MicroRNAs in systemic rheumatic diseases

MicroRNAs (miRNAs) are endogenous, non-coding, single-stranded RNAs about 21 nucleotides in length. miRNAs have been shown to regulate gene expression and thus influence a wide range of physiological and pathological processes. Moreover, they are detected in a variety of sources, including tissues, serum, and other body fluids, such as saliva. The role of miRNAs is evident in various malignant and nonmalignant diseases, and there is accumulating evidence also for an important role of miRNAs in systemic rheumatic diseases. Abnormal expression of miRNAs has been reported in autoimmune diseases, mainly in systemic lupus erythematosus and rheumatoid arthritis. miRNAs can be aberrantly expressed even in the different stages of disease progression, allowing miRNAs to be important biomarkers, to help understand the pathogenesis of the disease, and to monitor disease activity and effects of treatment. Different groups have demonstrated a link between miRNA expression and disease activity, as in the case of renal flares in lupus patients. Moreover, miRNAs are emerging as potential targets for new therapeutic strategies of autoimmune disorders. Taken together, recent data demonstrate that miRNAs can influence mechanisms involved in the pathogenesis, relapse, and specific organ involvement of autoimmune diseases. The ultimate goal is the identification of a miRNA target or targets that could be manipulated through specific therapies, aiming at activation or inhibition of specific miRNAs responsible for the development of disease.     

Frequent coexistence of anti-topoisomerase I and anti-U1RNP autoantibodies in African American patients associated with mild skin involvement: a retrospective clinical study

Introduction  

The presence of anti-topoisomerase I (topo I) antibodies is a classic scleroderma (SSc) marker presumably associated with a unique clinical subset. Here the clinical association of anti-topo I was reevaluated in unselected patients seen in a rheumatology clinic setting.     

Mapping and tagging of seed coat colour and the identification of microsatellite markers for marker-assisted manipulation of the trait in Brassica juncea

Microsatellite marker technology in combination with three doubled haploid mapping populations of Brassica juncea were used to map and tag two independent loci controlling seed coat colour in B. juncea. One of the populations, derived from a cross between a brown-seeded Indian cultivar, Varuna, and a Canadian yellow-seeded line, Heera, segregated for two genes coding for seed coat colour; the other two populations segregated for one gene each. Microsatellite markers were obtained from related Brassica species. Three microsatellite markers (Ra2-A11, Na10-A08 and Ni4-F11) showing strong association with seed coat colour were identified through bulk segregant analysis. Subsequent mapping placed Ra2-A11 and Na10-A08 on linkage group (LG) 1 at an interval of 0.6 cM from each other and marker Ni4-F11 on LG 2 of the linkage map of B. juncea published previously (Pradhan et al., Theor Appl Genet 106:607–614, 2003). The two seed coat colour genes were placed with markers Ra2-A11 and Na10-A08 on LG 1 and Ni4-F11 on LG 2 based on marker genotyping data derived from the two mapping populations segregating for one gene each. One of the genes (BjSC1) co-segregated with marker Na10-A08 in LG 1 and the other gene (BjSC2) with Ni4-F11 in LG 2, without any recombination in the respective mapping populations of 130 and 103 segregating plants. The identified microsatellite markers were studied for their length polymorphism in a number of yellow-seeded eastern European and brown-seeded Indian germplasm of B. juncea and were found to be useful for the diversification of yellow seed coat colour from a variety of sources into Indian germplasm.     

Effects of the core lipid on the energetics of binding of ApoA-I to model lipoprotein particles of different sizes

Interaction of apolipoproteins (apo) with lipid surfaces plays crucial roles in lipoprotein metabolism and cholesterol homeostasis. To elucidate the thermodynamics of binding of apoA-I to lipid, we used lipid emulsions composed of triolein (TO) and egg phosphatidylcholine (PC) as lipoprotein models. Determination of the level of binding of wild-type (WT) apoA-I and some deletion mutants to large (120 nm diameter; LEM) and small (35 nm diameter; SEM) emulsions indicated that N-terminal (residues 44-65) and C-terminal (residues 190-243 and 223-243) deletions have large effects on lipid interaction, whereas deletion of the central region (residues 123-166) has little effect. Substitution of amino acids at either L230 or L230, L233, and Y236 with proline residues also decreases the level of binding, indicating that an alpha-helix conformation in this C-terminal region is required for efficient lipid binding. Calorimetry showed that binding of WT apoA-I to SEM generates endothermic heat (DeltaH approximately 30 kcal/mol) in contrast to the exothermic heat (ca. -85 kcal/mol) generated upon binding to LEM and egg PC small unilamellar vesicles (SUV). This exothermic heat arises from an approximately 25% increase in alpha-helix content, and it drives the binding of apoA-I to LEM and SUV. There is a similar increase in alpha-helix content of apoA-I upon binding to either SEM or SUV, but the binding of apoA-I to SEM is an entropy-driven process. These results suggest that the presence of a core triglyceride modifies the highly curved SEM surface packing and thereby the thermodynamics of apoA-I binding in a manner that compensates for the exothermic heat generated by alpha-helix formation.     

Alpha-helix formation is required for high affinity binding of human apolipoprotein A-I to lipids

Apolipoprotein (apo) A-I is thought to undergo a conformational change during lipid association that results in the transition of random coil to alpha-helix. Using a series of deletion mutants lacking different regions along the molecule, we examined the contribution of alpha-helix formation in apoA-I to the binding to egg phosphatidylcholine (PC) small unilamellar vesicles (SUV). Binding isotherms determined by gel filtration showed that apoA-I binds to SUV with high affinity and deletions in the C-terminal region markedly decrease the affinity. Circular dichroism measurements demonstrated that binding to SUV led to an increase in alpha-helix content, but the helix content was somewhat less than in reconstituted discoidal PC.apoA-I complexes for all apoA-I variants, suggesting that the helical structure of apoA-I on SUV is different from that in discs. Isothermal titration calorimetry showed that the binding of apoA-I to SUV is accompanied by a large exothermic heat and deletions in the C-terminal regions greatly decrease the heat. Analysis of the rate of release of heat on binding, as well as the kinetics of quenching of tryptophan fluorescence by brominated PC, indicated that the opening of the N-terminal helix bundle is a rate-limiting step in apoA-I binding to the SUV surface. Significantly, the correlation of thermodynamic parameters of binding with the increase in the number of helical residues revealed that the contribution of alpha-helix formation upon lipid binding to the enthalpy and the free energy of the binding of apoA-I is -1.1 and -0.04 kcal/mol per residue, respectively. These results indicate that alpha-helix formation, especially in the C-terminal regions, provides the energetic source for high affinity binding of apoA-I to lipids.     

Structure-activity relationships of 111In- and 99mTc-labeled quinolin-4-one peptidomimetics as ligands for the vitronectin receptor: potential tumor imaging agents

The integrin receptor alpha(v)beta(3) is overexpressed on the endothelial cells of growing tumors and on some tumor cells themselves. Radiolabeled alpha(v)beta(3) antagonists have demonstrated potential application as tumor imaging agents and as radiotherapeutic agents. This report describes the total synthesis of eight new HYNIC and DOTA conjugates of receptor alpha(v)beta(3) antagonists belonging to the quinolin-4-one class of peptidomimetics, and their radiolabeling with (99m)Tc (for HYNIC) and (111)In (for DOTA). Tethering of the radionuclide-chelator complexes was achieved at two different sites on the quinolin-4-one molecule. All such derivatives maintained high affinity for receptor alpha(v)beta(3) and high selectivity versus receptors alpha(IIb)beta(3), alpha(v)beta(5), alpha(5)beta(1). Biodistribution of the radiolabeled compounds was evaluated in the c-neu Oncomouse mammary adenocarcinoma model. DOTA conjugate (111)In-TA138 presented the best biodistribution profile. Tumor uptake at 2 h postinjection was 9.39% of injected dose/g of tissue (%ID/g). Activity levels in selected organs was as follows: blood, 0.54% ID/g; liver, 1.94% ID/g; kidney, 2.33% ID/g; lung, 2.74% ID/g; bone, 1.56% ID/g. A complete biodistribution analysis of (111)In-TA138 and the other radiolabeled compounds of this study are presented and discussed. A scintigraphic imaging study with (111)In-TA138 showed a clear delineation of the tumors and rapid clearance of activity from nontarget tissues.     

Surface plasmon resonance analysis of the mechanism of binding of apoA-I to high density lipoprotein particles

The partitioning of apolipoprotein A-I (apoA-I) molecules in plasma between HDL-bound and -unbound states is an integral part of HDL metabolism. We used the surface plasmon resonance (SPR) technique to monitor in real time the reversible binding of apoA-I to HDL. Biotinylated human HDL₂ and HDL₃ were immobilized on a streptavidin-coated SPR sensor chip, and apoA-I solutions at different concentrations were flowed across the surface. The wild-type (WT) human and mouse apoA-I/HDL interaction involves a two-step process; apoA-I initially binds to HDL with fast association and dissociation rates, followed by a step exhibiting slower kinetics. The isolated N-terminal helix bundle domains of human and mouse apoA-I also exhibit a two-step binding process, consistent with the second slower step involving opening of the helix bundle domain. The results of fluorescence experiments with pyrene-labeled apoA-I are consistent with the N-terminal helix bundle domain interacting with proteins resident on the HDL particle surface. Dissociation constants (K_d) measured for WT human apoA-I interactions with HDL₂ and HDL₃ are about 10 µM, indicating that the binding is low affinity. This K_d value does not apply to all of the apoA-I molecules on the HDL particle but only to a relatively small, labile pool.Understanding the structure and function of HDL is significant because of the beneficial cardioprotective properties of this lipoprotein (1). The anti-atherogenic effects of HDL arise, in part, from its participation in the reverse cholesterol transport pathway where the principal HDL protein, apolipoprotein A-I (apoA-I), plays a central role (2). As a result, the structure-function relationships of apoA-I have been studied extensively (for reviews, see Refs. 3–5). Perhaps the most important characteristic of the apoA-I molecule is its ability to bind lipids; this interaction is mediated by the amphipathic α-helices present in the protein molecule (6). ApoA-I binds well to phospholipid (PL)-water interfaces and, under appropriate conditions, can solubilize the PL to create discoidal HDL particles (7, 8). The binding of apoA-I to a PL surface involves a two-step mechanism. First, α-helices in the C-terminal domain of the protein interact with the surface, and, second, the N-terminal helix bundle domain opens to allow more helix-lipid interactions to occur (5, 9). Although the binding of apoA-I to model PL particles has been studied extensively, the binding of apoA-I to HDL particles has not been investigated much because of the difficulty of separating free and bound apoA-I in this system. This lack of information about apoA-I/HDL interactions is significant because the cycling of apoA-I molecules on and off HDL particles occurs during the metabolism of HDL particles (10, 11), in particular to release apoA-I molecules into the preβ-HDL pool (10, 12). This recycling is consistent with the well-established ability of apolipoproteins, such as apoA-I, to exchange spontaneously between different populations of lipoprotein particles (13–16) and PL vesicles (17, 18). As a rule, any remodeling event that depletes HDL particles of PL induces particle fusion and dissociation of that fraction of the apoA-I molecules that is in a labile pool (19). At this stage, quantitative understanding of the kinetics of apoA-I interactions with HDL particles is unavailable.Here, we exploit surface plasmon resonance (SPR) to monitor in real time the association and dissociation reactions in the apoA-I/HDL system. SPR has been used to derive quantitative information about the binding of both lipoproteins (20) and apoE (21–23) to proteoglycans. As far as the application of SPR to the HDL system is concerned, the binding of several plasma remodeling factors to HDL immobilized on a sensor chip has been investigated successfully (24–26). Also, the conformation of apoA-I in HDL was explored by comparing the binding of HDL particles to anti-apoA-I monoclonal antibodies immobilized on an SPR chip (27). We have extended these approaches to study the binding of apoA-I to HDL particles. The results show that apoA-I can bind reversibly and with low affinity to HDL particles by a two-step mechanism.     

Flow-enhanced adhesion regulated by a selectin interdomain hinge

L-selectin requires a threshold shear to enable leukocytes to tether to and roll on vascular surfaces. Transport mechanisms govern flow-enhanced tethering, whereas force governs flow-enhanced rolling by prolonging the lifetimes of L-selectin-ligand complexes (catch bonds). Using selectin crystal structures, molecular dynamics simulations, site-directed mutagenesis, single-molecule force and kinetics experiments, Monte Carlo modeling, and flow chamber adhesion studies, we show that eliminating a hydrogen bond to increase the flexibility of an interdomain hinge in L-selectin reduced the shear threshold for adhesion via two mechanisms. One affects the on-rate by increasing tethering through greater rotational diffusion. The other affects the off-rate by strengthening rolling through augmented catch bonds with longer lifetimes at smaller forces. By forcing open the hinge angle, ligand may slide across its interface with L-selectin to promote rebinding, thereby providing a mechanism for catch bonds. Thus, allosteric changes remote from the ligand-binding interface regulate both bond formation and dissociation.     

Structural insights into the design of nonpeptidic isothiazolidinone-containing inhibitors of protein-tyrosine phosphatase 1B

Structural analyses of the protein-tyrosine phosphatase 1B (PTP1B) active site and inhibitor complexes have aided in optimization of a peptide inhibitor containing the novel (S)-isothiazolidinone (IZD) phosphonate mimetic. Potency and permeability were simultaneously improved by replacing the polar peptidic backbone of the inhibitor with nonpeptidic moieties. The C-terminal primary amide was replaced with a benzimidazole ring, which hydrogen bonds to the carboxylate of Asp(48), and the N terminus of the peptide was replaced with an aryl sulfonamide, which hydrogen bonds to Asp(48) and the backbone NH of Arg(47) via a water molecule. Although both substituents retain the favorable hydrogen bonding network of the peptide scaffold, their aryl rings interact weakly with the protein. The aryl ring of benzimidazole is partially solvent exposed and only participates in van der Waals interactions with Phe(182) of the flap. The aryl ring of aryl sulfonamide adopts an unexpected conformation and only participates in intramolecular pi-stacking interactions with the benzimidazole ring. These results explain the flat SAR for substitutions on both rings and the reason why unsubstituted moieties were selected as candidates. Finally, substituents ortho to the IZD heterocycle on the aryl ring of the IZD-phenyl moiety bind in a small narrow site adjacent to the primary phosphate binding pocket. The crystal structure of an o-chloro derivative reveals that chlorine interacts extensively with residues in the small site. The structural insights that have led to the discovery of potent benzimidazole aryl sulfonamide o-substituted derivatives are discussed in detail.     

ZNF198, a zinc finger protein rearranged in myeloproliferative disease, localizes to the PML nuclear bodies and interacts with SUMO-1 and PML

The ZNF198/FGFR1 fusion gene in atypical myeloproliferative disease produces a constitutively active cytoplasmic tyrosine kinase, unlike ZNF198 which is normally a nuclear protein. We have now shown that the ZNF198/FGFR1 fusion kinase interacts with the endogenous ZNF198 protein suggesting that the function of ZNF198 may be compromised in cells expressing it. Little is currently known about the endogenous function of ZNF198 and to investigate this further we performed a yeast two-hybrid analysis and identified SUMO-1 as a binding partner of ZNF198. These observations were confirmed using co-immunoprecipitation which demonstrated that ZNF198 is covalently modified by SUMO-1. Since many of the SUMO-1-modified proteins are targeted to the PML nuclear bodies we used confocal microscopy to show that SUMO-1, PML and ZNF198 colocalize to punctate structures, shown by immunocytochemistry to be PML bodies. Using co-immunoprecipitation we now show that PML and sumoylated ZNF198 can be found in a protein complex in the cell. Mutation of the SUMO-1 binding site in wild-type ZNF198 resulted in loss of distinct PML bodies, reduced PML levels and a more dispersed nuclear localization of the PML protein. In cells expressing ZNF198/FGFR1, which also lack the SUMO-1 binding site, SUMO-1 is preferentially localized in the cytoplasm, which is associated with loss of distinct PML bodies. Recently, arsenic trioxide (ATO) was proposed as an alternative therapy for APL that was resistant to traditional therapy. Treatment of cells expressing ZNF198/FGFR1 with ATO demonstrated reduced autophosphorylation of the ZNF198/FGFR1 protein and induced apoptosis, which is not seen in cells expressing wild-type ZNF198. Overall our results suggest that the sumoylation of ZNF198 is important for PML body formation and that the abrogation of sumoylation of ZNF198 in ZNF198/FGFR1 expressing cells may be an important mechanism in cellular transformation.