Identification of immunodominant regions of Brassica juncea glyoxalase I as potential antitumor immunomodulation targets

Glyoxalase I activity has been shown to be directly related to cancer and its inhibitors have been used as anti-cancer drugs. Immunochemical studies have shown immunochemical relatedness among animal and plant glyoxalase I, but its potential application for biomedical research has not been investigated. In order to understand the conserved immunochemical regions of the protein and to determine probable immunomodulation targets, a cellulose-bound scanning peptide library for Brassica juncea glyoxalase I was made using the spot synthesis method. Immuno-probing of the library, using B. juncea anti-glyoxalase I monospecific polyclonal antibodies, revealed three immunodominant regions, epitope I, II, and III. In the homology model of B. juncea glyoxalase I generated by threading its sequence onto the human glyoxalase I, the high accessible surface area and the hydrophilic nature of the epitopes confirmed their surface localization and hence their accessibility for antigen-antibody interaction. Epitopes I and II were specific to B. juncea glyoxalase I. Localizing the epitopes on available glyoxalase I sequences showed that epitope III containing the active site region was conserved across phyla. Therefore, this could be used as a potential immunomodulation target for cancer therapy. Moreover, as the most immunogenic epitopes were mapped on the surface of the protein, this method could be used to discover potential therapeutic targets. It is a simple and fast approach for such investigations. This study, to our knowledge, is the first in epitope mapping of glyoxalase I and has great biomedical potential.     

Crystal structure of the 65-kilodalton heat shock protein, chaperonin 60.2, of Mycobacterium tuberculosis

Chaperonin 60s are a ubiquitous class of proteins that promote folding and assembly of other cellular polypeptides in an ATP-dependent manner. The oligomeric state of chaperonin 60s has been shown to be crucial to their role as molecular chaperones. Chaperonin 60s are also known to be important stimulators of the immune system. Mycobacterium tuberculosis possesses a duplicate set of chaperonin 60s, both of which have been shown to be potent cytokine stimulators. The M. tuberculosis chaperonin 60s are present in the extracellular milieu at concentrations that are extremely low for the formation of an oligomer. Here we present the crystal structure of one of the chaperonin 60s of M. tuberculosis, also called Hsp65 or chaperonin 60.2, at 3.2-A resolution. We were able to crystallize the protein in its dimeric state. The unusual dimerization of the protein leads to exposure of certain hydrophobic patches on the surface of the protein, and we hypothesize that this might have relevance in binding to immunogenic peptides, as it does in the eukaryotic homologs.     

Effects of metachlopromide-induced hyperprolactinemia on serum testosterone and its response to hCG in adult male common marmosets (Callithrix jacchus)

Intramuscular administration of metachlopromide (2.5, 10, and 25 mg) induced increase in serum prolactin levels. Following 10 and 25 mg dose levels, prolactin levels were elevated at least for 8 hr. Single administration of metachlopromide failed to affect the nocturnal rise in circulating levels of testosterone. Daily treatment of metachlopromide (10 mg) for 30 days suppressed the nocturnal elevation of testosterone on day 28 of treatment. However, the hCG-stimulated testosterone production on day 30 was unaffected. The results of the present study demonstrate that metachlopromide-induced hyperprolactinemia in adult male common marmosets affects the hypothalamo-pituitary axis without any effect on testicular response to hCG.     

Genomewide discovery of DNA polymorphisms in rice cultivars with contrasting drought and salinity stress response and their functional relevance

Next‐generation sequencing technologies provide opportunities to understand the genetic basis of phenotypic differences, such as abiotic stress response, even in the closely related cultivars via identification of large number of DNA polymorphisms. We performed whole‐genome resequencing of three rice cultivars with contrasting responses to drought and salinity stress (sensitive IR64, drought‐tolerant Nagina 22 and salinity‐tolerant Pokkali). More than 356 million 90‐bp paired‐end reads were generated, which provided about 85% coverage of the rice genome. Applying stringent parameters, we identified a total of 1 784 583 nonredundant single‐nucleotide polymorphisms (SNPs) and 154 275 InDels between reference (Nipponbare) and the three resequenced cultivars. We detected 401 683 and 662 509 SNPs between IR64 and Pokkali, and IR64 and N22 cultivars, respectively. The distribution of DNA polymorphisms was found to be uneven across and within the rice chromosomes. One‐fourth of the SNPs and InDels were detected in genic regions, and about 3.5% of the total SNPs resulted in nonsynonymous changes. Large‐effect SNPs and InDels, which affect the integrity of the encoded protein, were also identified. Further, we identified DNA polymorphisms present in the differentially expressed genes within the known quantitative trait loci. Among these, a total of 548 SNPs in 232 genes, located in the conserved functional domains, were identified. The data presented in this study provide functional markers and promising target genes for salinity and drought tolerance and present a valuable resource for high‐throughput genotyping and molecular breeding for abiotic stress traits in rice.     

Candidate OP Phyla: Importance,Ecology and Cultivation Prospects

OP phyla were created in the domain bacteria, based on the group of 16S rRNA gene sequences recovered from the Obsidian Pool. However, due to the lack of cultured representative it is referred to as candidate phyla. Wider ecological occurrence was predicted for the OP phyla, especially OP3, OP10 and OP11. Recently, members of phylum OP5 and OP10 were cultured, providing clues to their cultivation prospects. At last the bioprospecting potentials of the OP members are discussed herein.     

A Forward Genetic Strategy Reveals Destabilizing Mutations in the Ebolavirus Glycoprotein That Alter Its Protease Dependence during Cell Entry

Ebolavirus (EBOV) entry into cells requires proteolytic disassembly of the viral glycoprotein, GP. This proteolytic processing, unusually extensive for an enveloped virus entry protein, is mediated by cysteine cathepsins, a family of endosomal/lysosomal proteases. Previous work has shown that cleavage of GP by cathepsin B (CatB) is specifically required to generate a critical entry intermediate. The functions of this intermediate are not well understood. We used a forward genetic strategy to investigate this CatB-dependent step. Specifically, we generated a replication-competent recombinant vesicular stomatitis virus bearing EBOV GP as its sole entry glycoprotein and used it to select viral mutants resistant to a CatB inhibitor. We obtained mutations at six amino acid positions in GP that independently confer complete resistance. All of the mutations reside at or near the GP1-GP2 intersubunit interface in the membrane-proximal base of the prefusion GP trimer. This region forms a part of the “clamp” that holds the fusion subunit GP2 in its metastable prefusion conformation. Biochemical studies suggest that most of the mutations confer CatB independence not by altering specific cleavage sites in GP but rather by inducing conformational rearrangements in the prefusion GP trimer that dramatically enhance its susceptibility to proteolysis. The remaining mutants did not show the preceding behavior, indicating the existence of multiple mechanisms for acquiring CatB independence during entry. Altogether, our findings suggest that CatB cleavage is required to facilitate the triggering of viral membrane fusion by destabilizing the prefusion conformation of EBOV GP.Filoviruses are enveloped, filamentous, nonsegmented negative-sense RNA viruses that can cause a deadly hemorrhagic fever with case fatality rates in excess of 90% (see references 4, 20, and 37 for recent reviews). All known filoviruses belong to one of two genera: Ebolavirus (EBOV), consisting of the five species Zaire (ZEBOV), Côte d''Ivoire, Sudan, Reston, and Bundibugyo (tentative); and Marburgvirus, consisting of the single Lake Victoria species (21, 62).Cell entry by filoviruses is mediated by their envelope glycoprotein, GP (60, 68). Mature GP is a trimer of three disulfide-linked GP1-GP2 heterodimers. GP1 and GP2 are generated by endoproteolytic cleavage of the GP0 precursor polypeptide by a furin-like protease during transport to the cell surface (31, 39, 63, 69). The membrane-distal subunit, GP1, mediates viral adhesion to host cells (10, 18, 38, 42, 56, 59) and regulates the activity of the transmembrane subunit, GP2, which catalyzes fusion of viral and cellular membrane bilayers (30, 39, 41, 64, 65). The consequence of membrane fusion is cytoplasmic delivery of the viral nucleocapsid cargo.Lee et al. (39) recently solved the crystal structure of a ZEBOV GP prefusion trimer lacking the heavily glycosylated GP1 mucin domain (Muc) and the GP2 transmembrane domain (see Fig. ​Fig.5).5). The three GP1 subunits together form a bowl-like structure encircled by sequences from the three GP2 subunits. The trimer is held together by GP1-GP2 and GP2-GP2 contacts; the hydrophobic GP2 fusion loop packs against the external surface of adjacent GP1 subunits, and each GP2 subunit contributes a strand to a trimeric α-helical coiled-coil stem. GP1 is organized into three subdomains. The base is intimately associated with GP2 and clamps it in its prefusion conformation. The head is proposed to mediate virus receptor binding during entry (10, 18, 38, 42). The glycan cap resides at the top of the trimer and is critical for GP assembly but must be removed during entry (see below) (31, 42). The base and glycan cap are connected by the β13-β14 loop, which was not visualized in the structure. The location and structure of the Muc domain are also unknown, but it is proposed to sheathe the top and/or sides of the prefusion GP trimer (39). Muc is dispensable for ZEBOV GP-dependent entry in tissue culture but may play roles in virus-cell adhesion and immune evasion in vivo (31, 42, 44, 56, 59).Open in a separate windowFIG. 5.CA074^R mutations localize at or near the GP1-GP2 interface in the GP prefusion crystal structure. In all diagrams, GP1 is depicted in blue, GP2 in red, GP1 CA074^R mutations in green, and GP2 CA074^R mutations in yellow. (A) Linear representation of the amino acid sequence of GPΔMuc. S-S indicates the intersubunit disulfide bond between C53 and C609. sp, signal peptide; fl, fusion loop; hr1 and hr2, heptad repeats; tm, transmembrane domain; N, N terminus; C, C terminus. (B) Structure of GP in a prefusion conformation (39). Cartoon representation of a GP1-GP2 monomer is shown. Remaining subunits are shown as a surface-shaded watermark. The boxed inset contains the membrane-proximal base of the trimer, in which the CA074^R mutations are located. The β13-β14 loop is modeled as a chain of blue circles. (C) Magnified view of the inset shown in panel B rotated by 90°. The side chains of D47, I584, K588, and their contacting residues are shown. Dashed pink lines connect atoms from different side chains separated by ≤3.9 Å. Other CA074^R residues are not shown for clarity. (D) View shown in panel C rotated by 90°. (E) Schematic diagram of the potential interactions made by residues mutated in the CA074^R viruses. Residues approaching ≤3.9 Å to each CA074^R residue are shown. Beige arcs, hydrophobic interactions; dashed lines, potential ionic interactions. Visualizations of the GP structures shown in panels B to D (Protein Data Bank accession no. 3CSY) were rendered in Pymol (Delano Scientific).Crystal structures of ZEBOV GP2 in its postfusion conformation indicate that filovirus GP is a “class I” viral membrane fusion protein (41, 65). Like the prototypic class I fusion proteins of human immunodeficiency virus and influenza virus, GP2 contains a hydrophobic fusion peptide near its N terminus and N- and C-terminal α-helical heptad repeat sequences (HR1 and HR2, respectively) (22, 28, 30, 39, 41, 64, 65, 67) (see Fig. ​Fig.5).5). GP2 drives membrane fusion by undergoing large-scale conformational changes; the prefusion HR1 helix-loop-helix rearranges to an unbroken α-helix, projecting the fusion loop into the endosomal membrane, and GP2 jackknifes on itself to form a hairpin-like structure in which the HR2s pack against grooves in the trimeric HR1 coiled coil (41, 65).The available GP structures make clear that the transition of GP2 from prefusion to postfusion conformation requires its release from its binding groove in the GP1 base subdomain. For all known class I fusion proteins, this transition is controlled by priming and triggering events. Priming typically involves a single endoproteolytic cleavage of the glycoprotein mediated by a cellular protease within the secretory pathway of the virus-producer cells (e.g., human immunodeficiency virus ENV → SU + TM by furin [27]). This cleavage is essential because it liberates an N-terminal fusion peptide and allows the glycoprotein to rearrange during fusion. Unusually for a class I fusion glycoprotein, however, ZEBOV GP does not require cleavage to GP1 and GP2 by a furin-like protease, even though this cleavage occurs efficiently (46, 69). Instead, the GP trimer is primed by extensive proteolytic remodeling during entry. This process is mediated by cysteine cathepsins, a class of papain superfamily cysteine proteases active within the cellular endosomal/lysosomal pathway (14, 54).The cysteine cathepsins B (CatB) and L (CatL) play essential and accessory roles, respectively, in ZEBOV entry into Vero cells (14). The functions of these enzymes in viral entry can be recapitulated in vitro. Incubation of vesicular stomatitis virus (VSV) pseudotypes bearing ZEBOV GP (VSV-GP) with a mixture of purified human CatL and CatB, or with the bacterial protease thermolysin (THL), results in the cleavage and removal of GP1 Muc and glycan cap sequences, leaving a stable ∼17-kDa N-terminal GP1 fragment and intact GP2 (see Fig. ​Fig.5)5) (18, 54). VSV particles containing this GP_17K intermediate no longer require CatB activity within cells, strongly suggesting that this protease plays a critical role in generating a related primed species during viral entry (54). Strikingly, incubation of VSV-GP with CatL alone (14, 54) or with bovine chymotrypsin (CHT) (this study) (Fig. ​(Fig.1;1; see also Fig. ​Fig.7)7) generates a similar but distinct GP_18K intermediate (containing a slightly larger ∼18-kDa GP1 fragment) that cannot bypass the requirement for CatB during entry. Therefore, the removal of a few residues from GP_18K by CatB is crucial for viral entry. The reason for this requirement is unknown. Finally, VSV-GP_17K particles cannot infect cells completely devoid of cysteine cathepsin activity, indicating the existence of at least one additional cysteine protease-dependent step during entry (34, 54; present study). The signal that acts on a fully primed GP intermediate to trigger membrane fusion remains unknown.Open in a separate windowFIG. 1.CatB activity is required for entry of ZEBOV GP-dependent entry, whereas CatL activity is dispensable. Vero cells were pretreated for 4 h with 1% (vol/vol) DMSO (vehicle), 0.5 μM FYdmk (CatL-selective inhibitor), 80 μM CA074 (CatB-selective inhibitor), 0.5 μM FYdmk plus 80 μM CA074, or 300 μM E64 (pan-cysteine cathepsin inhibitor). (A) The cells were then challenged with VSV-GPΔMuc, CHT-derived VSV-GP_18K (CHT-GP_18K), THL-derived VSV-GP_17K (THL-GP_17K), or VSV-G pseudotypes at a low MOI (0.02 to 0.1 eGFP-positive infectious units [iu] per cell) in the presence of drug, and viral titers (iu/ml) were determined at 18 h postinfection. CatB and CatL activities in extracts prepared from a parallel set of pretreated cells were measured by fluorogenic peptide turnover and are shown (bottom). Averages ± standard deviations (SD) for six trials from three independent experiments are shown. CatL activity below the detection threshold is indicated as zero without an accompanying SD. (B) Vero cells pretreated with protease inhibitors were challenged with VSV-GPΔMuc, cathepsin L-derived VSV-GP_18K (CatL-GP_18K), or cathepsin B-derived VSV-GP_17K (CatB-GP_17K), and viral infectivity was measured as described above. Averages ± SD for three trials from a representative experiment are shown.Open in a separate windowFIG. 7.rVSV-GPΔMuc mutants resemble the WT in cleavage to GP_18K and GP_17K intermediates. WT or mutant rVSV-GPΔMuc was incubated with the indicated protease(s) as described in Materials and Methods and then deglycosylated with PNGaseF (except for N40K and T42A, which lack the N40 glycan and do not require deglycosylation at this position). The resulting GP1 proteolytic fragments were resolved by SDS-PAGE and detected by Western blotting. Shorter protease incubation times were necessary to obtain cleavage intermediates for some mutants (see text for details). Positions of uncleaved GP1 and the ∼18-kDa and ∼17-kDa cleavage fragments are indicated on the left. *, partially cleaved GP fragment of unknown composition. Experimental samples shown on each gel (bold labels) were flanked by WT samples cleaved with CHT (WT 18K) or THL (WT 17K) to provide markers of band mobility.In this study, we used a forward genetic strategy to investigate the CatB-dependent step in ZEBOV entry. Specifically, we engineered and rescued a recombinant VSV (rVSV) encoding a mucin domain-deleted ZEBOV GP in place of the VSV glycoprotein G and used it to select viral mutants resistant to the CatB inhibitor CA074. Analysis of these viruses identified mutations in both GP1 and GP2 that allow CatB-independent cell entry. We found that GP_18K and/or GP_17K intermediates derived from some but not all of the mutant GPs are conformationally distinct from the wild type (WT), suggesting the existence of multiple mechanisms for CA074 resistance. Taken together, our results indicate that ZEBOV GP→GP_17K cleavage by CatB promotes fusion triggering and viral entry by destabilizing the prefusion conformation of GP.     

Bacterial bioreactors for high yield production of recombinant protein

We developed a new bacterial expression system that utilizes a combination of attributes (low temperature, induction of an mRNA-specific endoribonuclease causing host cell growth arrest, and culture condensation) to facilitate stable, high level protein expression, almost 30% of total cellular protein, without background protein synthesis. With the use of an optimized vector, exponentially growing cultures could be condensed 40-fold without affecting protein yields, which lowered sample labeling costs to a few percent of the cost of a typical labeling experiment. Because the host cells were completely growth-arrested, toxic amino acids such as selenomethionine and fluorophenylalanine were efficiently incorporated into recombinant proteins in the absence of cytotoxicity. Therefore, this expression system using Escherichia coli as a bioreactor is especially well suited to structural genomics, large-scale protein expressions, and the production of cytotoxic proteins.     

Multiple Gene Duplication and Rapid Evolution in the groEL Gene: Functional Implications

The chaperonins, GroEL and GroES, are present ubiquitously and provide a paradigm in the understanding of assisted protein folding. Due to its essentiality of function, GroEL exhibits high sequence conservation across species. Complete genome sequencing has shown the occurrence of duplicate or multiple copies of groEL genes in bacteria such as Mycobacterium tuberculosis and Corynebacterium glutamicum. Monophyly of each bacterial clade in the phylogenetic tree generated for the GroEL protein suggests a lineage-specific duplication. The duplicated groEL gene in Actinobacteria is not accompanied by the operonic groES despite the presence of upstream regulatory elements. Our analysis suggests that in these bacteria the duplicated groEL genes have undergone rapid evolution and divergence to function in a GroES-independent manner. Evaluation of multiple sequence alignment demonstrates that the duplicated genes have acquired mutations at functionally significant positions including those involved in substrate binding, ATP binding, and GroES binding and those involved in inter-ring and intra-ring interactions. We propose that the duplicate groEL genes in different bacterial clades have evolved independently to meet specific requirements of each clade. We also propose that the groEL gene, although essential and conserved, accumulates nonconservative substitutions to exhibit structural and functional variations. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Debashish Bhattacharya]