Recognition of nonstructural protein-peptides by cytotoxic t-lymphocytes raised against japanese encephalitis-virus

We have previously reported that Lyt(2+) cytotoxic T lymphocytes (CTL) can be raised against Japanese encephalitis virus (JEV) in BALB/c mice. In order to confirm the presence of H-2K(d)-restricted CTL and to examine their cross-recognition of West Wile virus (WNV), we tested the capacity of anti-JEV CTL to lyse uninfected syngeneic target cells that were pulsed with synthetic peptides. The sequence of the synthetic peptides was predicted based upon the H-2K(d) binding consensus motif. We show here that preincubation of uninfected syngeneic targets (P388D1) with JEV NS1- and NS3-derived peptides [NS1 (891-899) and NS3 (1804-1812)], but not with JEV NS5-derived peptide [NS5 (3370-3378)], partially sensitized them for lysis by polyclonal anti-JEV CTL. These results indicate the CTL recognition of NS1- and NS3-derived peptides of JEV.

Functional characterization of the precursor and spliced forms of RecA protein of Mycobacterium tuberculosis

The recA locus of pathogenic mycobacteria differs from that of nonpathogenic species because it contains large intervening sequences nested in the RecA homology region that are excised by an unusual protein-splicing reaction. In vivo assays indicated that Mycobacterium tuberculosis recA partially complemented Escherichia coli recA mutants for recombination and mutagenesis. Further, splicing of the 85 kDa precursor to 38 kDa MtRecA protein was necessary for the display of its activity, in vivo. To gain insights into the molecular basis for partial and lack of complementation by MtRecA and 85 kDa proteins, respectively, we purified both of them to homogeneity. MtRecA protein, but not the 85 kDa form, bound stoichiometrically to single-stranded DNA in the presence of ATP. MtRecA protein was cross-linked to 8-azidoadenosine 5'-triphosphate with reduced efficiency, and kinetic analysis of ATPase activity suggested that it is due to decreased affinity for ATP. In contrast, the 85 kDa form was unable to bind ATP, in the presence or absence of ssDNA and, consequently, was entirely devoid of ATPase activity. Molecular modeling studies suggested that the decreased affinity of MtRecA protein for ATP and the reduced efficiency of its hydrolysis might be due to the widening of the cleft which alters the hydrogen bonds and the contact area between the enzyme and the substrate and changes in the disposition of the amino acid residues around the magnesium ion and the gamma-phosphate. The formation of joint molecules promoted by MtRecA protein was stimulated by SSB when the former was added first. The probability of an association between the lack and partial levels of biological activity of RecA protein(s) to that of illegitimate recombination in pathogenic mycobacteria is considered.

Multivalent ligand presentation as a central concept to study intricate carbohydrate-protein interactions

Studying the weak binding affinities between carbohydrates and proteins has been a central theme in sustained efforts to uncover intricate details of this class of biomolecular interaction. The amphiphilic nature of most carbohydrates, the competing nature of the surrounding water molecules to a given protein receptor site and the receptor binding site characteristics led to the realization that carbohydrates are required to exert favorable interactions, primarily through clustering of the ligands. The clustering of sugar ligands has been augmented using many different innovative molecular scaffolds. The synthesis of clustered ligands also facilitates fine-tuning of the spatial and topological proximities between the ligands, so as to allow the identification of optimal molecular features for significant binding affinity enhancements. The kinetic and thermodynamic parameters have been delineated in many instances, thereby allowing an ability to correlate the multivalent presentation and the observed ligand-receptor interaction profiles. This critical review presents various multivalent ligands, synthetic and semisynthetic, and mechanisms by which the weak binding affinities are overcome, and the ligand-receptor complexation leads to significantly enhanced binding affinities (157 references).

Evaluating viewpoint entropy for ribbon representation of protein structure

While many measures of viewpoint goodness have been proposed in computer graphics, none have been evaluated for ribbon representations of protein secondary structure. To fill this gap, we conducted a user study on Amazon’s Mechanical Turk platform, collecting human viewpoint preferences from 65 participants for 4 representative su- perfamilies of protein domains. In particular, we evaluated viewpoint entropy, which was previously shown to be a good predictor for human viewpoint preference of other, mostly non-abstract objects. In a second study, we asked 7 molecular biology experts to find the best viewpoint of the same protein domains and compared their choices with viewpoint entropy. Our results show that viewpoint entropy overall is a significant predictor of human viewpoint preference for ribbon representations of protein secondary structure. However, the accuracy is highly dependent on the complexity of the structure: while most participants agree on good viewpoints for small, non-globular structures with few secondary structure elements, viewpoint preference varies considerably for complex structures. Finally, experts tend to choose viewpoints of both low and high viewpoint entropy to emphasize different aspects of the respective structure.

In vivo treatment of Heymann's Nephritis using a cytotoxic protein-toxin conjugate

In this study we investigated the possibility of treating Heymann's Nephritis (HN) by destroying antibody producing cells by targetting a toxin, gelonin - conjugated to gp330, the renal brush border antigen. HN was induced in rats by immunizing them with purified gp330. The gelonin-gp330 conjugate was administered 12 days after the antigenic challenge. Serum was screened for circulating antibodies. Proteinurea was estimated. The gp330-gelonin conjugate-treated animals had a circulating antibody titre in the serum much lower than that of diseased (untreated) animals. Proteinurea seen in diseased animals was not observed in treated animals. This work suggests the possibility of using a toxin-antigen conjugate for immunomodulating antibody mediated autoimmune renal disease.

Evaluation of anti-inflammatory potential of Chloranthus erectus (Buch.-Ham.) Verd. leaf extract in rats

Aim of the study: Chloranthus erectus (Buch.-Ham.) Verdcourt (Chloranthaceae) is a shrub native to tropical and temperate zone of Eastern Himalaya of India and South-East Asia and have traditionally been used as a folklore medicine to treat localised swelling, joint pain, skin inflammation, fever and bodyache. In this study, an attempthas been made to demonstrate the anti-inflammatory activity of methanol extract obtained from Chloranthus erectus leaves (MECEL) in acute, sub-acute and chronic mouse models. Materials and methods: Inflammation in the hind paw of Wistar albino rat was induced by carrageenan, histamine and serotonin, and tissue granuloma pouch was induced by cotton pellet method. Antiinflammatory drug-phenylbutazone was used as standard drug for comparison. Results: In acute carrageenan-induced rat hind paw edema, oral administration of MECEL at 200 mg/kg produced significant inhibition of edema by 38.34 % (p<0.01) while the histamine- and serotonin-induced sub-acute model, the inhibition of paw edema reached 52.54 % (p < 0.001) and 25.5 % (p < 0.01), respectively. in a 7-day study, MECEL at 20 and 50 mg/kg produced significant suppression of cotton pellet-induced tissue granuloma formation in rats. Conclusions: This preliminary study revealed that the methanol extract of Chloranthus erectus exhibited significant anti-inflammatory activity in the tested models, and may provide the scientific rationale for its popular folk medicine as anti-inflammatory agent.

The Mycobacterial MsDps2 Protein Is a Nucleoid-Forming DNA Binding Protein Regulated by Sigma Factors sigma(A) and sigma(B)

The Dps (DNA-binding protein from starved cells) proteins from Mycobacterium smegmatis MsDps1 and MsDps2 are both DNA-binding proteins with some differences. While MsDps1 has two oligomeric states, with one of them responsible for DNA binding, MsDps2 has only one DNA-binding oligomeric state. Both the proteins however, show iron-binding activity. The MsDps1 protein has been shown previously to be induced under conditions of starvation and osmotic stress and is regulated by the extra cellular sigma factors sigma(H) and sigma(F). We show here, that the second Dps homologue in M. smegmatis, namely MsDps2, is purified in a DNA-bound form and exhibits nucleoid-like structures under the atomic force microscope. It appears that the N-terminal sequence of Dps2 plays a role in nucleoid formation. MsDps2, unlike MsDps1, does not show elevated expression in nutritionally starved or stationary phase conditions; rather its promoter is recognized by RNA polymerase containing sigma(A) or sigma(B), under in vitro conditions. We propose that due to the nucleoid-condensing ability, the expression of MsDps2 is tightly regulated inside the cells.

Spontaneous and reversible self-assembly of a polypeptide fragment of insulin-like growth factor binding protein-2 into fluorescent nanotubular structures

In this communication, we report the spontaneous and reversible in vitro self-assembly of a polypeptide fragment derived from the C-terminal domain of Insulin-like Growth Factor Binding Protein (IGFBP-2) into soluble nanotubular structures several micrometres long via a mechanism involving inter-molecular disulfide bonds and exhibiting enhanced fluorescence.

Ligand Specificity of Group I Biotin Protein Ligase of Mycobacterium tuberculosis

Background: Fatty acids are indispensable constituents of mycolic acids that impart toughness & permeability barrier to the cell envelope of M. tuberculosis. Biotin is an essential co-factor for acetyl-CoA carboxylase (ACC) the enzyme involved in the synthesis of malonyl-CoA, a committed precursor, needed for fatty acid synthesis. Biotin carboxyl carrier protein (BCCP) provides the co-factor for catalytic activity of ACC. Methodology/Principal Findings: BPL/BirA (Biotin Protein Ligase), and its substrate, biotin carboxyl carrier protein (BCCP) of Mycobacterium tuberculosis (Mt) were cloned and expressed in E. coli BL21. In contrast to EcBirA and PhBPL, the similar to 29.5 kDa MtBPL exists as a monomer in native, biotin and bio-5'AMP liganded forms. This was confirmed by molecular weigt profiling by gel filtration on Superdex S-200 and Dynamic Light Scattering (DLS). Computational docking of biotin and bio-5'AMP to MtBPL show that adenylation alters the contact residues for biotin. MtBPL forms 11 H-bonds with biotin, relative to 35 with bio-5'AMP. Docking simulations also suggest that bio-5'AMP hydrogen bonds to the conserved `GRGRRG' sequence but not biotin. The enzyme catalyzed transfer of biotin to BCCP was confirmed by incorporation of radioactive biotin and by Avidin blot. The K-m for BCCP was similar to 5.2 mu M and similar to 420 nM for biotin. MtBPL has low affinity (K-b = 1.06 x 10(-6) M) for biotin relative to EcBirA but their K-m are almost comparable suggesting that while the major function of MtBPL is biotinylation of BCCP, tight binding of biotin/bio-5'AMP by EcBirA is channeled for its repressor activity. Conclusions/Significance: These studies thus open up avenues for understanding the unique features of MtBPL and the role it plays in biotin utilization in M. tuberculosis.

Recognition of Interaction Interface Residues in Low-Resolution Structures of Protein Assemblies Solely from the Positions of C alpha Atoms

Background: The number of available structures of large multi-protein assemblies is quite small. Such structures provide phenomenal insights on the organization, mechanism of formation and functional properties of the assembly. Hence detailed analysis of such structures is highly rewarding. However, the common problem in such analyses is the low resolution of these structures. In the recent times a number of attempts that combine low resolution cryo-EM data with higher resolution structures determined using X-ray analysis or NMR or generated using comparative modeling have been reported. Even in such attempts the best result one arrives at is the very course idea about the assembly structure in terms of trace of the C alpha atoms which are modeled with modest accuracy. Methodology/Principal Findings: In this paper first we present an objective approach to identify potentially solvent exposed and buried residues solely from the position of C alpha atoms and amino acid sequence using residue type-dependent thresholds for accessible surface areas of C alpha. We extend the method further to recognize potential protein-protein interface residues. Conclusion/Significance: Our approach to identify buried and exposed residues solely from the positions of C alpha atoms resulted in an accuracy of 84%, sensitivity of 83-89% and specificity of 67-94% while recognition of interfacial residues corresponded to an accuracy of 94%, sensitivity of 70-96% and specificity of 58-94%. Interestingly, detailed analysis of cases of mismatch between recognition of interface residues from C alpha positions and all-atom models suggested that, recognition of interfacial residues using C alpha atoms only correspond better with intuitive notion of what is an interfacial residue. Our method should be useful in the objective analysis of structures of protein assemblies when positions of only C alpha positions are available as, for example, in the cases of integration of cryo-EM data and high resolution structures of the components of the assembly.

Inhibition of Tyrosine Phosphorylation of Sperm Flagellar Proteins, Outer Dense Fiber Protein-2 and Tektin-2, Is Associated With Impaired Motility During Capacitation of Hamster Spermatozoa

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, tyrphostin-A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin-A47-treated spermatozoa exhibited circular motility, which was associated with a distinct hypo-tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000-60,000. In this study, we provide evidence on the localization of capacitation-associated tyrosine-phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo-tyrosine phosphorylated major proteins of tyrphostin-A47-treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein-2 and the 51 kDa protein as tektin-2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo-tyrosine-phosphorylation status of outer dense fiber protein-2 and tektin-2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR-tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa.

Functional diversity of human protein kinase splice variants marks significant expansion of human kinome

Background: Protein kinases are involved in diverse spectrum of cellular processes. Availability of draft version of the human genomic data in the year 2001 enabled recognition of repertoire of protein kinases. However, over the years the human genomic data is being refined and the current release of human genomic data has helped us to recognize a larger repertoire of over 900 human protein kinases represented mainly by splice variants. Results: Many of these identified protein kinases are alternatively spliced products. Interestingly, some of the human kinase splice variants appear to be significantly diverged in terms of their functional properties as represented by incorporation or absence of one or more domains. Many sets of protein kinase splice variants have substantially different domain organization and in a few sets of splice variants kinase domains belong to different subfamilies of kinases suggesting potential participation in different signal transduction pathways. Conclusions: Addition or deletion of a domain between splice variants of multi-domain kinases appears to be a means of generating differences in the functional features of otherwise similar kinases. It is intriguing that marked sequence diversity within the catalytic regions of some of the splice variant kinases result in kinases belonging to different subfamilies. These human kinase splice variants with different functions might contribute to diversity of eukaryotic cellular signaling.