal cortex of both group-housed and socially isolated mice decreased exponentially to 10%–20% of control values in about 30 min. The fractional rate constants (k h−1) of ALLO and 5α-DHP decline multiplied by the ALLO and 5α-DHP concentrations at any given steady-state estimate the rate of synthesis required to maintain that steady state. After 6 weeks of social isolation, ALLO and 5α-DHP biosynthesis rates were decreased to 30% of the values calculated in group-housed mice. Moreover, in socially isolated mice, the expression of 5α-reductase Type I mRNA and protein was approximately 50% lower than in group-housed mice whereas 3α-hydroxysteroid oxidoreductase mRNA expression was equal in the two groups. Protracted social isolation in mice may provide a model to investigate whether 5α-DHP by a genomic action, and ALLO by a nongenomic mechanism down-regulate the action of drugs acting as agonists, partial agonists, or positive allosteric modulators of the benzodiazepine recognition sites expressed by GABAA receptors.

An initial ATP-independent step in the unwinding of a herpes simplex virus type I origin of replication by a complex of the viral origin-binding protein and single-strand DNA-binding protein

Using a spectrophotometric assay that measures the hyperchromicity that accompanies the unwinding of a DNA duplex, we have identified an ATP-independent step in the unwinding of a herpes simplex virus type 1 (HSV-1) origin of replication, Oris, by a complex of the HSV-1 origin binding protein (UL9 protein) and the HSV-1 single-strand DNA binding protein (ICP8). The sequence unwound is the 18-bp A + T-rich segment that links the two high-affinity UL9 protein binding sites, boxes I and II of Oris. P1 nuclease sensitivity of Oris and single-strand DNA-dependent ATPase measurements of the UL9 protein indicate that, at 37°C, the A + T-rich segment is sufficiently single stranded to permit the binding of ICP8. Binding of the UL9 protein to boxes I and II then results in the formation of the UL9 protein–ICP8 complex, that can, in the absence of ATP, promote unwinding of the A + T-rich segment. On addition of ATP, the helicase activity of the UL9 protein–ICP8 complex can unwind boxes I and II, permitting access of the replication machinery to the Oris sequences.

Herpes simplex virus DNA packaging sequences adopt novel structures that are specifically recognized by a component of the cleavage and packaging machinery

The product of the herpes simplex virus type 1 UL28 gene is essential for cleavage of concatemeric viral DNA into genome-length units and packaging of this DNA into viral procapsids. To address the role of UL28 in this process, purified UL28 protein was assayed for the ability to recognize conserved herpesvirus DNA packaging sequences. We report that DNA fragments containing the pac1 DNA packaging motif can be induced by heat treatment to adopt novel DNA conformations that migrate faster than the corresponding duplex in nondenaturing gels. Surprisingly, these novel DNA structures are high-affinity substrates for UL28 protein binding, whereas double-stranded DNA of identical sequence composition is not recognized by UL28 protein. We demonstrate that only one strand of the pac1 motif is responsible for the formation of novel DNA structures that are bound tightly and specifically by UL28 protein. To determine the relevance of the observed UL28 protein–pac1 interaction to the cleavage and packaging process, we have analyzed the binding affinity of UL28 protein for pac1 mutants previously shown to be deficient in cleavage and packaging in vivo. Each of the pac1 mutants exhibited a decrease in DNA binding by UL28 protein that correlated directly with the reported reduction in cleavage and packaging efficiency, thereby supporting a role for the UL28 protein–pac1 interaction in vivo. These data therefore suggest that the formation of novel DNA structures by the pac1 motif confers added specificity on recognition of DNA packaging sequences by the UL28-encoded component of the herpesvirus cleavage and packaging machinery.

Raft-partitioning of the ubiquitin ligases Cbl and Nedd4 upon IgE-triggered cell signaling

The high affinity receptor for IgE, FcɛRI on mast cells and basophils plays an essential role in immunological defense. Upon multivalent antigen binding, FcɛRI becomes phoshorylated by the protein-tyrosine kinase Lyn, as a result of receptor clustering in lipid rafts. FcɛRI has been shown to be ubiquitinated. Ubiquitination can lead to degradation by proteasomes, but it can also act as a sorting signal to internalize proteins destined to the endosomal/lysosomal pathway. We have analyzed whether FcɛRI ubiquitination takes place within rafts. We report biochemical and imaging evidence in rat basoleukemia cells for the presence of ubiquitinated FcɛRI in clustered rafts upon receptor activation. Moreover, we demonstrated that the ubiquitin ligases Cbl and Nedd4 colocalize with FcɛRI patches and showed that both ligases become associated with lipid rafts after activation of IgE signaling. Because Cbl is known to interact with the FcɛRI signaling complex, ubiquitination is likely to be an important parameter regulating IgE-triggered signaling occurring in rafts.

Site-specific mutations in the mature form of human IL-18 with enhanced biological activity and decreased neutralization by IL-18 binding protein

IL-18 can be considered a proinflammatory cytokine mediating disease as well as an immunostimulatory cytokine that is important for host defense against infection and cancer. The high-affinity, constitutively expressed, and circulating IL-18 binding protein (IL-18BP), which competes with cell surface receptors for IL-18 and neutralizes IL-18 activity, may act as a natural antiinflammatory as well as immunosuppressive molecule. In the present studies, the IL-18 precursor caspase-1 cleavage site was changed to a factor Xa site, and, after expression in Escherichia coli, mature IL-18 was generated by factor Xa cleavage. Mature IL-18 generated by factor Xa cleavage was fully active. Single point mutations in the mature IL-18 peptide were made, and the biological activities of the wild-type (WT) IL-18 were compared with those of the mutants. Mutants E42A and K89A exhibited 2-fold increased activity compared with WT IL-18. A double mutant, E42A plus K89A, exhibited 4-fold greater activity. Unexpectedly, IL-18BP failed to neutralize the double mutant E42A plus K89A compared with WT IL-18. The K89A mutant was intermediate in being neutralized by IL-18BP, whereas neutralization of the E42A mutant was comparable to that in the WT IL-18. The identification of E42 and K89 in the mature IL-18 peptide is consistent with previous modeling studies of IL-18 binding to IL-18BP and explains the unusually high affinity of IL-18BP for IL-18.

Local osmotic gradients drive the water flux associated with Na+/glucose cotransport

It recently was proposed [Loo, D. D. F., Zeuthen, T., Chandy, G. & Wright, E. M. (1996) Proc. Natl. Acad. Sci. USA 93, 13367–13370] that SGLT1, the high affinity intestinal and renal sodium/glucose cotransporter carries water molecules along with the cosubstrates with a strict stoichiometry of two Na+, one glucose, and ≈220 water molecules per transport cycle. Using electrophysiology together with sensitive volumetric measurements, we investigated the nature of the driving force behind the cotransporter-mediated water flux. The osmotic water permeability of oocytes expressing human SGLT1 (Lp ± SE) averaged 3.8 ± 0.3 × 10−4 cm⋅s−1 (n = 15) and addition of 100 μM phlorizin (a specific SGLT1 inhibitor) reduced the permeability to 2.2 ± 0.2 × 10−4 cm⋅s−1 (n = 15), confirming the presence of a significant water permeability closely associated with the cotransporter. Addition of 5 mM α-methyl-glucose (αMG) induced an average inward current of 800 ± 10 nA at −50 mV and a water influx reaching 120 ± 20 pL cm−2 ⋅s−1 within 5–8 min. After rapidly inhibiting the Na+/glucose cotransport with phlorizin, the water flux remained significantly elevated, clearly indicating the presence of a local osmotic gradient (Δπ) estimated at 16 ± 2 mOsm. In short-term experiments, a rapid depolarization from −100 to 0 mV in the presence of αMG decreased the cotransport current by 94% but failed to produce a comparable reduction in the swelling rate. A mathematical model depicting the intracellular accumulation of transported osmolytes can accurately account for these observations. It is concluded that, in SGLT1-expressing oocytes, αMG-dependent water influx is induced by a local osmotic gradient by using both endogenous and SGLT1-dependent water permeability.

Toward rules for 1:1 polyamide:DNA recognition

Polyamides composed of four amino acids, imidazole (Im), pyrrole (Py), hydroxypyrrole (Hp), and β-alanine (β), are synthetic ligands that form highly stable complexes in the minor groove of DNA. Although specific pairing rules within the 2:1 motif can be used to distinguish the four Watson⋅Crick base pairs, a comparable recognition code for 1:1 polyamide:DNA complexes had not been described. To set a quantitative baseline for the field, the sequence specificities of Im, Py, Hp, and β for the four Watson⋅Crick base pairs were determined for two polyamides, Im-β-ImPy-β-Im-β-ImPy-β-Dp (1, for Im, Py, and β) and Im-β-ImHp-β-Im-β-ImPy-β-Dp (2, for Hp), in a 1:1 complex within the DNA sequence context 5′-AAAGAGAAGAG-3′. Im residues do not distinguish G,C from A,T but bind all four base pairs with high affinity. Py and β residues exhibit ≥10-fold preference for A,T over G,C base pairs. The Hp residue displays a unique preference for a single A⋅T base pair with an energetic penalty.

Immucillin H, a powerful transition-state analog inhibitor of purine nucleoside phosphorylase, selectively inhibits human T lymphocytes

Transition-state theory has led to the design of Immucillin-H (Imm-H), a picomolar inhibitor of purine nucleoside phosphorylase (PNP). In humans, PNP is the only route for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T cell-mediated immunosuppression. This study reports the biological effects and mechanism of action of Imm-H on malignant T cell lines and on normal activated human peripheral T cells. Imm-H inhibits the growth of malignant T cell leukemia lines with the induction of apoptosis. Imm-H also inhibits activated normal human T cells after antigenic stimulation in vitro. However, Imm-H did not inhibit malignant B cells, colon cancer cell lines, or normal human nonstimulated T cells, demonstrating the selective activity of Imm-H. The effects on leukemia cells were mediated by the cellular phosphorylation of deoxyguanosine and the accumulation of dGTP, an inhibitor of ribonucleotide diphosphate reductase. Cells were protected from the toxic effects of Imm-H when deoxyguanosine was absent or when deoxycytidine was present. Guanosine incorporation into nucleic acids was selectively blocked by Imm-H with no effect on guanine, adenine, adenosine, or deoxycytidine incorporation. Imm-H may have clinical potential for treatment of human T cell leukemia and lymphoma and for other diseases characterized by abnormal activation of T lymphocytes. The design of Imm-H from an enzymatic transition-state analysis exemplifies a powerful approach for developing high-affinity enzyme inhibitors with pharmacologic activity.

Role of MEKK2-MEK5 in the regulation of TNF-α gene expression and MEKK2-MKK7 in the activation of c-Jun N-terminal kinase in mast cells

Cross-linking of the high-affinity IgE receptor (FcɛRI) on mast cells with IgE and multivalent antigen triggers mitogen-activated protein (MAP) kinase activation and cytokine gene expression. We report here that MAP kinase kinase 4 (MKK4) gene disruption does not affect either MAP kinase activation or cytokine gene expression in response to cross-linking of FcɛRI in embryonic stem cell-derived mast cells. MKK7 is activated in response to cross-linking of FcɛRI, and this activation is inhibited by MAP/ERK kinase (MEK) kinase 2 (MEKK2) gene disruption. In addition, expression of kinase-inactive MKK7 in the murine mast cell line MC/9 inhibits c-Jun NH2-terminal kinase (JNK) activation in response to cross-linking of FcɛRI, whereas expression of kinase-inactive MKK4 does not affect JNK activation by this stimulus. However, FcɛRI-induced activation of the tumor necrosis factor-α (TNF-α) gene promoter is not affected by expression of kinase-inactive MKK7. We describe an alternative pathway by which MEKK2 activates MEK5 and big MAP kinase1/extracellular signal-regulated kinase 5 in addition to MKK7 and JNK, and interruption of this pathway inhibits TNF-α promoter activation. These findings suggest that JNK activation by antigen cross-linking is dependent on the MEKK2-MKK7 pathway, and cytokine production in mast cells is regulated in part by the signaling complex MEKK2-MEK5-ERK5.

Purification and Characterization of a NADPH-Dependent Aldehyde Reductase from Mung Bean That Detoxifies Eutypine, a Toxin from Eutypa lata1

Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by Eutypa lata, the causal agent of eutypa dieback in the grapevine (Vitis vinifera). Eutypine is enzymatically converted by numerous plant tissues into eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), a metabolite that is nontoxic to grapevine. We report a four-step procedure for the purification to apparent electrophoretic homogeneity of a eutypine-reducing enzyme (ERE) from etiolated mung bean (Vigna radiata) hypocotyls. The purified protein is a monomer of 36 kD, uses NADPH as a cofactor, and exhibits a Km value of 6.3 μm for eutypine and a high affinity for 3- and 4-nitro-benzaldehyde. The enzyme failed to catalyze the reverse reaction using eutypinol as a substrate. ERE detoxifies eutypine efficiently over a pH range from 6.2 to 7.5. These data strongly suggest that ERE is an aldehyde reductase that could probably be classified into the aldo-keto reductase superfamily. We discuss the possible role of this enzyme in eutypine detoxification.

Arg-302 facilitates deprotonation of Glu-325 in the transport mechanism of the lactose permease from Escherichia coli

A mechanistic model for lactose/H+ symport via the lactose permease of Escherichia coli proposed recently indicates that the permease must be protonated to bind ligand with high affinity. Moreover, in the ground state, the symported H+ is shared between His-322 (helix X) and Glu-269 (helix VIII), whereas Glu-325 (helix X) is charge-paired with Arg-302 (helix IX). Substrate binding at the outer surface induces a conformational change that leads to transfer of the H+ to Glu-325 and reorientation of the binding site to the inner surface. After release of the substrate, Glu-325 is deprotonated on the inside because of rejuxtapositioning with Arg-302. To test the role of Arg-302 in the mechanism, the catalytic properties of mutants Arg-302→Ala and Arg-302→Ser were studied. Both mutants are severely defective in active lactose transport, as well as in efflux or influx down a concentration gradient, translocation modes that involve net H+ movement. In marked contrast, the mutants catalyze equilibrium exchange of lactose and bind ligand with high affinity. These characteristics are remarkably analogous to those of permease mutants with neutral replacements for Glu-325, a residue that plays a direct role in H+ translocation. These observations lend strong support for the argument that Arg-302 interacts with Glu-325 to facilitate deprotonation of the carboxylic acid during turnover.