Characterization of the metabolic phenotype of chronically activated lymphocytes.

Activated lymphocytes proliferate, secrete cytokines, and can make antibodies. Normally activated B and T cells meet the bioenergetic demand for these processes by up-regulating aerobic glycolysis. In contrast, several lines of evidence suggest that pathogenic lymphocytes in autoimmune diseases like lupus meet ATP demands through oxidative phosphorylation. Using (13)C-glucose as a stable tracer, we found that splenocytes from mice with lupus derive the same fraction of lactate from glucose as control animals, suggesting comparable levels of glycolysis and non-oxidative ATP production. However, lupus splenocytes increase glucose oxidation by 40% over healthy control animals. The ratio between pentose phosphate cycle (PPC) activity and glycolysis is the same for each group, indicating that increased glucose oxidation is due to increased activity of the TCA cycle in lupus splenocytes. Repetitive stimulation of cultured human T cells was used to model chronic lymphocyte activation, a phenotype associated with lupus. Chronically activated T cells rely primarily on oxidative metabolism for ATP synthesis suggesting that chronic antigen stimulation may be the basis for the metabolic findings observed in lupus mice. Identification of disease-related bioenergetic phenotypes should contribute to new diagnostic and therapeutic strategies for immune diseases.

Sequence specific recognition of ssDNA by a lupus autoantibody: kinetics and mechanism of binding.

11F8 is a pathogenic anti-ssDNA monoclonal autoantibody isolated from a lupus-prone mouse. Previous studies have established that 11F8 is sequence specific. To determine the basis for the observed binding specificity, stopped-flow fluorescence spectroscopy was used to measure the kinetic parameters and establish the mechanisms for the association of 11F8 with its target sequence, noncognate, and nonspecific ssDNA ligands. The data revealed that sequence-specific binding follows a two-step mechanism where the initial association step is second order. Values of k(1) are fast and above the modified Smoluchowski limit for a diffusion limited interaction (10(5)-10(6)M(-1)s(-1)). The dependency of k(1) on [salt] and solvent polarity indicates that electrostatic steering is responsible for this rapid association rate. The second association step is rate limiting and is characteristic of an isomerization process during which binding interfaces are optimized. This step apparently is driven by the desolvation of hydrophobic surfaces within the binding interface. The differences in the rate of dissociation for the various DNA ligands suggest that specificity is governed primarily through the dissociation of the final complexes.

Signaling pathways and effector mechanisms pre-programmed cell death.

Apoptosis is a complex biochemical process that involves all aspects of the cell from the plasma membrane to the nucleus. Apoptosis stimuli are mediated by many different cellular processes including protein synthesis and degradation, the alteration in protein phosphorylation states, the activation of lipid second messenger systems, and disruption of normal mitochondrial function. Despite this diversity in signal transduction, all apoptotic pathways are believed to converge ultimately with the activation of caspases leading to the characteristic morphological changes of apoptosis. In this review, we discuss what is known about these pathways and its implication for normal cellular function.

Thermodynamic basis for sequence-specific recognition of ssDNA by an autoantibody.

11F8 is a sequence-specific DNA binding monoclonal autoantibody previously isolated from an autoimmune lupus-prone mouse [Stevens, S. Y., and Glick, G. D. (1999) Biochemistry 38, 560-568]. This antibody, like many other lupus anti-DNAs, localizes to kidney tissue and eventually leads to renal damage through a process that first involves the binding of DNA antigens. A series of experiments were conducted to investigate the thermodynamic and structural basis by which this antibody discriminates between specific, noncognate, and nonspecific sequences. Sequence-specific binding occurs with a minimal dependence on the polyelectrolyte effect along with a favorable binding enthalpy reflecting the presence of base stacking and contacts to DNA bases. This favorable binding enthalpy apparently is derived from desolvation at the binding interface and is consistent with recent models of the nonclassical hydrophobic effect. Noncognate recognition is also driven by the nonclassical hydrophobic effect, but is accompanied by highly unfavorable entropies that are responsible for reduced affinity relative to the high-affinity consensus sequence. Nonspecific recognition is driven completely by the polyelectrolyte effect involving extensive electrostatic interactions with the phosphate backbone. Collectively, the data demonstrate the ability of 11F8 to adapt its mode of binding to the available DNA surface and provide a thermodynamic model for sequence-specific recognition of single-stranded DNA. The salient features of this model employ the paradigms invoked to explain protein.dsDNA, protein.RNA, and antibody.antigen binding.

The hairpin ribozyme substrate binding-domain: a highly constrained D-shaped conformation.

The two domains of the hairpin ribozyme-substrate complex, usually depicted as straight structural elements, must interact with one another in order to form an active conformation. Little is known about the internal geometry of the individual domains in an active docked complex. Using various crosslinking and structural approaches in conjunction with molecular modeling (constraint-satisfaction program MC-SYM), we have investigated the conformation of the substrate-binding domain in the context of the active docked ribozyme-substrate complex. The model generated by MC-SYM showed that the domain is not straight but adopts a bent conformation (D-shaped) in the docked state of the ribozyme, indicating that the two helices bounding the internal loop are closer than was previously assumed. This arrangement rationalizes the observed ability of hairpin ribozymes with a circularized substrate-binding strand to cleave a circular substrate, and provides essential information concerning the organization of the substrate in the active conformation. The internal geometry of the substrate-binding strand places G8 of the substrate-binding strand near the cleavage site, which has allowed us to predict the crucial role played by this nucleotide in the reaction chemistry.

Rapid magnesium chelation as a method to study real-time tertiary unfolding of RNA.

This unit describes a method to measure the unfolding of RNA tertiary structure on a millisecond time scale. A stopped-flow spectrophotometer is used to measure the rate of unfolding induced by the addition of EDTA to an RNA whose tertiary structure has been stabilized in the presence of magnesium ions. Using this methodology, rate constants for unfolding of tertiary or secondary structure can be obtained over a range of temperatures, and these values can be used to construct Arrhenius and Eyring plots, from which activation energy, Arrhenius pre-exponential factor, and enthalpy and entropy of activation can be obtained. These data provide information about the energy of the transition state and the energy barriers between secondary and tertiary structure, which is necessary for predicting RNA tertiary structure from secondary structure.

Engineering disulfide cross-links in RNA via air oxidation.

This unit presents protocols for the synthesis of alkylthiol-modified ribonucleosides, their incorporation into synthetic RNA, and the formation of intramolecular disulfide bonds in RNA by air oxidation. The disulfide bonds can be formed in quantitative yields between thiols positioned in close proximity by virtue of either the secondary or tertiary structure of the RNA. Disulfide cross-links are useful tools to probe solution structures of RNA, to monitor dynamic motion, to stabilize folded RNAs, and to study the process of tertiary structure folding.

Anti-DNA autoantibodies and systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that affects most of the organs and tissues of the body, causing glomerulonephritis, arthritis, and cerebritis. SLE can be fatal with nephritis, in particular, predicting a poor outcome for patients. In this review, we highlight what has been learned about SLE from the study of mouse models, and pay particular attention to anti-DNA autoantibodies, both as pathological agents of lupus nephritis and as DNA-binding proteins. We summarize the current approaches used to treat SLE and discuss the targeting of anti-DNA autoantibodies as a new treatment for lupus nephritis.

Evidence for sequence-specific recognition of DNA by anti-single-stranded DNA autoantibodies.

Anti-DNA autoantibodies are a serological hallmark of the autoimmune disorder systemic lupus erythematosus. In a process involving antigen recognition, these antibodies are also believed to mediate the kidney inflammation that results in much of the morbidity and mortality associated with lupus. However, the nature of specific DNA antigens recognized by anti-DNA and the way in which anti-DNA interact with these molecules remains poorly understood. As a first step in defining the molecular basis of anti-DNA interactions, binding site selection experiments were conducted using three clonally related murine monoclonal anti-ssDNA autoantibodies previously isolated from a lupus prone MRL-lpr mouse (Swanson, P. C., Ackroyd, P. C., and Glick, G. D. (1996) Biochemistry 35, 1624-1633). Studying the interaction of these autoantibodies with the selected sequences (and variants) through affinity measurements and footprinting experiments provides evidence for sequence-specific binding of ssDNA. However, despite the similarity in amino acid sequence between the three mAbs, only mAb 11F8 appears to possess sequence specificity. The salient features of the interaction between 11F8 and its selected sequence (e.g., limited dependence of ionic strength upon binding, cross-reactivity, and conformational complementarity) are best described by combining the paradigms invoked to explain protein.nucleic acid and antibody.antigen recognition.

Design, synthesis, and analysis of conformationally constrained nucleic acids.

In this review I discuss straightforward and general methods to modify nucleic acid structure with disulfide cross-links. A motivating factor in developing this chemistry was the notion that disulfide bonds would be excellent tools to probe the structure, dynamics, thermodynamics, folding, and function of DNA and RNA, much in the way that cystine cross-links have been used to study proteins. The chemistry described has been used to synthesize disulfide cross-linked hairpins and duplexes, higher order structures like triplexes, nonground-state conformations, and tRNAs. Since the cross-links form quantitatively by mild air oxidation and do not perturb either secondary or tertiary structure, this modification should prove quite useful for the study of nucleic acids.

Conformational transitions of an unmodified tRNA: implications for RNA folding.

Unmodified tRNAs are powerful systems to study the effects of posttranscriptional modifications and site-directed mutations on both the structure and function of these ribonucleic acids. To define the general limitations of synthetic constructs as models for native tRNAs, it is necessary to elucidate the conformational states of unmodified tRNAs as a function of solution conditions. Here we report the conformational properties of unmodified yeast tRNAPhe as a function of ionic strength, [Mg2+], and temperature using a combination of spectroscopic measurements along with chemical and enzymatic probes. We find that in low [Na+] buffer at low temperature, native yeast tRNAPhe adopts tertiary structure in the absence of Mg2+. By contrast, tertiary folding of unmodified yeast tRNAPhe has an absolute requirement for Mg2+. Below the melting temperature of the cloverleaf, unmodified yeast tRNAPhe exists in a Mg2+-dependent equilibrium between secondary and tertiary structure. Taken together, our findings suggest that although the tertiary structures of tRNAs are broadly comparable, the intrinsic stability of the tertiary fold, the conformational properties of intermediate states, and the stability of intermediate states can differ significantly between tRNA sequences. Thus, the use of unmodified tRNAs as models for native constructs can have significant limitations. Broad conclusions regarding "tRNA folding" as a whole must be viewed cautiously, particularly in cases where structural changes occur, such as during protein synthesis.

Characterization of a unique anti-DNA hybridoma.

We have previously described the isolation and in vitro binding properties of eight anti-DNA monoclonal antibodies (MAbs) from an MRL-lpr mouse. In light of recent reports that have indicated it is possible to isolate multiple MAbs from a single hybridoma, our pathogenic hybridoma, 11F8, was examined for evidence of similar phenomena. Chromosome counting suggested that 11F8 cells are unusual and might indeed be expressing multiple heavy and/or light chains. PCR, cloning, and sequencing of immunoglobulin heavy and light chains indicate that 11F8 displays expression of both gamma 2a and gamma 3 heavy chains at the DNA level. Flow cytometry and amino acid sequencing reveals that expression of multiple isotypes also occurs at the protein level but only a single heavy- and light-chain sequence is able to bind DNA. Based on these results, we conclude that 11F8 is an unusual hybridoma that secretes two distinct heavy and at least one light chain from a single cell, and may represent a trioma, a stable three-cell fusion.

Probing structural elements in RNA using engineered disulfide cross-links.

Three analogs of unmodified yeast tRNAPhe, each possessing a single disulfide cross-link, have been designed and synthesized. One cross-link is between G1 and C72 in the amino acid acceptor stem, a second cross-link is in the central D region of yeast tRNAPhe between C11 and C25 and the third cross-link bridges U16 and C60 at the D loop/T loop interface. Air oxidation to form the cross-links is quantitative and analysis of the cross-linked products by native and denaturing PAGE, RNase T1 mapping, Pb(II) cleavage, UV cross-linking and thermal denaturation demonstrates that the disulfide bridges do not alter folding of the modified tRNAs relative to the parent sequence. The finding that cross-link formation between thiol-derivatized residues correlates with the position of these groups in the crystal structure of native yeast tRNAPhe and that the modifications do not significantly perturb native structure suggests that this methodology should be applicable to the study of RNA structure, conformational dynamics and folding pathways.

Use of cross-links to study the conformational dynamics of triplex DNA.

The conformational dynamics of a 34-base-long pyrimidine purine-pyrimidine motif intramolecular DNA triple helix possessing three cytosine residues in the Hoogsteen strand (1) and a disulfide cross-linked analog (2) were studied by two-dimensional exchange and NOE spectroscopy and by measuring base-catalyzed imino proton exchange rates. Under acidic conditions that stabilize triplexes containing Hoogsteen strand cytosines (pH 6.0 and 1 degree C), sequences 1 and 2 exhibit a small and identical degree of conformational heterogeneity. However, at a higher temperature (pH 6.0 and 37 degrees C), 1 exhibits much more extensive conformational heterogeneity than 2. The exchange times for Watson-Crick imino protons are approximately 1 h for both triplexes. However, the Hoogsteen base-pair lifetimes of 1 could not be measured because this sequence is conformationally labile under the alkaline conditions necessary to conduct these experiments. Because of the extraordinary pH stability conferred by the cross-link, it is possible to estimate the base-pair lifetimes for 2. The lifetimes of the Hoogsteen base pairs range from about 3 to 370 ms, and in all cases are shorter than that of the Watson-Crick base pair contained in the same triplet. These experiments represent the first measurement of base-pair lifetimes within Hoogsteen triplets. The ability to measure individual base-pair lifetimes may prove useful in studies that attempt to modulate triplex properties through rational design.

A new method to monitor the rate of conformational transitions in RNA.

Many RNAs need Mg2+to produce stable tertiary structures. Here we describe a simple method to measure the rate and activation parameters of tertiary structure unfolding that exploits this Mg2+dependence. Our approach is based on mixing an RNA solution with excess EDTA in a stopped-flow instrument equipped with an absorbance detector, under conditions of temperature and ionic strength where, after chelation of Mg2+, tertiary structure unfolds. We have demonstrated the utility of this method by studying phenylalanine-specific transfer RNA from yeast (tRNAPhe) because the unfolding rates and the corresponding activation parameters have been determined previously and provide a benchmark for our technique. We find that within error, our stopped-flow method reproduces both the rate and activation enthalpy for tertiary unfolding of yeast tRNAPhe measured previously by temperature-jump relaxation kinetics. Since many different RNAs require divalent magnesium for tertiary structure stabilization, this technique should be applicable to study the folding of other RNAs.

The effect of cross-links on the conformational dynamics of duplex DNA.

The base pair lifetimes and apparent dissociation constants of a 21 base DNA hairpin and an analog possessing a disulfide cross-link bridging the 3'- and 5'-terminal bases were determined by measuring imino proton exchange rates as a function of exchange catalyst concentration and temperature. A comparison of the lifetimes and apparent dissociation constants for corresponding base pairs of the two hairpins indicates that the cross-link neither increases the number of base pairs involved in fraying nor alters the lifetime, dissociation constant, or the opened structure from which exchange occurs for the base pairs that are not frayed. The cross-link does, however, stabilize the frayed penultimate base pair of the stem duplex. Significantly, it appears that the disulfide cross-link is more effective at preventing fraying of the penultimate base pair than is the 5 base hairpin loop. Because this disulfide cross-link can be incorporated site specifically, and does not adversely affect static or dynamic properties of DNA, it should prove very useful in studies of nucleic acid structure and function.

Thermodynamic properties of a conformationally constrained intramolecular DNA triple helix.

We describe the thermodynamic properties of an intramolecular triple helix with two all-thymine linker loops in which the Hoogsteen strand is covalently crosslinked to the underlying Watson-Crick hairpin duplex by means of a disulfide bridge. We compare these properties to those of the corresponding intramolecular triplex without the disulfide crosslink. Optical and calorimetric measurements reveal that the uncrosslinked parent triplex melts in a biphasic manner above pH 6, with the initial triplex to duplex transition (Hoogsteen strand release) occurring at lower temperatures than subsequent melting of the hairpin helix. By contrast, crosslinking increases the thermal stability of the Hoogsteen transition such that the triplex and underlying hairpin duplex melt as a single transition under all conditions studied. Model independent thermodynamic data obtained by differential scanning calorimetry reveals the crosslink-induced increase in triplex thermal stability corresponds to a free energy stabilization of about 3 kcal/mol, with this stabilization being entirely entropic in origin. In other words, the crosslink is enthalpically neutral, but nevertheless, induces a triplex stabilization of 3 kcal/mol due to a reduction in the entropy change associated with triplex melting. In an effort to define the origin(s) of this entropic impact, we measured the pH and ionic strength dependence of the melting transitions. From a comparison of the melting transitions at different pH values and ionic strengths, we estimate that 0.4 more protons are associated with the crosslinked triplex state than with the uncrosslinked triplex, and 1.3 fewer counterions are released on melting the crosslinked triplex. We discuss how such crosslink-induced changes in proton binding and counterion release, in conjunction with potential changes in hydration and conformational freedom, could combine to give rise to the observed changes in entropy.

Murine glomerulotropic monoclonal antibodies are highly oligoclonal and exhibit distinctive molecular features.

We recently produced a panel of seven glomerular-binding mAbs from a nephritic MRL-lpr mouse that bind to histones/nucleosomes (group I) or DNA (group II) adherent to glomerular basement membrane. To elucidate the molecular basis of their binding and ontogeny, we sequenced their variable (V) regions, analyzed the apparent somatic mutations, and predicted their three-dimensional structures. There were two clonally related sets (3 of 4 in group I, 3 of 3 in group II) both of the VHJ1558 family, and one mAb of the VH 7183 family. V region somatic mutations within clonally related sets had little effect on glomerular binding and did not appear to be selected for based on glomerular binding. The VH regions were most homologous with those from autoantibodies to histones, DNA, or IgG (i.e., rheumatoid factors), the Vkappa regions, with those from autoantibodies to small nuclear ribonucleoproteins (snRNP). The VH regions also exhibited an unusual VD junction (in the group I clonally related set) and an overall high content of charged amino acids (arginine, aspartic acid) in complementarity-determining regions (CDRs), particularly in CDR3. Molecular modeling studies suggested that the Fv regions of these mAbs converge to form a flat, open surface with a net positive charge. The CDR arginines in group I mAbs; appear to be located in Ag contact regions of the binding cleft. In sum, these data suggest that glomerulotropic mAbs are a highly restricted set of Abs with distinctive molecular features that may mediate their binding to glomeruli.