Structural and functional studies of the abundant tegument protein ORF52 from murine gammaherpesvirus 68

The tegument is a layer of proteins between the nucleocapsid and the envelope of herpesviruses. The functions of most tegument proteins are still poorly understood. In murine gammaherpesvirus 68, ORF52 is an abundant tegument protein of 135 residues that is required for the assembly and release of infectious virus particles. To help understand the molecular basis for the function of this protein, we have determined its crystal structure at 2.1 A resolution. The structure reveals a dimeric association of this protein. Interestingly, an N-terminal alpha-helix that assumes different conformation in the two monomers of the dimer mediates the formation of an asymmetrical tetramer and contains many highly conserved residues. Structural and sequence analyses suggest that this helix is more likely involved in interactions with other components of the tegument or nucleocapsid of the virus and that ORF52 functions as a symmetrical dimer. The asymmetrical tetramer of ORF52 may be a "latent" form of the protein, when it is not involved in virion assembly. The self-association of ORF52 has been confirmed by co-immunoprecipitation and fluorescence resonance energy transfer experiments. Deletion of the N-terminal alpha-helix, as well as mutation of the conserved Arg(95) residue, abolished the function of ORF52. The results of the functional studies are fully consistent with the structural observations and indicate that the N-terminal alpha-helix is a crucial site of interaction for ORF52.     

JUZBOX: A web server for extracting biomedical words from the protein sequence

The recognition of gene/protein names in literature is one of the pivotal steps in the processing of biological literatures for information extraction or data mining. We have compiled a lexicon of biomedical words (conserved patterns/ potential motifs) which has the combination of only 20 alphabets of amino acids. The remaining 6 letters of the English alphabets (B, J, O, U, X, Z) are treated as invalid amino acid characters (to our context), We have jumbled the 6 letters for the sake of usage and convenience and termed as ’JUZBOX‘ and these characters were filtered in the biomedical lexicon. Undoubtedly, the generation of biomedical words from protein sequence using JUZBOX have applications specific for functional annotation.

Availability

JUZBOX is available freely at http://www.spices.res.in/juzbox     

Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling

Background  

Heat stress (HS) and related illnesses are a major concern in military, sports, and fire brigadiers. HS results in physiologic responses of increased temperature, heart rate and sweating. In heat stroke, inflammatory response plays an important role and it is evidenced that turpentine (T) induced circulating inflammatory cytokines reduced survival rate and duration at 42°C. Here we report the alteration in the protein expression in liver cells upon HS with and without T treatment using two dimensional gel electrophoresis (2-DE), tryptic in-gel digestion and MALDI-TOF-MS/MS approaches     

Biofilm formation by Pseudoalteromonas ruthenica and its removal by chlorine

The distribution of a recently described marine bacterium, SBT 033 GenBank Accession No. AY723742), Pseudoalteromonas ruthenica, at the seawater intake point, outfall and mixing point of an atomic power plant is described, and its ability to form biofilm was investigated. The effectiveness of the antifouling biocide chlorine in the inactivation of planktonic as well as biofilm cells of P. ruthenica was studied in the laboratory. The results show that the planktonic cells were more readily inactivated than the cells enclosed in a biofilm matrix. Viable counting showed that P. ruthenica cells in biofilms were up to 10 times more resistant to chlorine than those in liquid suspension. Using confocal laser scanning microscopy it was shown that significant detachment of P. ruthenica biofilm developed on a glass substratum could be accomplished by treatment with a dose of 1 mg l-1 chlorine. Chlorine-induced detachment led to a significant reduction in biofilm thickness (up to 69%) and substratum coverage (up to 61%), after 5-min contact time. The results show that P. ruthenica has a remarkable ability to form biofilms but chlorine, a common biocide, can be used to effectively kill and detach these biofilms.     

p21-activated kinase 1 regulates microtubule dynamics by phosphorylating tubulin cofactor B

p21-activated kinase 1 (Pak1) induces cytoskeleton reorganization in part by regulating microtubule dynamics through an elusive mechanism. Using a yeast two-hybrid screen, we identified tubulin cofactor B (TCoB) (a cofactor in the assembly of the alpha/beta-tubulin heterodimers) as an interacting substrate of Pak1. Pak1 directly phosphorylated TCoB in vitro and in vivo on serines 65 and 128 and colocalized with TCoB on newly polymerized microtubules and on centrosomes. TCoB interacted with the GTPase-binding domain of Pak1 and activated Pak1 in vitro and in vivo. In contrast to wild-type TCoB, an S65A, S128A double mutant and knock-down of the endogenous TCoB or Pak1 reduced microtubule polymerization, suggesting that Pak1 phosphorylation is necessary for normal TCoB function. Overexpression of TCoB dramatically increased the number of gamma-tubulin-containing microtubule-organizing centers, a phenotype reminiscent of cells overexpressing Pak1. TCoB was overexpressed and phosphorylated in breast tumors. These findings reveal a novel role for TCoB and Pak1 in regulating microtubule dynamics.     

Estrogen receptor activation at serine 305 is sufficient to upregulate cyclin D1 in breast cancer cells

Recent studies have shown that p21-activated kinase 1 (Pak1) phosphorylates estrogen receptor-alpha (ER alpha) at Ser 305 and also promotes its transactivation function. Here, we sought to investigate whether substitution of serine 305 in ER with glutamic acid (ER alpha-S305E), which mimics the phosphorylation state, would influence the status of ER-target genes. To explore this possibility, we generated clones overexpressing ER alpha-S305E in ER-negative MDA-MB-231 cells and analyzed the status of ER-regulated genes using a gene array. Results indicated that the expression of ER alpha-S305E is sufficient to upregulate the expression of a few but not all ER-regulated genes, i.e., cyclin D1 and zinc finger protein 147 (estrogen-responsive finger protein), while there was no significant change in the expression of remaining genes on the array. In addition, we found an increased expression as well as nuclear accumulation of cyclin D1 protein in MDA-MB-231 cells expressing ER alpha-S305E as compared to the level of cyclin D1 in MDA-MB-231 cells expressing WT-ER alpha or pcDNA. Furthermore, ER alpha-S305E, but not mutation of ER alpha-S305 to alanine, enhanced the cyclin D1 promoter activity. These findings suggest that ER alpha activation at S305 is sufficient to upregulate the expression of cyclin D1, an ER-regulated gene that is implicated in the progression of breast cancer. Phosphorylation of ER alpha by Pak1 or its upstream regulators could upregulate the expression of a subset of ER-target genes in a ligand-independent manner and hence, might contribute toward the development of hormone independence in breast cancer cells.     

From genomes to systems

A report on the 2nd Conference of the Consortium for Post-Genome Science (CPGS) 'Genomes to Systems', Manchester, UK, 1-3 September 2004.     

Bioenergetic consequences of opening the ATP-sensitive K(+) channel of heart mitochondria

There is an emerging consensus that pharmacological opening of the mitochondrial ATP-sensitive K(+) (K(ATP)) channel protects the heart against ischemia-reperfusion damage; however, there are widely divergent views on the effects of openers on isolated heart mitochondria. We have examined the effects of diazoxide and pinacidil on the bioenergetic properties of rat heart mitochondria. As expected of hydrophobic compounds, these drugs have toxic, as well as pharmacological, effects on mitochondria. Both drugs inhibit respiration and increase membrane proton permeability as a function of concentration, causing a decrease in mitochondrial membrane potential and a consequent decrease in Ca(2+) uptake, but these effects are not caused by opening mitochondrial K(ATP) channels. In pharmacological doses (<50 microM), both drugs open mitochondrial K(ATP) channels, and resulting changes in membrane potential and respiration are minimal. The increased K(+) influx associated with mitochondrial K(ATP) channel opening is approximately 30 nmol. min(-1). mg(-1), a very low rate that will depolarize by only 1-2 mV. However, this increase in K(+) influx causes a significant increase in matrix volume. The volume increase is sufficient to reverse matrix contraction caused by oxidative phosphorylation and can be observed even when respiration is inhibited and the membrane potential is supported by ATP hydrolysis, conditions expected during ischemia. Thus opening mitochondrial K(ATP) channels has little direct effect on respiration, membrane potential, or Ca(2+) uptake but has important effects on matrix and intermembrane space volumes.