Stochastic description of the ligand-receptor interaction of biologically active substances at extremely low doses

Signalling molecules can be effective at extraordinarily low concentrations (down to attomolar levels). To handle such cases, probabilistic methods have been used to describe the formal kinetics of action of biologically active substances in these low doses, although it has been necessary to review what is meant by such a term. The mean numbers of transformed/degraded molecules and their dispersions were calculated for the possible range of ligand-receptor binding schemes. We used both analytical equations and numerical simulations to calculate the coefficients of variation (ratio of standard deviation to mean) and demonstrated that the distribution of the coefficient is highly dependent on the reaction scheme. It may, therefore, be used as an additional factor for discriminating between cooperative and noncooperative models of ligand-receptor interaction over extreme ranges of ligand dilution. The relevance to signalling behaviour is discussed.     

Dopamine depletion and subsequent treatment with L-DOPA, but not the long-lived dopamine agonist pergolide, enhances activity of the Akt pathway in the rat striatum

Dysregulation of signaling pathways is believed to contribute to Parkinson's disease pathology and l-DOPA-induced motor complications. Long-lived dopamine (DA) agonists are less likely to cause motor complications by virtue of continuous stimulation of DA receptors. In this study, we compared the effects of the unilateral 6-hydroxydopamine lesion and subsequent treatment with l-DOPA and DA agonist pergolide on signaling pathways in rats. Pergolide caused less pronounced behavioral sensitization than l-DOPA (25 mg/kg, i.p., 10 days), particularly at lower dose (0.5 and 0.25 mg/kg, i.p.). Pergolide, but not l-DOPA, reversed lesion-induced up-regulation of preproenkephalin and did not up-regulate preprodynorphine or DA D3 receptor in the lesioned hemisphere. Pergolide was as effective as l-DOPA in reversing the lesion-induced elevation of ERK2 phosphorylation in response to acute apomorphine administration (0.05 mg/kg, s.c.). Chronic l-DOPA significantly elevated the level of Akt phosphorylation at both Thr(308) and Ser(473) and concentration of phosphorylated GSK3alpha, whereas pergolide suppressed the lesion- and/or challenge-induced supersensitive Akt responses. The data indicate that l-DOPA, unlike pergolide, exacerbates imbalances in the Akt pathway caused by the loss of DA. The results support the hypothesis that the Akt pathway is involved in long-term actions of l-DOPA and may be linked to l-DOPA-induced dyskinesia.     

Structure and function of the visual arrestin oligomer

A distinguishing feature of rod arrestin is its ability to form oligomers at physiological concentrations. Using visible light scattering, we show that rod arrestin forms tetramers in a cooperative manner in solution. To investigate the structure of the tetramer, a nitroxide side chain (R1) was introduced at 18 different positions. The effects of R1 on oligomer formation, EPR spectra, and inter-spin distance measurements all show that the structures of the solution and crystal tetramers are different. Inter-subunit distance measurements revealed that only arrestin monomer binds to light-activated phosphorhodopsin, whereas both monomer and tetramer bind microtubules, which may serve as a default arrestin partner in dark-adapted photoreceptors. Thus, the tetramer likely serves as a 'storage' form of arrestin, increasing the arrestin-binding capacity of microtubules while readily dissociating to supply active monomer when it is needed to quench rhodopsin signaling.     

Assembly of Slx4 signaling complexes behind DNA replication forks

Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress.     

Determination of Fc function with frozen red blood cells.

The Fc function of immunoglobulins is commonly determined by an assay based on monitoring immunoglobulin induced, complement mediated red cell lysis. This assay requires a continuous source of fresh red cells. We have shown that the assay can be successfully performed with frozen red cells. The possibility of access to a stored standard stock of red cells will improve the convenience of performing the assay and could contribute to improved assay reproducibility.     

Dopamine D1 receptor interaction with arrestin3 in neostriatal neurons

Dopamine D1 receptor interactions with arrestins have been characterized using heterologously expressed D1 receptor and arrestins. The purpose of this study was to investigate the interaction of the endogenous D1 receptor with endogenous arrestin2 and 3 in neostriatal neurons. Endogenous arrestin2 and 3 in striatal homogenates bound to the C-terminus of the D1 receptor in a glutathione-S-transferase (GST) pulldown assay, with arrestin3 binding more strongly. The D1 C-terminus and, to a lesser extent, the third cytoplasmic loop also bound purified arrestin2 and 3. In neostriatal neurons, 2, 5, and 20 min agonist treatment increased the colocalization of the D1 receptor and arrestin3 immunoreactivity without altering the colocalization of the D1 receptor and arrestin2. Further, agonist treatment for 5 and 20 min caused translocation of arrestin3, but not arrestin2, to the membrane. The binding of arrestin3, but not arrestin2, to the D1 receptor was increased as assessed by coimmunoprecipitation after agonist treatment for 5 and 20 min. Agonist treatment of neurons induced D1 receptor internalization (35-45%) that was maximal within 2-5 min, a time-course similar to that of the increase in colocalization of the D1 receptor with arrestin3. These data indicate that the D1 receptor preferentially interacts with arrestin3 in neostriatal neurons.     

Mapping the arrestin-receptor interface. Structural elements responsible for receptor specificity of arrestin proteins

Arrestins selectively bind to phosphorylated activated forms of their cognate G protein-coupled receptors. Arrestin binding prevents further G protein activation and often redirects signaling to other pathways. The comparison of the high-resolution crystal structures of arrestin2, visual arrestin, and rhodopsin as well as earlier mutagenesis and peptide inhibition data collectively suggest that the elements on the concave sides of both arrestin domains most likely participate in receptor binding directly, thereby dictating its receptor preference. Using comparative binding of visual arrestin/arrestin2 chimeras to the preferred target of visual arrestin, light-activated phosphorylated rhodopsin (PRh*), and to the arrestin2 target, phosphorylated activated m2 muscarinic receptor (P-m2 mAChR*), we identified the elements that determine the receptor specificity of arrestins. We found that residues 49-90 (beta-strands V and VI and adjacent loops in the N-domain) and 237-268 (beta-strands XV and XVI in the C-domain) in visual arrestin and homologous regions in arrestin2 are largely responsible for their receptor preference. Only 35 amino acids (22 of which are nonconservative substitutions) in the two elements are different. Simultaneous exchange of both elements between visual arrestin and arrestin2 fully reverses their receptor specificity, demonstrating that these two elements in the two domains of arrestin are necessary and sufficient to determine their preferred receptor targets.     

The Role of Arrestin α-Helix I in Receptor Binding

Arrestins rapidly bind phosphorylated activated forms of their cognate G protein-coupled receptors, thereby preventing G protein coupling and often switching signaling to other pathways. Amphipathic α-helix I (residues 100-111) has been implicated in receptor binding, but the mechanism of its action has not been determined yet. Here we show that several mutations in the helix itself and in adjacent hydrophobic residues in the body of the N-domain reduce arrestin1 binding to light-activated phosphorylated rhodopsin (P-Rh^?). On the background of phosphorylation-independent mutants that bind with high affinity to both P-Rh^? and light-activated unphosphorylated rhodopsin, these mutations reduce the stability of the arrestin complex with P-Rh^?, but not with light-activated unphosphorylated rhodopsin. Using site-directed spin labeling, we found that the local structure around α-helix I changes upon binding to rhodopsin. However, the intramolecular distances between α-helix I and adjacent β-strand I (or the rest of the N-domain), measured using double electron-electron resonance, do not change, ruling out relocation of the helix due to receptor binding. Collectively, these data demonstrate that α-helix I plays an indirect role in receptor binding, likely keeping β-strand I, which carries several phosphate-binding residues, in a position favorable for its interaction with receptor-attached phosphates.     

Bacterial leaching of manganese ores

Leaching of various types of ores, containing 12-30% manganese, by the thiobacterium Acidithiobacillus ferrooxidans was studied. Leaching of reduced ores (manganocalcite and manganiferous limestone) was mediated mainly by degradation of manganiferous minerals (by sulfuric acid produced in the course of bacterial oxidation of pyrite or sulfur). Bacterial treatment of the ores for 144 and 192 h allowed solubilization of 96-98% of manganese. Inoculation of bacteria into pulp with pyrite increased the rate of leaching of oxide ore (psilomelane) by 37%, and the degree of its extraction within 180 h increased from 80 to 97%.     

Emerging roles for TNIP1 in regulating post-receptor signaling

A vast number of cellular processes and signaling pathways are regulated by various receptors, ranging from transmembrane to nuclear receptors. These receptor-mediated processes are modulated by a diverse set of regulatory proteins. TNFα-induced protein 3-interacting protein 1 is such a protein that inhibits both transduction by transmembrane receptors, such as TNFα-receptor, EGF-R, and TLR, and nuclear receptors' PPAR and RAR activity. These receptors play key roles in regulating inflammation and inflammatory diseases. A growing number of references have implicated TNIP1 through GWAS and expression studies in chronic inflammatory diseases such as psoriasis and rheumatoid arthritis, although TNIP1s exact role has yet been determined. In this review, we aim to integrate the current knowledge of TNIP1s functions with the diseases in which it has been associated to potentially elucidate the role this regulator has in promoting or alleviating these inflammatory diseases.