Purification and functional analysis of recombinant Acholeplasma laidlawii histone-like HU protein

HU is a most abundant DNA-binding protein in bacteria. This protein is conserved either in its heterodimeric form or in one of its homodimeric forms in all bacteria, in plant chloroplasts, and in some viruses. HU protein non-specifically binds and bends DNA as a hetero- or homodimer and can participate in DNA supercoiling and DNA condensation. It also takes part in some DNA functions such as replication, recombination, and repair. HU does not recognize any specific sequences but shows some specificity to cruciform DNA and to repair intermediates, e.g., nick, gap, bulge, 3′-overhang, etc. To understand the features of HU binding to DNA and repair intermediates, a fast and easy HU proteins purification procedure is required. Here we report overproduction and purification of the HU homodimers. The method of HU purification allows obtaining a pure recombinant non-tagged protein cloned in Escherichia coli. We applied this method for purification of Acholeplasma laidlawii HU and demonstrated that this protein possesses a DNA - binding activity and is free of contaminating nuclease activity. Besides that we have shown that expression of A. laidlawii ihf_hu gene in a slow-growing hupAB E. coli strain restores the wild-type growth indicating that aclHU can perform the basic functions of E. coli HU in vivo.     

Complete genome and proteome of Acholeplasma laidlawii

We present the complete genome sequence and proteogenomic map for Acholeplasma laidlawii PG-8A (class Mollicutes, order Acholeplasmatales, family Acholeplasmataceae). The genome of A. laidlawii is represented by a single 1,496,992-bp circular chromosome with an average G+C content of 31 mol%. This is the longest genome among the Mollicutes with a known nucleotide sequence. It contains genes of polymerase type I, SOS response, and signal transduction systems, as well as RNA regulatory elements, riboswitches, and T boxes. This demonstrates a significant capability for the regulation of gene expression and mutagenic response to stress. Acholeplasma laidlawii and phytoplasmas are the only Mollicutes known to use the universal genetic code, in which UGA is a stop codon. Within the Mollicutes group, only the sterol-nonrequiring Acholeplasma has the capacity to synthesize saturated fatty acids de novo. Proteomic data were used in the primary annotation of the genome, validating expression of many predicted proteins. We also detected posttranslational modifications of A. laidlawii proteins: phosphorylation and acylation. Seventy-four candidate phosphorylated proteins were found: 16 candidates are proteins unique to A. laidlawii, and 11 of them are surface-anchored or integral membrane proteins, which implies the presence of active signaling pathways. Among 20 acylated proteins, 14 contained palmitic chains, and six contained stearic chains. No residue of linoleic or oleic acid was observed. Acylated proteins were components of mainly sugar and inorganic ion transport systems and were surface-anchored proteins with unknown functions.     

Small molecule inhibitors of protein interaction with glycosaminoglycans (SMIGs), a novel class of bioactive agents with anti-inflammatory properties

Background

Small molecule inhibitors of biologically important protein–glycosaminoglycan (GAG) interactions have yet to be identified.

Methods

Compound libraries were screened in an assay of L-selectin–IgG binding to heparin (a species of heparan sulfate [HS-GAG]). Hits were validated, IC-50s established and direct binding of hits to HS-GAGs was investigated by incubating compounds alone with heparin. Selectivity of inhibitors was assessed in 11 different protein-GAG binding assays. Anti-inflammatory activity of selected compounds was evaluated in animal models.

Results

Screening identified a number of structurally-diverse planar aromatic cationic amines. Scaffolds similar to known GAG binders, chloroquine and tilorone, were also identified. Inhibitors displayed activity also against bovine kidney heparan sulfate. Direct binding of compounds to GAGs was verified by incubating compounds with heparin alone. Selectivity of inhibitors was demonstrated in a panel of 11 heparin binding proteins, including selectins, chemokines (IL-8, IP-10), Beta Amyloid and cytokines (VEGF, IL-6). A number of selected lead compounds showed dose-dependent efficacy in peritonitis, paw edema and delayed type hypersensitivity.

Conclusions

A new class of compounds, SMIGs, inhibits protein–GAG interaction by direct binding to GAGs. Although their IC-50s were in the low micro-molar range, SMIGs binding to HS-GAGs appeared to be stable in physiological conditions, indicating high avidity binding. SMIGs may interfere with major checkpoints for inflammatory and autoimmune events.

General significance

SMIGs are a class of structurally-diverse planar aromatic cationic amines that have an unusual mode of action — inhibiting protein–GAG interactions via direct and stable binding to GAGs. SMIGs may have therapeutic potential in inflammatory and autoimmune disorders.     

The last rRNA methyltransferase of E. coli revealed: The yhiR gene encodes adenine-N6 methyltransferase specific for modification of A2030 of 23S ribosomal RNA

The ribosomal RNA (rRNA) of Escherichia coli contains 24 methylated residues. A set of 22 methyltransferases responsible for modification of 23 residues has been described previously. Herein we report the identification of the yhiR gene as encoding the enzyme that modifies the 23S rRNA nucleotide A2030, the last methylated rRNA nucleotide whose modification enzyme was not known. YhiR prefers protein-free 23S rRNA to ribonucleoprotein particles containing only part of the 50S subunit proteins and does not methylate the assembled 50S subunit. We suggest renaming the yhiR gene to rlmJ according to the rRNA methyltransferase nomenclature. The phenotype of yhiR knockout gene is very mild under various growth conditions and at the stationary phase, except for a small growth advantage at anaerobic conditions. Only minor changes in the total E. coli proteome could be observed in a cell devoid of the 23S rRNA nucleotide A2030 methylation.     

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 2. Treatment of some ROS- and Age-related diseases (heart arrhythmia, heart infarctions, kidney ischemia, and stroke)

Effects of 10-(6′-plastoquinonyl) decyltriphenylphosphonium (SkQ1) and 10-(6′-plastoquinonyl) decylrhod-amine 19 (SkQR1) on rat models of H₂O₂- and ischemia-induced heart arrhythmia, heart infarction, kidney ischemia, and stroke have been studied ex vivo and in vivo. In all the models listed, SkQ1 and/or SkQR1 showed pronounced protective effect. Supplementation of food with extremely low SkQ1 amount (down to 0.02 nmol SkQ1/kg per day for 3 weeks) was found to abolish the steady heart arrhythmia caused by perfusion of isolated rat heart with H₂O₂ or by ischemia/reperfusion. Higher SkQ1 (125–250 nmol/kg per day for 2–3 weeks) was found to decrease the heart infarction region induced by an in vivo ischemia/reperfusion and lowered the blood levels of lactate dehydrogenase and creatine kinase increasing as a result of ischemia/reperfusion. In single-kidney rats, ischemia/reperfusion of the kidney was shown to kill the majority of the animals in 2–4 days, whereas one injection of SkQ1 or SkQR1 (1 μmol/kg a day before ischemia) saved lives of almost all treated rats. Effect of SkQR1 was accompanied by decrease in ROS (reactive oxygen species) level in kidney cells as well as by partial or complete normalization of blood creatinine and of some other kidney-controlled parameters. On the other hand, this amount of SkQ1 (a SkQ derivative of lower membrane-penetrating ability than SkQR1) saved the life but failed to normalize ROS and creatinine levels. Such an effect indicates that death under conditions of partial kidney dysfunction is mediated by an organ of vital importance other than kidney, the organ in question being an SkQ1 target. In a model of compression brain ischemia/reperfusion, a single intraperitoneal injection of SkQR1 to a rat (1 μmol/kg a day before operation) effectively decreased the damaged brain area. SkQ1 was ineffective, most probably due to lower permeability of the blood-brain barrier to this compound. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 12, pp. 1607–1621.     

Naturally occurring disulfide-bound dimers of three-fingered toxins: a paradigm for biological activity diversification

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.