PE_PGRS Antigens of Mycobacterium tuberculosis Induce Maturation and Activation of Human Dendritic Cells

Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, infects one-third of the world's population. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). DCs are sentinels of the immune system and are important for eliciting both primary and secondary immune responses to pathogens. In this context, to understand the molecular pathogenesis of tuberculosismand host response to mycobacteria and to conceive prospective vaccine candidates, it is important to understand how cell wall Ags of M. tuberculosis and, in particular, the proline-glutamic acid-polymorphic guanine-cytosine-rich sequence (PE_PGRS) family of proteins modulate DC maturation and function. In this study, we demonstrate that two cell wall-associated/secretory PE_PGRS proteins, PE_PGRS 17 (Rv0978c) and PE_PGRS 11 (Rv0754), recognize TLR2, induce maturation and activation of human DCs, and enhance the ability of DCs to stimulate CD4(+) T cells. We further found that PE_PGRS protein-mediated activation of DCs involves participation of ERK1/2, p38 MAPK, and NF-kappa B signaling pathways. Priming of human DCs with IFN-gamma further augmented PE_PGRS 17 or PE_PGRS 11 Ag-induced DC maturation and secretion of key proinflammatory cytokines. Our results suggest that by activating DCs, PE_PGRS proteins, important mycobacterial cell wall Ags, could potentially contribute in the initiation of innate immune responses during tuberculosis infection and hence regulate the clinical course of tuberculosis. The Journal of Immunology, 2010, 184: 3495-3504.

Rotavirus nonstructural proteins: a structural perspective

Rotavirus is a major cause of acute infantile diarrhoea worldwide. The virus genome consists of 11 segments of double-stranded RNA that codesfor six structural proteins (VP1-6) and six non-structural proteins(NSP1-6). NSPs are proteins expressed from the virus genome in the infected cell, but are not incorporated into the mature virus article. NSPs play an essential role in virus replication, morphogenesis and pathogenesis, and most of them exhibit multifunctional properties. Structure-function analysis of the NSPs is essential for understanding the molecular mechanisms by which the virus circumvents host innate immune responses, inhibits cellular protein synthesis, hijacks the protein synthetic machinery for its own propagation and manifests the disease process. Because of their essential roles in virus biology, NSPs represent potential targets for the development of antiviral agents. Determination of the three-dimensional structure of NSPs has been hindered due to low-level expression and aggregation. To date, the complete three-dimensional structure of only NSP2 has been determined. The structures of the N- and C-terminal domains of NSP3 and the diarrhoea-inducing domain of NSP4 have also been determined. This review primarily covers the structural and biological functions of the NSPs whose three-dimensional structural aspects have been fully or partially understood, but provides a brief account of other NSPs and the structural features of the mature virion as determined by electron cryomicroscopy.

Structural plasticity and enzyme action: Crystal structures of Mycobacterium tuberculosis peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA in order to avoid the toxicity resulting from its accumulation and to free the tRNA available for further rounds in protein synthesis. The structure of the enzyme from Mycobacteritan tuberculosis has been determined in three crystal forms. This structure and the structure of the enzyme frorn Escherichia coli in its crystal differ substantially on account of the binding of the C terminus of the E. coli enzyme to the peptide-binding site of a neighboring molecule in the crystal. A detailed examination of this difference led to an elucidation of the plasticity of the binding site of the enzyme. The peptide-binding site of the enzyme is a cleft between the body, of the molecule and a polypepticle Y stretch involving a loop and a helix. This stretch is in the open conformation when the enzyme is in the free state as in the crystals of M. tuberculosis peptidyl-tRNA hydrolase. Furthermore, there is no physical continuity between the tRNA and the peptide-binding sites. The molecule in the E. coli crystal mimics the peptide-bound enzyme molecule. The peptide stretch referred to earlier now closes on the bound peptide. Concurrently, a channel connecting the tRNA and the peptide-binding site opens primarily through the concerted movement of two residues. Thus, the crystal structure of M. tuberculosis peptidyl-tRNA hydrolase when compared with the crystal structure of the E. coli enzyme, leads to a model of structural changes associated with enzyme action on the basis of the plasticity of the molecule. (c) 2007 Elsevier Ltd. All rights reserved.

Structural plasticity and enzyme action: Crystal structures of Mycobacterium tuberculosis peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA in order to avoid the toxicity resulting from its accumulation and to free the tRNA available for further rounds in protein synthesis. The structure of the enzyme from Mycobacteritan tuberculosis has been determined in three crystal forms. This structure and the structure of the enzyme frorn Escherichia coli in its crystal differ substantially on account of the binding of the C terminus of the E. coli enzyme to the peptide-binding site of a neighboring molecule in the crystal. A detailed examination of this difference led to an elucidation of the plasticity of the binding site of the enzyme. The peptide-binding site of the enzyme is a cleft between the body, of the molecule and a polypepticle Y stretch involving a loop and a helix. This stretch is in the open conformation when the enzyme is in the free state as in the crystals of M. tuberculosis peptidyl-tRNA hydrolase. Furthermore, there is no physical continuity between the tRNA and the peptide-binding sites. The molecule in the E. coli crystal mimics the peptide-bound enzyme molecule. The peptide stretch referred to earlier now closes on the bound peptide. Concurrently, a channel connecting the tRNA and the peptide-binding site opens primarily through the concerted movement of two residues. Thus, the crystal structure of M. tuberculosis peptidyl-tRNA hydrolase when compared with the crystal structure of the E. coli enzyme, leads to a model of structural changes associated with enzyme action on the basis of the plasticity of the molecule. (c) 2007 Elsevier Ltd. All rights reserved.

Novel insertion and deletion mutants of RpoB that render Mycobacterium smegmatis RNA polymerase resistant to rifampicin-mediated inhibition of transcription

The startling increase in the occurrence of rifampicin (Rif) resistance in the clinical isolates of Mycobacterium tuberculosis worldwide is posing a serious concern to tuberculosis management. The majority of Rif resistance in bacteria arises from mutations in the RpoB subunit of the RNA polymerase. We isolated M. smegmatis strains harbouring either an insertion (6 aa) or a deletion (10 aa) in their RpoB proteins. Although these strains showed a compromised fitness for growth in 7H9 Middlebrook medium, their resistance to Rif was remarkably high. The attenuated growth of the strains correlated with decreased specific activities of the RNA polymerases from the mutants. While the RNA polymerases from the parent or a mutant strain (harbouring a frequently occurring mutation, H442Y, in RpoB) were susceptible to Rif-mediated inhibition of transcription from calf thymus DNA, those from the insertion and deletion mutants were essentially refractory to such inhibition. Three-dimensional structure modelling revealed that the RpoB amino acids that interact with Rif are either deleted or unable to interact with Rif due to their unsuitable spatial positioning in these mutants. We discuss possible uses of the RpoB mutants in studying transcriptional regulation in mycobacteria and as potential targets for drug design.

Salmonella typhimurium mutants affecting establishment of lysogeny

Salmonella typhimurium mutants have been isolated in which phage P22 failsto establish lysogeny. These appear to be defective in cAMP metabolism. A phage mutation overcoming the bacterial defect has been mapped between gene c 1 and gene

Identification of Specific DNA Binding Residues in the TCP Family of Transcription Factors in Arabidopsis

The TCP transcription factors control multiple developmental traits in diverse plant species. Members of this family share an similar to 60-residue-long TCP domain that binds to DNA. The TCP domain is predicted to form a basic helix-loop-helix ( bHLH) structure but shares little sequence similarity with canonical bHLH domain. This classifies the TCP domain as a novel class of DNA binding domain specific to the plant kingdom. Little is known about how the TCP domain interacts with its target DNA. We report biochemical characterization and DNA binding properties of a TCP member in Arabidopsis thaliana, TCP4. We have shown that the 58-residue domain of TCP4 is essential and sufficient for binding to DNA and possesses DNA binding parameters comparable to canonical bHLH proteins. Using a yeast-based random mutagenesis screen and site-directed mutants, we identified the residues important for DNA binding and dimer formation. Mutants defective in binding and dimerization failed to rescue the phenotype of an Arabidopsis line lacking the endogenous TCP4 activity. By combining structure prediction, functional characterization of the mutants, and molecular modeling, we suggest a possible DNA binding mechanism for this class of transcription factors.

Biochemical characterization of the intracellular domain of the human guanylyl cyclase C receptor provides evidence for a catalytically active homotrimer

Guanylyl cyclase C (GCC) is the receptor for the family of guanylin peptides and bacterial heat-stable enterotoxins (ST). The receptor is composed of an extracellular, ligand-binding domain and an intracellular domain with a region of homology to protein kinases and a guanylyl cyclase catalytic domain. We have expressed the entire intracellular domain of GCC in insect cells and purified the recombinant protein, GCC-IDbac, to study its catalytic activity and regulation. Kinetic properties of the purified protein were similar to that of full-length GCC, and high activity was observed when MnGTP was used as the substrate. Nonionic detergents, which stimulate the guanylyl cyclase activity of membrane-associated GCC, did not appreciably increase the activity of GCC-IDbac, indicating that activation of the receptor by Lubrol involved conformational changes that required the transmembrane and/or the extracellular domain. The guanylyl cyclase activity of GCC-IDbac was inhibited by Zn2+, at concentrations shown to inhibit adenylyl cyclase, suggesting a structural homology between the two enzymes. Covalent crosslinking of GCC-IDbac indicated that the protein could associate as a dimer, but a large fraction was present as a trimer. Gel filtration analysis also showed that the major fraction of the protein eluted at a molecular size of a trimer, suggesting that the dimer detected by cross-linking represented subtle differences in the juxtaposition of the individual polypeptide chains. We therefore provide evidence that the trimeric state of GCC is catalytically active, and sequences required to generate the trimer are present in the intracellular domain of GCC.

The fate of the initiator tRNAs is sensitive to the critical balance between interacting proteins

Formylation of the initiator tRNA is essential for normal growth of Escherichia coil, The initiator tRNA containing the U35A36 mutation (CUA anticodon) initiates from UAG codon, However, an additional mutation at position 72 (72A --> G) renders the tRNA (G72/U35A36) inactive in initiation because it is defective in formylation, In this study, we isolated U1G72/U35A36 tRNA containing a wobble base pair at 1-72 positions as an intragenic suppressor of the G72 mutation. The U1G72/U35A36 tRNA is formylated and participates in initiation. More importantly, we show that the mismatch at 1-72 positions of the initiator tRNA, which was thus far thought to be the hallmark of the resistance of this tRNA against peptidyl-tRNA hydrolase (PTH), is not sufficient, The amino acid attached to the initiator tRNA is also important in conferring protection against PTH. Further, we show that the relative levels of PTH and IF2 influence the path adopted by the initiator tRNAs in protein synthesis. These findings provide an important clue to understand the dual function of the single tRNA(Met) in initiation and elongation, in the mitochondria of various organisms.

Structure of uracil-DNA glycosylase from Mycobacterium tuberculosis: insights into interactions with ligands

Uracil N-glycosylase (Ung) is the most thoroughly studied of the group of uracil DNA-glycosylase (UDG) enzymes that catalyse the first step in the uracil excision-repair pathway. The overall structure of the enzyme from Mycobacterium tuberculosis is essentially the same as that of the enzyme from other sources. However, differences exist in the N- and C-terminal stretches and some catalytic loops. Comparison with appropriate structures indicate that the two-domain enzyme closes slightly when binding to DNA, while it opens slightly when binding to the proteinaceous inhibitor Ugi. The structural changes in the catalytic loops on complexation reflect the special features of their structure in the mycobacterial protein. A comparative analysis of available sequences of the enzyme from different sources indicates high conservation of amino-acid residues in the catalytic loops. The uracil-binding pocket in the structure is occupied by a citrate ion. The interactions of the citrate ion with the protein mimic those of uracil, in addition to providing insights into other possible interactions that inhibitors could be involved in.

Characterization of foot-and-mouth disease virus types Ο and Asia 1 RNA

Foot-and-mouth disease is an acute and highly contagious febrile disease affecting cloven-footed animals. Identification of the foot-and-mouth disease virus (FMDV), the causative agent of the disease, posed problems because of the occurrence of many types and subtypes of the virus. A molecular approach based on oligonucleotide mapping of FMDV RNA has been used for the identification and characterization of virus isolates obtained in a disease outbreak (King et al., 1981). One-dimensional oligonucleotide mapping was used for rapid analysis of FMDV RNA (LaTorre et al., 1982). FMDV types Ο and Asia 1 of Indian origin are being routinely used for vaccine production in India. This report presents the differences between FMDV types Ο and Asia 1 at molecular level based on one-dimensional oligonucleotide mapping of virus-induced poly (A) RNA.

Glioblastoma-Specific Protein Interaction Network Identifies PP1A and CSK21 as Connecting Molecules between Cell Cycle-Associated Genes

Glioblastoma (GBM; grade IV astrocytoma) is a very aggressive form of brain cancer with a poor survival and few qualified predictive markers. This study integrates experimentally validated genes that showed specific upregulation in GBM along with their protein-protein interaction information. A system level analysis was used to construct GBM-specific network. Computation of topological parameters of networks showed scale-free pattern and hierarchical organization. From the large network involving 1,447 proteins, we synthesized subnetworks and annotated them with highly enriched biological processes. A careful dissection of the functional modules, important nodes, and their connections identified two novel intermediary molecules CSK21 and protein phosphatase 1 alpha (PP1A) connecting the two subnetworks CDC2-PTEN-TOP2A-CAV1-P53 and CDC2-CAV1-RB-P53-PTEN, respectively. Real-time quantitative reverse transcription-PCR analysis revealed CSK21 to be moderately upregulated and PP1A to be overexpressed by 20-fold in GBM tumor samples. Immunohistochemical staining revealed nuclear expression of PP1A only in GBM samples. Thus, CSK21 and PP1A, whose functions are intimately associated with cell cycle regulation, might play key role in gliomagenesis. Cancer Res; 70(16); 6437-47. (C)2010 AACR.

Recycling of the Posttermination Complexes of Mycobacterium smegmatis and Escherichia coli Ribosomes Using Heterologous Factors

In eubacteria, ribosome recycling factor (RRF) and elongation factor G (EFG) function together to dissociate posttermination ribosomal complexes. Earlier studies, using heterologous factors from Mycobacterium tuberculosis in Escherichia coli revealed that specific interactions between RRF and EFG are crucial for their function in ribosome recycling. Here, we used translation factors from E.coli,Mycobacterium smegmatis and M. tuberculosis, and polysomes from E. coli and M. smegmatis, and employed in vivo and in vitro experiments to further understand the role of EFG in ribosome recycling. We show thatE. coli EFG (EcoEFG) recycles E. coli ribosomes with E. coli REF (EcoRRF), but not with mycobacterial RRFs. Also, EcoEFG fails to recycle M. smegmatis ribosomes with either EcoRRF or mycobacterial RRFs. On the other hand, mycobacterial EFGs recycle both E. coli and M. smegmatis ribosomes with either of the RRFs. These observations suggest that EFG establishes distinct interactions with REF and the ribosome to carry out ribosome recycling. Furthermore, the EFG chimeras generated by swapping domains betweenmycobacterial EFGs and EcoEFG suggest that while the residues needed to specify the EFG interaction with REF arelocated in domains IV and V. those required to specify its interaction with the ribosome are located throughout the molecule. (C) 2010 Elsevier Ltd. All rights reserved.

Rapid isolation of lysyl-tRNA synthetase from rat liver

The high molecular weight aminoacyl-tRNA synthetase complex (the 24S complex) was isolated from rat liver by ultracentrifugation. The lysyl-tRNA synthetase (E.C. 6.1.1.6) was selectively dissociated by hydrophobic interaction chromatography on 1,6 diaminohexyl agarose followed by hydroxylapatite chromatography and DEAE chromatography. The lysyl-tRNA synthetase dissociated from the 24S synthetase complex was purified approximately to 2700 fold with 14% yield.