A Fully Automated Multiplex Assay for Diagnosis of Lyme Disease with High Specificity and Improved Early Sensitivity.

Lyme borreliosis is a tick-borne disease caused by the Borrelia burgdorferisensu lato complex. Bio-Rad Laboratories has developed a fully automated multiplex bead-based assay for the detection of IgM and IgG antibodies to B. burgdorferi The BioPlex 2200 Lyme Total assay exhibits an improved rate of seropositivity in patients with early Lyme infection. Asymptomatic subjects from endemic and nonendemic origins demonstrated a seroreactivity rate of approximately 4% that was similar to other commercial assays evaluated in this study. Coupled to this result was the observation that the Lyme Total assay retained a high first-tier specificity of 96% while demonstrating a relatively high sensitivity of 91% among a well-characterized CDC Premarketing Lyme serum panel. The Lyme Total assay also performs well under a modified two-tier algorithm (sensitivity, 84.4 to 88.9%; specificity, 98.4 to 99.5%). Furthermore, the new assay is able to readily detect early Lyme infection in patient samples from outside North America.

NFkappaB selectivity of estrogen receptor ligands revealed by comparative crystallographic analyses.

Our understanding of how steroid hormones regulate physiological functions has been significantly advanced by structural biology approaches. However, progress has been hampered by misfolding of the ligand binding domains in heterologous expression systems and by conformational flexibility that interferes with crystallization. Here, we show that protein folding problems that are common to steroid hormone receptors are circumvented by mutations that stabilize well-characterized conformations of the receptor. We use this approach to present the structure of an apo steroid receptor that reveals a ligand-accessible channel allowing soaking of preformed crystals. Furthermore, crystallization of different pharmacological classes of compounds allowed us to define the structural basis of NFkappaB-selective signaling through the estrogen receptor, thus revealing a unique conformation of the receptor that allows selective suppression of inflammatory gene expression. The ability to crystallize many receptor-ligand complexes with distinct pharmacophores allows one to define structural features of signaling specificity that would not be apparent in a single structure.

The two major isoforms of thyroid hormone receptor, TRα1 and TRß1, preferentially partner with distinct panels of auxiliary proteins.

Thyroid hormone receptors (TRs) are expressed primarily as two major isoforms, TRα1 and TRß1, which are expressed at different times in development and at different tissue abundances in the adult. The transcription properties and biological properties of TRα1 and TRß1 can differ. We report here that although overlapping, TRα1 and TRß1 recruit distinct panels of partner proteins that may account for their divergent biological functions, and which appear to explain their distinct target gene regulatory properties.

Research resource: identification of novel coregulators specific for thyroid hormone receptor-ß2.

Thyroid hormone receptors (TRs) are expressed as a series of interrelated isoforms that perform distinct biological roles. The TRß2 isoform is found predominantly in the hypothalamus, pituitary, retina, and cochlea and displays unique transcriptional properties relative to the other TR isoforms. To more fully understand the isoform-specific biological and molecular properties of TRß2, we have identified a series of previously unrecognized proteins that selectively interact with TRß2 compared with the more widely expressed TRß1. Several of these proteins preferentially enhance the transcriptional activity of TRß2 when coexpressed in cells and are likely to represent novel, isoform-specific coactivators. Additional proteins were also identified in our screen that bind equally to TRß1 and TRß2 and may function as isoform-independent auxiliary proteins for these and/or other nuclear receptors. We propose that a combination of isoform-specific recruitment and tissue-specific expression of these newly identified coregulator candidates serves to customize TR function for different biological purposes in different cell types.

Regulation of SMRT corepressor dimerization and composition by MAP kinase phosphorylation.

The SMRT (Silencing Mediator of Retinoid and Thyroid hormone receptors) corepressor mediates gene repression by nuclear receptors and other transcriptional factors. The SMRT protein serves as a key nucleating core that organizes the assembly of a larger corepressor complex. We report here that SMRT interacts with itself to form a protein dimer, and that Erk2, a mitogen-activated protein (MAP) kinase, disrupts this SMRT self-dimerization in vitro and in vivo. Notably Erk2 phosphorylation also results in a re-organization of the overall corepressor complex, characterized by a reduced sedimentation coefficient, partial release of HDAC3, TBL-1, and TBLR-1, and inhibition of transcriptional repression. We propose that SMRT dimers form the central platform on which additional corepressor components assemble, and that kinase signaling modifies the architecture, composition, and function of this complex. These observations contribute to our understanding of how the SMRT corepressor complex assembles and is regulated during cell proliferation and differentiation.

Estrogen receptors recruit SMRT and N-CoR corepressors through newly recognized contacts between the corepressor N terminus and the receptor DNA binding domain.

Estrogen receptors (ERs) are hormone-regulated transcription factors that regulate key aspects of reproduction and development. ERs are unusual in that they do not typically repress transcription in the absence of hormone but instead possess otherwise cryptic repressive functions that are revealed upon binding to certain hormone antagonists. The roles of corepressors in the control of these aspects of ER function are complex and incompletely understood. We report here that ERs recruit SMRT through an unusual mode of interaction involving multiple contact surfaces. Two surfaces of SMRT, located at the N- and C-terminal domains, contribute to the recruitment of the corepressor to ERs in vitro and are crucial for the corepressor modulation of ER transcriptional activity in cells. These corepressor surfaces contact the DNA binding domain of the receptor, rather than the hormone binding domain previously elucidated for other corepressor/nuclear receptor interactions, and are modulated by the ER's recognition of cognate DNA binding sites. Several additional nuclear receptors, and at least one other corepressor, N-CoR, share aspects of this novel mode of corepressor recruitment. Our results highlight a molecular mechanism that helps explain several previously paradoxical aspects of ER-mediated transcriptional antagonism, which may have a broader significance for an understanding of target gene repression by other nuclear receptors.

The p160 coactivator PAS-B motif stabilizes nuclear receptor binding and contributes to isoform-specific regulation by thyroid hormone receptors.

Thyroid hormone receptors (TRs) are hormone-regulated transcription factors that play multiple roles in vertebrate endocrinology and development. TRs are expressed as a series of distinct receptor isoforms that mediate different biological functions. The TRbeta2 isoform is expressed primarily in the hypothalamus, pituitary, cochlea, and retina, and displays an enhanced response to hormone agonist relative to the other TR isoforms. We report here that the unusual transcriptional properties of TRbeta2 parallel the ability of this isoform to bind p160 coactivators cooperatively through multiple contact surfaces; the more broadly expressed TRbeta1 isoform, in contrast, utilizes a single contact mechanism. Intriguingly, the PAS-B domain in the p160 N terminus plays a previously unanticipated role in permitting TRbeta2 to recruit coactivator at limiting triiodothyronine concentrations. The PAS-B sequences also play an important role in coactivator binding by estrogen receptor-alpha. We propose that the PAS-B domain of the p160 coactivators is an important modulator of coactivator recruitment for a specific subset of nuclear receptors, permitting stronger transcriptional activation at lower hormone concentrations than would otherwise occur, and allowing isoform-specific mRNA splicing to customize the hormone response in different tissues.