Isolation of compensatory inhibitor domain mutants to novel activation domain variants using the split-ubiquitin screen.

The control of transcription factor function plays an important role in the development of many processes in eukaryotes, such as drug resistance in fungi and human tumours undergoing chemotherapy. Detailed molecular mapping of the interactions between transcription factors and their protein partners can give important information about their mechanisms of action and reveal potential therapeutic targets. We devised a genetic screening system for mapping the interaction site between the Saccharomyces cerevisiae transcription factor-inhibitor pair Gal4p and Gal80p. A novel Gal4p activation domain mutant, L868K, was produced, which prevented it interacting with Gal80p. The split-ubiquitin system was used with a mutant GAL80 library in order to screen for compensatory mutants in Gal80p which would restore binding with L868K. Five single amino acid residue compensatory mutations in Gal80p which restored the interaction with Gal4p(L868K) were isolated. These compensatory mutations were specific to L868K as they were unable to restore the interaction with two other Gal4p mutants that were incapable of interacting with Gal80p. Mutations within Gal80p that were capable of compensating for Gal4p (L868K) clustered inside a Gal80p surface cleft, supporting the idea that this area is important for Gal4p binding. Our data suggest a way to generate information about interaction sites that should be applicable to any transcription factor.

Evaluating antirheumatic treatments using synovial biopsy: a recommendation for standardisation to be used in clinical trials.

Inflammation of synovium is one of the hallmarks of rheumatoid arthritis (RA). Analysis of synovial tissue has increased our understanding of RA pathogenesis, aided in identifying potential therapeutic targets and has been used in the response and mechanistic evaluation of antirheumatic treatments. In addition, studies are ongoing, aimed at the identification of diagnostic and prognostic biomarkers in the synovium. This paper outlines the currently used procedures for sampling and processing of synovial tissue, and presents a standardised recommendation to support multicentre translational research.

The effect of ligand binding on the galactokinase activity of yeast Gal1p and its ability to activate transcription.

The galactokinase from Saccharomyces cerevisiae (ScGal1p) is a bifunctional protein. It is an enzyme responsible for the conversion of alpha-D-galactose into galactose 1-phosphate at the expense of ATP but can also function as a transcriptional inducer of the yeast GAL genes. For both of these activities, the protein requires two ligands; a sugar (galactose) and a nucleotide (ATP). Here we investigate the effect of these ligands on the stability and conformation of ScGal1p to determine how the ligands alter protein function. We show that nucleotide binding increases the thermal stability of ScGal1p, whereas binding of galactose alone had no effect on the stability of the protein. This nucleotide stabilization effect is also observed for the related proteins S. cerevisiae Gal3p and Kluyveromyces lactis Gal1p and suggests that nucleotide binding results in the formation of, or the unmasking of, the galactose-binding site. We also show that the increase in stability of ScGal1p does not result from a large conformational change but is instead the result of a smaller more energetically favorable stabilization event. Finally, we have used mutant versions of ScGal1p to show that the galactokinase and transcriptional induction functions of the protein are distinct and separable. Mutations resulting in constitutive induction do not function by mimicking the more stable active conformation but have highlighted a possible site of interaction between ScGal1p and ScGal80p. These data give significant insights into the mechanism of action of both a galactokinase and a transcriptional inducer.

Effect of the early use of the anti-tumor necrosis factor adalimumab on the prevention of job loss in patients with early rheumatoid arthritis.

OBJECTIVE: To compare work disability and job loss in early rheumatoid arthritis (RA) patients receiving adalimumab plus methotrexate (adalimumab + MTX) versus MTX alone. METHODS: In this multicenter, randomized, controlled trial, patients with RA for <2 years who had never taken MTX and who self-reported work impairment were randomized to adalimumab + MTX or placebo + MTX for 56 weeks. Primary outcome was job loss of any cause and/or imminent job loss at or after week 16. Secondary outcomes included disease activity, function (Health Assessment Questionnaire [HAQ] score), and RA quality of life (RAQoL) questionnaire score. Work was evaluated with work diaries and the RA Work Instability Scale. RESULTS: Although job loss during the 56-week study was significantly lower with adalimumab + MTX (14 of 75 patients) compared with MTX alone (29 of 73 patients; P=0.005), the primary end point was not met (12 of 75 versus 20 of 73 patients; P=0.092), likely owing to early drop out in the MTX group. There were significant improvements in American College of Rheumatology 20% response criteria, 28-joint Disease Activity Score, DeltaHAQ, DeltaRAQoL, and working time lost in the adalimumab + MTX group. Twenty-four serious adverse events were reported in 17 participants, with no differences between groups. CONCLUSION: Adalimumab + MTX reduced job loss and improved productivity in early RA when compared with MTX alone, which supports the early use of anti-tumor necrosis factor therapy and suggests its cost efficacy.

The interaction between an acidic transcriptional activator and its inhibitor. The molecular basis of Gal4p recognition by Gal80p.

The GAL genes, which encode the enzymes required for normal galactose metabolism in yeast, are transcriptionally regulated by three proteins: Gal4p, an activator; Gal80p, an inhibitor; and Gal3p, a galactose sensor. These proteins control the switch between inert and active gene expression. The transcriptional activation function of Gal4p is rendered inactive in the presence of Gal80p. Here we present the three-dimensional structure of a complex between the acidic activation domain of Gal4p and Gal80p. The transactivation domain initiates with an extended region of polypeptide chain followed by two turns of an amphipathic alpha-helix. It fits into and across a deep cleft within the Gal80p dimer with the protein-protein interface defined primarily by hydrophobic interactions. A disordered loop in the apo-Gal80p structure (Asp-309 to Ser-316) becomes well-defined upon binding of the transactivation domain. This investigation provides a new molecular scaffold for understanding previous biochemical and genetic studies.

Arthroscopy as a research tool: a review.

Arthroscopy continues to experience a growth in interest from the rheumatology community reflecting a common desire to gain better understanding of the underlying processes in inflammatory and degenerative joint diseases. Arthroscopy provides the ability to assess the internal appearances of a joint in a well tolerated and repeatable manner, to obtain tissue samples from the principle site of pathology within the joint and thus confers on it the role of "gold standard" amongst currently available imaging techniques. The evolution of arthroscopy is reviewed together with an overview of the evidence obtained from its research application in the rheumatology. Methodology for the conduct of arthroscopy and synovial biopsy is described.

Therapy of patients with rheumatoid arthritis: outcome of infliximab failures switched to etanercept.

OBJECTIVE: The role of alternative tumor necrosis factor (TNF) antagonist therapies in the context of failure of initial TNF antagonist therapy in patients with rheumatoid arthritis (RA) has yet to be clearly defined. The goal of this study was to determine the efficacy of etanercept in patients who failed to respond to infliximab. METHODS: Ninety-five patients with RA who failed to respond to infliximab and methotrexate were treated with etanercept (with continuation of concomitant methotrexate). Thirty-four patients never achieved a response to infliximab (primary nonresponse), 38 had an initial response to infliximab but relapsed (secondary nonresponse), and 23 demonstrated toxicity. Disease Activity Score in 28 joints (DAS28), European League Against Rheumatism (EULAR) response, and American College of Rheumatology (ACR) response were determined after 12 weeks of etanercept. RESULTS: After 12 weeks of etanercept, 38% of patients achieved an ACR 20% response (ACR20) on etanercept. Of these, 24% and 15% achieved ACR50 and ACR70 responses, respectively. In the primary infliximab nonresponse group, 42%, 30%, and 15% achieved ACR20, ACR50, and ACR70 responses, respectively; the percentages for the secondary nonresponse group were 34%, 21%, and 14%, respectively. Significant DAS28 reductions were observed in the entire cohort and nonresponse subtype groups. Sixty-one percent of the cohort achieved either a moderate or good EULAR score (67% of primary and 56% of secondary infliximab failures). No toxicity was observed in patients who stopped infliximab due to intolerance; 19 of 23 continued etanercept after week 12. CONCLUSION: This study confirms that etanercept is effective in patients who fail to respond to infliximab and suggests a higher response in patients who have never had a response to infliximab.

Uncoupling of endothelial nitric oxidase synthase by hypochlorous acid: role of NAD(P)H oxidase-derived superoxide and peroxynitrite.

OBJECTIVE: The aim of the present study is to determine whether hypochlorous acid (HOCl), the major oxidant of leukocyte-derived myeloperoxidase (MPO), oxidizes the zinc-thiolate center of endothelial nitric oxide synthase (eNOS) and uncouples the enzyme. METHODS AND RESULTS: Exposure of purified recombinant eNOS to HOCl (> or = 100 micromol/L) released zinc and disrupted the enzyme-active eNOS dimers. In parallel with increased detections of both O2*- and ONOO-, clinically relevant concentrations of HOCl disrupted eNOS dimers in cultured human umbilical vein endothelial cells (HUVEC) at concentration 10- to 100-fold lower than those required for recombinant eNOS. In HUVEC, HOCl increased the translocation of both p67(phox) and p47(phox) of NAD(P)H oxidase and the phosphorylation of atypical protein kinase C-zeta. Further, genetic or pharmacological inhibition of either NAD(P)H oxidase-derived O2*- or PKC-zeta or NOS abolished the effects of HOCl on eNOS dimers. Consistently, HOCl increased both O2*- and ONOO- and eNOS dimer oxidation in isolated mouse aortas from C57BL/6 but less in those of gp91(phox) knock-out mice. Finally, in human carotid atherosclerotic arteries, eNOS predominantly existed as monomers in parallel with increased staining of both MPO and 3-nitrotyrosine. CONCLUSIONS: We conclude that HOCl uncouples eNOS by ONOO- generated from PKC-zeta-dependent NAD(P)H oxidase.

Molecular structure of Saccharomyces cerevisiae Gal1p, a bifunctional galactokinase and transcriptional inducer.

Gal1p of Saccharomyces cerevisiae is capable of performing two independent cellular functions. First, it is a key enzyme in the Leloir pathway for galactose metabolism where it catalyzes the conversion of alpha-d-galactose to galactose 1-phosphate. Second, it has the capacity to induce the transcription of the yeast GAL genes in response to the organism being challenged with galactose as the sole source of carbon. This latter function is normally performed by a highly related protein, Gal3p, but in its absence Gal1p can induce transcription, albeit inefficiently, both in vivo and in vitro. Here we report the x-ray structure of Gal1p in complex with alpha-d-galactose and Mg-adenosine 5'-(beta,gamma-imido)triphosphate (AMPPNP) determined to 2.4 Angstrom resolution. Overall, the enzyme displays a marked bilobal appearance with the active site being wedged between distinct N- and C-terminal domains. Despite being considerably larger than other galactokinases, Gal1p shares a similar molecular architecture with these enzymes as well as with other members of the GHMP superfamily. The extraordinary levels of similarity between Gal1p and Gal3p ( approximately 70% amino acid identity and approximately 90% similarity) have allowed a model for Gal3p to be constructed. By identifying the locations of mutations of Gal3p that result in altered transcriptional properties, we suggest potential models for Gal3p function and mechanisms for its interaction with the transcriptional inhibitor Gal80p. The GAL genetic switch has long been regarded as a paradigm for the control of gene expression in eukaryotes. Understanding the manner in which two of the proteins that function in transcriptional regulation interact with one another is an important step in determining the overall molecular mechanism of this switch.

Molecular structure of human galactose mutarotase.

Galactose mutarotase catalyzes the conversion of beta-d-galactose to alpha-d-galactose during normal galactose metabolism. The enzyme has been isolated from bacteria, plants, and animals and is present in the cytoplasm of most cells. Here we report the x-ray crystallographic analysis of human galactose mutarotase both in the apoform and complexed with its substrate, beta-d-galactose. The polypeptide chain folds into an intricate array of 29 beta-strands, 25 classical reverse turns, and 2 small alpha-helices. There are two cis-peptide bonds at Arg-78 and Pro-103. The sugar ligand sits in a shallow cleft and is surrounded by Asn-81, Arg-82, His-107, His-176, Asp-243, Gln-279, and Glu-307. Both the side chains of Glu-307 and His-176 are in the proper location to act as a catalytic base and a catalytic acid, respectively. These residues are absolutely conserved among galactose mutarotases. To date, x-ray models for three mutarotases have now been reported, namely that described here and those from Lactococcus lactis and Caenorhabditis elegans. The molecular architectures of these enzymes differ primarily in the loop regions connecting the first two beta-strands. In the human protein, there are six extra residues in the loop compared with the bacterial protein for an approximate longer length of 9 A. In the C. elegans protein, the first 17 residues are missing, thereby reducing the total number of beta-strands by one.

Substrate specificity and mechanism from the structure of Pyrococcus furiosus galactokinase.

Galactokinase (GalK) catalyses the first step of the Leloir pathway of galactose metabolism, the ATP-dependent phosphorylation of galactose to galactose-1-phosphate. In man, defects in galactose metabolism can result in disorders with severe clinical consequences, and deficiencies in galactokinase have been linked with the development of cataracts within the first few months of life. The crystal structure of GalK from Pyrococcus furiosus in complex with MgADP and galactose has been determined to 2.9 A resolution to provide insights into the substrate specificity and catalytic mechanism of the enzyme. The structure consists of two domains with the active site in a cleft at the domain interface. Inspection of the substrate binding pocket identifies the amino acid residues involved in galactose and nucleotide binding and points to both structural and mechanistic similarities with other enzymes of the GHMP kinase superfamily to which GalK belongs. Comparison of the sequence of the Gal3p inducer protein, which is related to GalK and which forms part of the transcriptional activation of the GAL gene cluster in the yeast Saccharomyces cerevisiae, has led to an understanding of the molecular basis of galactose and nucleotide recognition. Finally, the structure has enabled us to further our understanding on the functional consequences of mutations in human GalK which cause galactosemia.

Turnover of type II collagen and aggrecan in cartilage matrix at the onset of inflammatory arthritis in humans: relationship to mediators of systemic and local inflammation.

OBJECTIVE: To determine in vivo the extent of damage to, and changes in turnover of, articular cartilage type II collagen (CII) and the proteoglycan aggrecan following the onset of inflammatory arthritis in humans, and to examine the hypothesis that there are direct relationships between cartilage biomarkers of damage/turnover and clinical, histologic, and molecular markers of inflammation. METHODS: Synovial fluid (SF) and synovial membrane (SM) were obtained by arthroscopy, and a synovitis score was determined, in 32 patients with rheumatoid arthritis (RA) (13 with early untreated disease, 19 with established disease), 18 with psoriatic arthritis (PsA), and 10 with osteoarthritis (OA). Systemic disease activity markers were recorded, and SM CD3+ T cells, CD4+ T cells, CD68+ macrophages, and lining layer hyperplasia were quantified. SF levels of tumor necrosis factor alpha (TNFalpha), interleukin-10 (IL-10), matrix metalloproteinase 1 (MMP-1), MMP-3, Col2-3/4C(Long mono) neoepitope (C2C) (reflecting collagenase cleavage of cartilage CII), C-propeptide of type II procollagen (PIICP) (a biosynthesis marker), keratan sulfate (KS), and the 846 epitope of aggrecan (turnover) were measured by enzyme-linked immunosorbent assay or radioimmunoassay. RESULTS: Levels of cartilage degradation products in early RA or early PsA were not elevated above levels in OA, although in early inflammatory arthritis, TNFalpha and MMP-1 levels were similar to those observed in late inflammatory disease and higher than those in OA. PIICP was reduced in early RA. Correlations were observed between the SF C2C neoepitope level and the Health Assessment Questionnaire score, C-reactive protein level, plasma viscosity, synovitis score, and SF TNFalpha and MMP-1 levels. KS epitope content was reduced in direct relation to SM macrophage infiltration in the sublining and lining layers and in the presence of elevated SF MMP-3. Both SF MMP-1 and SF MMP-3 levels correlated with CD4+ T cell infiltration and lining layer hyperplasia in the SM, and MMP-1 levels correlated with lining layer CD68 levels, but TNFalpha and IL-10 levels did not. CONCLUSION: Except for CII synthesis, there were no significant changes in extracellular matrix turnover of aggrecan or CII in the early stages of human inflammatory arthritis. However, the direct correlation between the increases in TNFalpha and MMP-1 production and collagen degradation suggests that collagenase cleavage of cartilage collagen is related to the activities of TNFalpha and MMP-1. The reduction in CII synthesis in early RA may contribute to the developing pathology, since a lack of synthesis of this molecule would inhibit maintenance of cartilage matrix.

Angiopoietins, growth factors, and vascular morphology in early arthritis.

OBJECTIVE: To examine angiogenic growth factors in patients with early, untreated inflammatory arthritides and controls. METHODS: Synovial membrane (SM) infiltrate and Ang1, Ang2, and vascular endothelial growth factor (VEGF) mRNA and protein expression were examined using immunohistochemistry and in situ hybridization. Synovial fluid (SF) VEGF, transforming growth factor-beta (TGF-beta 1), and tumor necrosis factor-alpha (TNF-alpha) protein were measured by ELISA. Vascular morphology was assessed at arthroscopy. RESULTS: Ang2 mRNA and protein expression was observed in early psoriatic arthritis (PsA) and rheumatoid arthritis (RA) SM. Expression of Ang2 and VEGF was significantly greater in early PsA SM and correlated strongly. SF VEGF and TGF-beta 1 concentrations were also significantly higher in early PsA compared to RA. Distinct vascular morphology, with tortuous vessels in PsA, correlated with microscopic vascular scores (r = 0.54, p = 0.005) and VEGF levels (r = 0.51, p = 0.01). Ang1 mRNA and protein expression was observed, but concentrations were markedly lower than for Ang2 and VEGF. Clinical disease activity, SM infiltration, and SF TNF-alpha concentrations were similar in both groups. CONCLUSION: This is the first report of angiopoietin expression in early inflammatory arthritis. There is a close relationship between angiopoietins, VEGF, TGF-beta, and vascular morphology. There is differential angiogenesis at an early stage of inflammation, with major pathogenic and therapeutic implications.

Comparison of synovial tissues from the knee joints and the small joints of rheumatoid arthritis patients: Implications for pathogenesis and evaluation of treatment.

OBJECTIVE: Serial synovial biopsy samples are increasingly being used for the evaluation of novel therapies for rheumatoid arthritis (RA). Most studies have used tissues from knee biopsies, but technical improvements have made serial small joint arthroscopy feasible as well. Theoretically, there could be differences in the features of synovial inflammation between various joints as a result of mechanical factors, differences in innervation, and other factors. We therefore undertook this study to compare the cell infiltrate in paired synovial biopsy samples from inflamed knee joints and paired inflamed small joints of patients with RA. METHODS: Nine RA patients with both an inflamed knee joint and an inflamed small joint (wrist or metacarpophalangeal joint) underwent an arthroscopic synovial biopsy of both joints on the same day. Multiple biopsy specimens were collected and stained for macrophages, T cells, plasma cells, fibroblast-like synoviocytes, and interleukin-6 (IL-6) by immunohistochemistry. Sections were evaluated by digital image analysis. RESULTS: There were no significant differences in mean cell numbers for all markers investigated in samples from the knee joint compared with samples from the small joints. We detected statistically significant correlations for the numbers of sublining macrophages, T cells, and plasma cells, as well as for IL-6 expression, between the knee joint and the small joints. However, there was no significant correlation between different joints for the numbers of intimal macrophages or fibroblast-like synoviocytes. CONCLUSION: The results of this study show that the inflammation in one inflamed joint is generally representative of that in other inflamed joints. Therefore, it is possible to use serial samples from the same joint, selecting either large or small joints, for the evaluation of antirheumatic therapies.

Kinetic analysis of yeast galactokinase: implications for transcriptional activation of the GAL genes.

Galactokinase (EC 2.7.1.6) catalyses the first step in the catabolism of galactose. Yeast galactokinase, Gal1p, and the closely related but catalytically inactive Gal3p, also function as ligand sensors in the GAL genetic switch. In the presence of galactose and ATP (the substrates of the reaction catalysed by Gal1p) Gal1p or Gal3p can bind to Gal80p, a transcriptional repressor. This relieves the inhibition of a transcriptional activator, Gal4p, and permits expression of the GAL genes. In order to learn more about the mechanism of ligand sensing by Gal3p and Gal1p, we studied the kinetics of the reaction catalysed by Gal1p. Galactose-1-phosphate, a product of the reaction, is a mixed inhibitor both with respect to galactose and to ATP suggesting that the reaction proceeds via a compulsory, ordered, ternary complex mechanism. There is little variation in either the turnover number or the specificity constants in the pH range 6.0-9.5, implying that no catalytic base is required in the reaction. These data are discussed both in the context of galactokinase enzymology and their implications for the mechanism of transcriptional induction.