Expression of error-prone polymerases in BL2 cells activated for Ig somatic hypermutation

High affinity antibodies are generated in mice and humans by means of somatic hypermutation (SHM) of variable (V) regions of Ig genes. Mutations with rates of 10−5–10−3 per base pair per generation, about 106-fold above normal, are targeted primarily at V-region hot spots by unknown mechanisms. We have measured mRNA expression of DNA polymerases ι, η, and ζ by using cultured Burkitt's lymphoma (BL)2 cells. These cells exhibit 5–10-fold increases in heavy-chain V-region mutations targeted only predominantly to RGYW (R = A or G, Y = C or T, W = T or A) hot spots if costimulated with T cells and IgM crosslinking, the presumed in vivo requirements for SHM. An ∼4-fold increase pol ι mRNA occurs within 12 h when cocultured with T cells and surface IgM crosslinking. Induction of pols η and ζ occur with T cells, IgM crosslinking, or both stimuli. The fidelity of pol ι was measured at RGYW hot- and non-hot-spot sequences situated at nicks, gaps, and double-strand breaks. Pol ι formed T⋅G mispairs at a frequency of 10−2, consistent with SHM-generated C to T transitions, with a 3-fold increased error rate in hot- vs. non-hot-spot sequences for the single-nucleotide overhang. The T cell and IgM crosslinking-dependent induction of pol ι at 12 h may indicate an SHM “triggering” event has occurred. However, pols ι, η, and ζ are present under all conditions, suggesting that their presence is not sufficient to generate mutations because both T cell and IgM stimuli are required for SHM induction.

Toward a new generation of vaccines: The anti-cytokine therapeutic vaccines

Pathological conditions, such as cancers, viral infections, and autoimmune diseases, are associated with abnormal cytokine production, and the morbidity associated with many medical disorders is often directly a result of cytokine production. Because of the absence of negative feedback control occurring in some pathophysiologic situations, a given cytokine may flood and accumulate in the extracellular compartment of tissues or tumors thereby impairing the cytokine network homeostasis and contributing to local pathogenesis. To evaluate whether the rise of anti-cytokine Abs by vaccination is an effective way to treat these pathological conditions without being harmful to the organism, we have analyzed each step of the cytokine process (involving cytokine production, target response, and feedback regulation) and have considered them in the local context of effector–target cell microenvironment and in the overall context of the macroenvironment of the immune system of the organism. In pathologic tissues, Abs of high affinity, as raised by anti-cytokine vaccination, should neutralize the pool of cytokines ectopically accumulated in the extracellular compartment, thus counteracting their pathogenic effects. In contrast, the same Abs should not interfere with cytokine processes occurring in normal tissues, because under physiologic conditions cytokine production by effector cells (induced by activation but controlled by negative feedback regulation) does not accumulate in the extracellular compartment. These concepts are consistent with results showing that following animal and human anti-cytokine vaccination, induction of high-affinity Abs has proven to be safe and effective and encourages this approach as a pioneering avenue of therapy.

The Human Cytomegalovirus US28 Protein Is Located in Endocytic Vesicles and Undergoes Constitutive Endocytosis and Recycling

Genes encoding chemokine receptor-like proteins have been found in herpes and poxviruses and implicated in viral pathogenesis. Here we describe the cellular distribution and trafficking of a human cytomegalovirus (HCMV) chemokine receptor encoded by the US28 gene, after transient and stable expression in transfected HeLa and Cos cells. Immunofluorescence staining indicated that this viral protein accumulated intracellularly in vesicular structures in the perinuclear region of the cell and showed overlap with markers for endocytic organelles. By immunogold electron microscopy US28 was seen mostly to localize to multivesicular endosomes. A minor portion of the protein (at most 20%) was also expressed at the cell surface. Antibody-feeding experiments indicated that cell surface US28 undergoes constitutive ligand-independent endocytosis. Biochemical analysis with the use of iodinated ligands showed that US28 was rapidly internalized. The high-affinity ligand of US28, the CX3C-chemokine fractalkine, reduced the steady-state levels of US28 at the cell surface, apparently by inhibiting the recycling of internalized receptor. Endocytosis and cycling of HCMV US28 could play a role in the sequestration of host chemokines, thereby modulating antiviral immune responses. In addition, the distribution of US28 mainly on endosomal membranes may allow it to be incorporated into the viral envelope during HCMV assembly.

DNA bending by an adenine–thymine tract and its role in gene regulation

To gain insight into the structural basis of DNA bending by adenine–thymine tracts (A-tracts) and their role in DNA recognition by gene-regulatory proteins, we have determined the crystal structure of the high-affinity DNA target of the cancer-associated human papillomavirus E2 protein. The three independent B-DNA molecules of the crystal structure determined at 2.2-Å resolution are examples of A-tract-containing helices where the global direction and magnitude of curvature are in accord with solution data, thereby providing insights, at the base pair level, into the mechanism of DNA bending by such sequence motifs. A comparative analysis of E2–DNA conformations with respect to other structural and biochemical studies demonstrates that (i) the A-tract structure of the core region, which is not contacted by the protein, is critical for the formation of the high-affinity sequence-specific protein–DNA complex, and (ii) differential binding affinity is regulated by the intrinsic structure and deformability encoded in the base sequence of the DNA target.

Location and properties of metal-binding sites on the human prion protein

Although a functional role in copper binding has been suggested for the prion protein, evidence for binding at affinities characteristic of authentic metal-binding proteins has been lacking. By presentation of copper(II) ions in the presence of the weak chelator glycine, we have now characterized two high-affinity binding sites for divalent transition metals within the human prion protein. One is in the N-terminal octapeptide-repeat segment and has a Kd for copper(II) of 10−14 M, with other metals (Ni2+, Zn2+, and Mn2+) binding three or more orders of magnitude more weakly. However, NMR and fluorescence data reveal a previously unreported second site around histidines 96 and 111, a region of the molecule known to be crucial for prion propagation. The Kd for copper(II) at this site is 4 × 10−14 M, whereas nickel(II), zinc(II), and manganese(II) bind 6, 7, and 10 orders of magnitude more weakly, respectively, regardless of whether the protein is in its oxidized α-helical (α-PrP) or reduced β-sheet (β-PrP) conformation. A role for prion protein (PrP) in copper metabolism or transport seems likely and disturbance of this function may be involved in prion-related neurotoxicity.

A mutant cholera toxin B subunit that binds GM1- ganglioside but lacks immunomodulatory or toxic activity

GM1-ganglioside receptor binding by the B subunit of cholera toxin (CtxB) is widely accepted to initiate toxin action by triggering uptake and delivery of the toxin A subunit into cells. More recently, GM1 binding by isolated CtxB, or the related B subunit of Escherichia coli heat-labile enterotoxin (EtxB), has been found to modulate leukocyte function, resulting in the down-regulation of proinflammatory immune responses that cause autoimmune disorders such as rheumatoid arthritis and diabetes. Here, we demonstrate that GM1 binding, contrary to expectation, is not sufficient to initiate toxin action. We report the engineering and crystallographic structure of a mutant cholera toxin, with a His to Ala substitution in the B subunit at position 57. Whereas the mutant retained pentameric stability and high affinity binding to GM1-ganglioside, it had lost its immunomodulatory activity and, when part of the holotoxin complex, exhibited ablated toxicity. The implications of these findings on the mode of action of cholera toxin are discussed.

In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei

Most eukaryotic telomeres contain a repeating motif with stretches of guanine residues that form a 3′-terminal overhang extending beyond the telomeric duplex region. The telomeric repeat of hypotrichous ciliates, d(T4G4), forms a 16-nucleotide 3′-overhang. Such sequences can adopt parallel-stranded as well as antiparallel-stranded quadruplex conformations in vitro. Although it has been proposed that guanine-quadruplex conformations may have important cellular roles including telomere function, recombination, and transcription, evidence for the existence of this DNA structure in vivo has been elusive to date. We have generated high-affinity single-chain antibody fragment (scFv) probes for the guanine-quadruplex formed by the Stylonychia telomeric repeat, by ribosome display from the Human Combinatorial Antibody Library. Of the scFvs selected, one (Sty3) had an affinity of Kd = 125 pM for the parallel-stranded guanine-quadruplex and could discriminate with at least 1,000-fold specificity between parallel or antiparallel quadruplex conformations formed by the same sequence motif. A second scFv (Sty49) bound both the parallel and antiparallel quadruplex with similar (Kd = 3–5 nM) affinity. Indirect immunofluorescence studies show that Sty49 reacts specifically with the macronucleus but not the micronucleus of Stylonychia lemnae. The replication band, the region where replication and telomere elongation take place, was also not stained, suggesting that the guanine-quadruplex is resolved during replication. Our results provide experimental evidence that the telomeres of Stylonychia macronuclei adopt in vivo a guanine-quadruplex structure, indicating that this structure may have an important role for telomere functioning.

4-Hydroxytamoxifen binds to and deactivates the estrogen-related receptor γ

The estrogen-related receptors (ERRα, ERRβ, and ERRγ) form a family of orphan nuclear receptors that share significant amino acid identity with the estrogen receptors, but for which physiologic roles remain largely unknown. By using a peptide sensor assay, we have identified the stilbenes diethylstilbestrol (DES), tamoxifen (TAM), and 4-hydroxytamoxifen (4-OHT) as high-affinity ligands for ERRγ. In direct binding assays, 4-OHT had a Kd value of 35 nM, and both DES and TAM displaced radiolabeled 4-OHT with Ki values of 870 nM. In cell-based assays, 4-OHT binding caused a dissociation of the complex between ERRγ and the steroid receptor coactivator-1, and led to an inhibition of the constitutive transcriptional activity of ERRγ. ERRα did not bind 4-OHT, but replacing a single amino acid predicted to be in the ERRα ligand-binding pocket with the corresponding ERRγ residue allowed high-affinity 4-OHT binding. These results demonstrate the existence of high-affinity ligands for the ERR family of orphan receptors, and identify 4-OHT as a molecule that can regulate the transcriptional activity of ERRγ.

Binding of the cellulose-binding domain of exoglucanase Cex from Cellulomonas fimi to insoluble microcrystalline cellulose is entropically driven.

Isothermal titration microcalorimetry is combined with solution-depletion isotherm data to analyze the thermodynamics of binding of the cellulose-binding domain (CBD) from the beta-1,4-(exo)glucanase Cex of Cellulomonas fimi to insoluble bacterial microcrystalline cellulose. Analysis of isothermal titration microcalorimetry data against two putative binding models indicates that the bacterial microcrystalline cellulose surface presents two independent classes of binding sites, with the predominant high-affinity site being characterized by a Langmuir-type Ka of 6.3 (+/-1.4) x 10(7) M-1 and the low-affinity site by a Ka of 1.1 (+/-0.6) x 10(6) M-1. CBDCex binding to either site is exothermic, but is mainly driven by a large positive change in entropy. This differs from protein binding to soluble carbohydrates, which is usually driven by a relatively large exothermic standard enthalpy change for binding. Differential heat capacity changes are large and negative, indicating that sorbent and protein dehydration effects make a dominant contribution to the driving force for binding.

Silencing of human fetal globin expression is impaired in the absence of the adult beta-globin gene activator protein EKLF.

Globin genes are subject to tissue-specific and developmental stage-specific regulation. A switch from human fetal (gamma)-to adult (beta)-globin expression occurs within erythroid precursor cells of the adult lineage. Previously we and others showed by targeted gene disruption that the zinc finger gene, erythroid Krüppel-like factor (EKLF), is required for expression of the beta-globin gene in mice, presumably through interaction with a high-affinity binding site in the proximal promoter. To examine the role of EKLF in the developmental regulation of the human gamma-globin gene we interbred EKLF heterozygotes (+/-) with mice harboring a human beta-globin yeast artificial chromosome transgene. We find that in the absence of EKLF, while human beta-globin expression is dramatically reduced, gamma-globin transcripts are elevated approximately 5-fold. Impaired silencing of gamma-globin expression identifies EKLF as the first transcription factor participating quantitatively in the gamma-globin to beta-globin switch. Our findings are compatible with a competitive model of switching in which EKLF mediates an adult stage-specific interaction between the beta-globin gene promoter and the locus control region that excludes the gamma-globin gene.

Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene expression.

Glucose is the preferred carbon source for most eukaryotic cells and has profound effects on many cellular functions. How cells sense glucose and transduce a signal into the cell is a fundamental, unanswered question. Here we describe evidence that two unusual glucose transporters in the yeast Saccharomyces cerevisiae serve as glucose sensors that generate an intracellular glucose signal. The Snf3p high-affinity glucose transporter appears to function as a low glucose sensor, since it is required for induction of expression of several hexose transporter (HXT) genes, encoding glucose transporters, by low levels of glucose. We have identified another apparent glucose transporter, Rgt2p, that is strikingly similar to Snf3p and is required for maximal induction of gene expression in response to high levels of glucose. This suggests that Rgt2p is a high glucose-sensing counterpart to Snf3p. We identified a dominant mutation in RGT2 that causes constitutive expression of several HXT genes, even in the absence of the inducer glucose. This same mutation introduced into SNF3 also causes glucose-independent expression of HXT genes. Thus, the Rgt2p and Snf3p glucose transporters appear to act as glucose receptors that generate an intracellular glucose signal, suggesting that glucose signaling in yeast is a receptor-mediated process.

Suppression of trkB expression by antisense oligonucleotides alters a neuronal phenotype in the rod pathway of the developing rat retina.

trkB is the high-affinity receptor for brain-derived neurotrophic factor (BDNF), a trophic molecule with demonstrated effects on the survival and differentiation of a wide variety of neuronal populations. In the mammalian retina, trkB is localized to both ganglion cells and numerous cells in the inner nuclear layer. Much information on the role of BDNF in neuronal development has been derived from the study of trkB- and BDNF-deficient mutant mice. This includes an attenuation of the numbers of cortical neurons immunopositive for the calcium-binding proteins, parvalbumin, and calbindin. Unfortunately, these mutant animals typically fail to survive for > 24-48 hr after birth. Since most retinal neuronal differentiation occurs postnatally, we have devised an alternative scheme to suppress the expression of trkB in the retina to examine the role of BDNF on the postnatal development of neurons of the inner retina. Neonatal rats were treated with intraocular injection of an antisense oligonucleotide (1-2 microliters of 10-100 microM solution) targeted to the trkB mRNA. Immunohistochemistry with a polyclonal antibody to trkB showed that the expression of trkB in retinal neurons was suppressed 48-72 hr following a single injection. Northern blot analysis demonstrated that antisense treatment had no effect on the level of trkB mRNA, even after multiple injections. This suggests an effect of trkB antisense treatment on protein translation, but not on RNA transcription. No alterations were observed in the thickness of retinal cellular or plexiform layers, suggesting that BDNF is not the sole survival factor for these neurons. There were, however, alterations in the patterns of immunostaining for parvalbumin, a marker for the narrow-field, bistratified AII amacrine cell-a central element of the rod (scotopic) pathway. This was evidenced by a decrease in both the number of immunostained somata (> 50%) and in the intensity of immunolabeling. However, the immunostaining pattern of calbindin was not affected. These studies suggest that the ligands for trkB have specific effects on the neurochemical phenotypic expression of inner retinal neurons and in the development of a well-defined retinal circuit.

Early events in poliovirus infection: virus-receptor interactions.

The interaction of poliovirus with its cell receptor initiates conformational changes that lead to uncoating of the viral RNA. Three types of genetic analyses have been used to study the poliovirus-receptor interaction: (i) mutagenesis of the poliovirus receptor (PVR), (ii) selection of viral mutants resistant to neutralization with soluble PVR, and (iii) selection of viral variants adapted to use mutant PVRs. The results of these studies show that a small portion of the first immunoglobulin-like domain of PVR contacts viral residues within a deep depression on the surface of the capsid that encircles the fivefold axis of symmetry. Viral capsid residues that influence the interaction with PVR are also found in locations such as the capsid interior that cannot directly contact PVR. These mutations might influence the ability of the capsid to undergo receptor-mediated conformational transitions that are necessary for high-affinity interactions with PVR.

RNA replication by Q beta replicase: a working model.

Two classes of RNA ligands that bound to separate, high affinity nucleic acid binding sites on Q beta replicase were previously identified. RNA ligands to the two sites, referred to as site I and site II, were used to investigate the molecular mechanism of RNA replication employed by the four-subunit replicase. Replication inhibition by site I- and site II-specific ligands defined two subsets of replicatable RNAs. When provided with appropriate 3' ends, ligands to either site served as replication templates. UV crosslinking experiments revealed that site I is associated with the S1 subunit, site II with elongation factor Tu, and polymerization with the viral subunit of the holoenzyme. These results provide the framework for a three site model describing template recognition and product strand initiation by Q beta replicase.

Synthesis and characterization of a photoactivatable analog of corticotropin-releasing factor for specific receptor labeling.

A novel photoactivatable analog of ovine corticotropin-releasing factor (ovine photoCRF) has been synthesized and characterized. A diazirine group, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl residue, was covalently bound to the amino terminus of ovine CRF (oCRF), which was N-terminally extended by a tyrosyl residue for radioactive labeling with 125I. Under mild conditions, photolysis yielded highly reactive carbenes, responsible for the formation of covalent bonds to the CRF receptor. Ovine photoCRF was shown to bind to the high-affinity site of the CRF receptor with a similar Kd value as oCRF. When radioactively iodinated ovine photoCRF (ovine 125I-photoCRF) was covalently linked to rat CRF receptor, type 1 (rCRFR1), permanently transfected into human embryonic kidney (HEK) 293 cells, a highly glycosylated 75-kDa protein was identified with SDS/PAGE. The specificity of ovine 125I-photoCRF was demonstrated by the finding that this analog could be displaced from the receptor by oCRF, but not other unrelated peptides such as vasoactive intestinal peptide. The observed size of the 75-kDa cross-link was in agreement with the molecular weight reported earlier for native CRFR1 from rat brain. Deglycosylation of the 75-kDa cross-link with peptide:N-glycosidase (PNGase) yielded a 46-kDa protein, in agreement with the molecular weight estimated from cDNA coding for rat CRFR1. The developed CRF analog, photoCRF, is expected to facilitate future biochemical and physiological analysis of CRF receptors and--by analogous strategies--of other peptide receptors.