The co-operonic PE25/PPE41 protein complex of Mycobacterium tuberculosis elicits increased humoral and cell mediated immune response

Background

Many of the PE/PPE proteins are either surface localized or secreted outside and are thought to be a source of antigenic variation in the host. The exact role of these proteins are still elusive. We previously reported that the PPE41 protein induces high B cell response in TB patients. The PE/PPE genes are not randomly distributed in the genome but are organized as operons and the operon containing PE25 and PPE41 genes co-transcribe and their products interact with each other.

Methodology/Principal Finding

We now describe the antigenic properties of the PE25, PPE41 and PE25/PPE41 protein complex coded by a single operon. The PPE41 and PE25/PPE41 protein complex induces significant (p<0.0001) B cell response in sera derived from TB patients and in mouse model as compared to the PE25 protein. Further, mice immunized with the PE25/PPE41 complex and PPE41 proteins showed significant (p<0.00001) proliferation of splenocyte as compared to the mice immunized with the PE25 protein and saline. Flow cytometric analysis showed 15–22% enhancement of CD8⁺ and CD4⁺ T cell populations when immunized with the PPE41 or PE25/PPE41 complex as compared to a marginal increase (8–10%) in the mice immunized with the PE25 protein. The PPE41 and PE25/PPE41 complex can also induce higher levels of IFN-γ, TNF-α and IL-2 cytokines.

Conclusion

While this study documents the differential immunological response to the complex of PE and PPE vis-à-vis the individual proteins, it also highlights their potential as a candidate vaccine against tuberculosis.     

Dual role of JNK1-mediated phosphorylation of Bcl-2 in autophagy and apoptosis regulation

Autophagy and apoptosis are fundamental cellular pathways that are both regulated by JNK-mediated Bcl-2 phosphorylation. Several years ago, JNK-mediated Bcl-2 phosphorylation was shown to interfere with its binding to proapoptotic BH3 domain-containing proteins such as Bax and recently, our laboratory demonstrated that JNK1-mediated Bcl-2 phosphorylation interferes with its binding to the proautophagy BH3 domain-containing protein Beclin 1. Here, we examined the kinetic relationship between Bcl-2 phosphorylation, Bcl-2-Beclin 1 interactions, Bcl-2-Bax interactions, and caspase 3 activation during nutrient starvation. We found that after a short period of nutrient deprivation (4 hours), a small amount of Bcl-2 phosphorylation dissociates Bcl-2 from the Bcl-2-Beclin 1 complex but not from the Bcl-2-Bax complex. After 16 hours of nutrient deprivation, Bcl-2 phosphorylation reaches maximal levels, the Bcl-2-Bax complex is disrupted, and active caspase 3 is detected, indicating the initiation of apoptosis. Based on this result, we propose a speculative model for understanding the interrelationship between autophagy and apoptosis regulated by JNK1-mediated Bcl-2 phosphorylation. According to this model, rapid Bcl-2 phosphorylation may occur initially to promote cell survival by disrupting the Bcl-2-Beclin 1 complex and activating autophagy. At a certain point when autophagy is no longer able to keep the cell alive, Bcl-2 phosphorylation might then serve to inactivate its antiapoptotic function.     

Lattice stabilization and enhanced diffraction in human p38α crystals by protein engineering

Mitogen-activated protein (MAP) kinase p38α is activated in response to environmental stress and cytokines, and plays a significant role in inflammatory responses. For these reasons, it is an important target for the treatment of a wide range of inflammatory and autoimmune diseases. The crystals of p38α that we obtained by published procedures were usually small, quite mosaic, and difficult to reproduce and thus posed a difficulty for the intensive high-resolution studies required for a structure-guided drug discovery approach. Based on crystallographic and biochemical evidences, we prepared a single point mutation of a surface cysteine (C162S) and found that it prevents aggregation and improves the homogeneity and stability of the enzyme. This mutation also facilitates the crystallization process and increases the diffracting power of p38α crystals. Surprisingly, we found that the mutation induces a change in the conformation of a nearby surface loop resulting in stronger lattice interactions, consistent with the improved crystal quality. The mutant protein, because of its improved stability and strengthened lattice interactions, thus provides a significantly improved reagent for use in structure-based drug design for this important disease target.     

Insulin-like growth factor I and glucagon-like peptide-2 responses to fasting followed by controlled or ad libitum refeeding in rats

Luminal nutrients stimulate structural and functional regeneration in the intestine through mechanisms thought to involve insulin-like growth factor I (IGF-I) and glucagon-like peptide-2 (GLP-2). We investigated the relationship between IGF-I and GLP-2 responses and mucosal growth in rats fasted for 48 h and then refed for 2 or 4 days by continuous intravenous or intragastric infusion or ad libitum feeding. Fasting induced significant decreases in body weight, plasma concentrations of IGF-I and bioactive GLP-2, jejunal mucosal cellularity (mass, protein, DNA, and villus height), IGF-I mRNA, and ileal proglucagon mRNA. Plasma IGF-I concentration was restored to fed levels with 2 days of ad libitum refeeding but not with 4 days of intravenous or intragastric refeeding. Administration of an inhibitor of endogenous GLP-2 (rat GLP-2 3-33) during ad libitum refeeding partially attenuated mucosal growth and prevented the increase in plasma IGF-I to fed levels; however, plasma GLP-2 and jejunal IGF-I mRNA were restored to fed levels. Intragastric refeeding restored intestinal cellularity and functional capacity (sucrase activity and sodium-glucose transporter-1 expression) to fed levels, whereas intravenous refeeding had no effect. Intestinal regeneration after 4 days of intragastric or 2 days of ad libitum refeeding was positively associated with increases in plasma concentrations of GLP-2 and jejunal IGF-I mRNA. These data suggest that luminal nutrients stimulate intestinal growth, in part, by increased expression of both GLP-2 and IGF-I.     

Enteral nutrients potentiate the intestinotrophic action of glucagon-like peptide-2 in association with increased insulin-like growth factor-I responses in rats

Glucagon-like peptide-2 (GLP-2) is a nutrient-dependent, intestinotrophic hormone derived from posttranslational processing of proglucagon in the distal bowel. GLP-2 is thought to act through indirect mediators, such as IGF-I. We investigated whether intestinal expression of GLP-2 and IGF-I system components are increased with the mucosal growth induced by enteral nutrient (EN) and/or a low dose of GLP-2 in parenterally fed rats. Rats were randomized to four treatment groups using a 2 x 2 design and maintained with parenteral nutrition (PN) for 7 days: PN alone, EN, GLP-2, and EN+GLP-2; n = 7-9. The two main treatment effects are +/-GLP-2 (100 microg.kg body wt(-1).day(-1)) and +/-EN (43% of energy needs, days 4-6). Combination treatment with EN+GLP-2 induced synergistic intestinal growth in ileum, resulting in greater mucosal cellularity, sucrase segmental activity, and gain of body weight (ENxGLP-2, P < 0.04). In addition, EN+GLP-2 induced a significant 28% increase in plasma concentration of bioactive GLP-2, a significant 102% increase in ileal proglucagon mRNA with no change in ileal dipeptidyl peptidase-IV (DPP-IV) specific activity, and significantly reduced plasma DPP-IV activity compared with GLP-2. This indicates that EN potentiates the intestinotrophic action of GLP-2. Proliferation of enterocytes due to GLP-2 infusion was associated with greater expression of ileal proglucagon, GLP-2 receptor, IGF-I, IGF binding protein-3 mRNAs, and greater IGF-I peptide concentration in ileum (P < 0.032). Ileal IGF-I mRNA was positively correlated with expression of proglucagon, GLP-2R, and IGFBP-5 mRNAs (R2 = 0.43-0.56, P < 0.0001). Our findings support the hypothesis that IGF-I is one of the downstream mediators of GLP-2 action in a physiological model of intestinal growth.     

Structure of Seneca Valley Virus-001: an oncolytic picornavirus representing a new genus

The crystal structure of Seneca Valley Virus-001 (SVV-001), the representative member of a new genus, Senecavirus, is reported at 2.3A resolution. SVV-001 is the first naturally occurring nonpathogenic picornavirus shown to mediate selective cytotoxicity towards tumor cells with neuroendocrine cancer features. The nonsegmented (+) ssRNA genome of SVV-001 shares closest sequence similarity with the genomes of the members of Cardiovirus. The overall tertiary structure of VP1-VP4 subunits is conserved with the exception of loops, especially those of VP1 that show large deviations relative to the members of the cardioviruses. The surface loops of VP1 and VP2 are predicted to mediate cell tropism of SVV-001. In addition, the organization of the packaged nucleic acid density indicates that certain regions of VP2 and VP4 interact closely with the packaged nucleic acid.     

Lipases catalyzed enantioselective hydrolysis of (R,S)-methyl 1,4-benzodioxan-2-carboxylate intermediate for (S)-doxazosin mesylate

(S)-1,4-benzodioxan-2-carboxylic acid-1 is used as starting compound for the production of the more effective (S) enantiomer of the drug doxazosin mesylate. The catalytic ability of some commercial lipases for preparations of (S) enantiomer of 1 from (±) methyl 1,4-benzodioxin-2-carboxylate-2 is reported. Lipases from bacterial sources were more successful in resolving the ester than those from the yeast lipases. About 85% enantiomerically pure ester was achieved by lipase from alcaligenes sp.     

IGF-I treatment facilitates transition from parenteral to enteral nutrition in rats with short bowel syndrome

The goal of growth factor treatment in patients with short bowel syndrome (SBS) is to facilitate transition from parenteral to enteral feedings. Ideal use of growth factors would be acute treatment that produces sustained effects. We investigated the ability of acute insulin-like growth factor I (IGF-I) treatment to facilitate weaning from total parenteral nutrition (TPN) to enteral feeding in a rat model of SBS. After a 60% jejunoileal resection + cecectomy, rats treated with IGF-I or vehicle were maintained exclusively with TPN for 4 days and transitioned to oral feeding. TPN and IGF-I were stopped 7 days after resection, and rats were maintained with oral feeding for 10 more days. In IGF-I-treated rats, serum concentration of IGF-I and final body weight were significantly greater because of a proportionate increase in carcass lean body mass than in vehicle-treated rats. Acute IGF-I treatment induced sustained jejunal hyperplasia on the basis of significantly greater concentrations of jejunal mucosal protein and DNA without a change in histology or sucrase activity. These results demonstrate that acute IGF-I facilitates weaning from parenteral to enteral nutrition in association with maintenance of a greater body weight and serum IGF-I concentration in rats with SBS.     

The genomic organization of mouse resistin reveals major differences from the human resistin: functional implications

The resistin gene is a potential candidate for the etiology of insulin resistance and type 2 diabetes and has been implicated as the molecular link between type 2 diabetes and obesity. Unlike the mouse resistin, expression of the human resistin appears to be regulated differently. We report comparative analyses of the mouse and human genomic fragments encoding the resistin gene. At the amino acid level the two proteins exhibit 59% identity. While at the mRNA level the human resistin shows 64.4% sequence identity with its mouse counterpart, the mouse resistin genomic sequence displays only 46.7% sequence identity with the human resistin and is almost three times bigger than the human resistin. The intronic sequences per se displayed the least identities (28.7%), however the intron boundaries were highly conserved between human and mouse. The mouse resistin carries a very large intron in the 3' UTR, which has a number of regulatory sequences possibly involved in differential gene expression. Of particular significance is the presence of a PPAR/RXR heterodimer binding site within intron X (IntX-PPRE) which may possibly confer TZD responsiveness. Oligonucleotides carrying the authentic PPAR/RXR binding element (Aco-PPRE) as well as IntX-PPRE specifically bound factors (PPAR/RXR heterodimers) present in differentiated 3T3-L1 adipocyte cells in an electrophoretic mobility shift assay. IntX-PPRE oligonucleotide modulated the expression of the luciferase reporter gene in transient transfection assays using 3T3-L1 cells.