Functional Identification of Regulatory Elements within the Promoter Region of Platelet-Derived Growth Factor 2

Human platelet-derived growth factor (PDGF) is composed of two polypeptide chains, PDGF-1 and PDGF-2,the human homolog of the v-sis oncogene. Deregulation of PDGF-2 expression can confer a growth advantage to cells possessing the cognate receptor and, thus, may contribute to the malignant phenotype. We investigated the regulation of PDGF-2 mRNA expression during megakaryocytic differentiation of K562 cells. Induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) led to a greater than 200-fold increase in PDGF-2 transcript levels in these cells. Induction was dependent on protein synthesis and was not enhanced by cycloheximide exposure.In our initial investigation of the PDGF-2 promoter, a minimal promoter region, which included sequences extending only 42 base pairs upstream of the TATA signal, was found to be as efficient as 4 kilobase pairs upstream of the TATA signal in driving expression of a reporter gene in uninduced K562 cells. We also functionally identified different regulatory sequence elements of the PDGF-2 promoter in TPA-induced K562 cells. One region acted as a transcriptional silencer, while another region was necessary for maximal activity of the promoter in megakaryoblasts. This region was shown to bind nuclear factors and was the target for trans-activation in normal and tumor cells. In one tumor cell line, which expressed high PDGF-2 mRNA levels, the presence of the positive regulatory region resulted in a 30-fold increase in promoter activity. However, the ability of the minimal PDGF-2 promoter to drive reporter gene expression in uninduced K562 cells and normal fibroblasts, which contained no detectable PDGF-2 transcripts, implies the existence of other negative control mechanisms beyond the regulation of promoter activity.

The Multifunctional PE_PGRS11 Protein from Mycobacterium tuberculosis Plays a Role in Regulating Resistance to Oxidative Stress

Mycobacterium tuberculosis utilizes unique strategies to survive amid the hostile environment of infected host cells. Infection-specific expression of a unique mycobacterial cell surface antigen that could modulate key signaling cascades can act as a key survival strategy in curtailing host effector responses like oxidative stress. We demonstrate here that hypothetical PE_PGRS11 ORF encodes a functional phosphoglycerate mutase. The transcriptional analysis revealed that PE_PGRS11 is a hypoxia-responsive gene, and enforced expression of PE_PGRS11 by recombinant adenovirus or Mycobacterium smegmatis imparted resistance to alveolar epithelial cells against oxidative stress. PE_PGRS11-induced resistance to oxidative stress necessitated the modulation of genetic signatures like induced expression of Bcl2 or COX-2. This modulation of specific antiapoptotic molecular signatures involved recognition of PE_PGRS11 by TLR2 and subsequent activation of the PI3K-ERK1/ 2-NF-kappa B signaling axis. Furthermore, PE_PGRS11 markedly diminished H2O2-induced p38 MAPK activation. Interestingly, PE_PGRS11 protein was exposed at the mycobacterial cell surface and was involved in survival of mycobacteria under oxidative stress. Furthermore, PE_PGRS11 displayed differential B cell responses during tuberculosis infection. Taken together, our investigation identified PE_PGRS11 as an in vivo expressed immunodominant antigen that plays a crucial role in modulating cellular life span restrictions imposed during oxidative stress by triggering TLR2-dependent expression of COX-2 and Bcl2. These observations clearly provide a mechanistic basis for the rescue of pathogenic Mycobacterium-infected lung epithelial cells from oxidative stress.

Inhibition of peroxidase-catalyzed protein tyrosine nitration by antithyroid drugs and their analogues

In this paper, we describe the effect of some commonly used thiourea-based antithyroid drugs and their analogues on the peroxidase-catalyzed nitration reactions. The nitration of bovine serum albumin (BSA) and cytochrome c was studied using the antibody against 3-nitro-L-tyrosine. This study reveals that the thione-based antithyroid drugs effectively inhibit lactoperoxidase (LPO)-catalyzed nitration of BSA. These compounds show very weak inhibition towards the nitration of cytochrome c. Some of these compounds also inhibit myeloperoxidase (MPO)-catalyzed nitration of L-tyrosine. A structure-activity correlation study on the peroxidase-catalyzed nitration of L-tyrosine reveals that the presence of thione/selone moiety is important for the inhibition. Although the presence of a free N-H group adjacent to C=S moiety is necessary for most of the thiones to inhibit the LPO-catalyzed nitration, the corresponding selones do not require the presence of any free N-H group for their activity. Furthermore, experiments with different concentrations of H2O2 suggest that the antithyroid drugs and related thiones inhibit the nitration reaction mainly by coordinating to the Fe(III)-center of the enzyme active site as previously proposed for the inhibition of peroxidase-catalyzed iodination. On the other hand, the selenium compounds inhibit the nitration by scavenging H2O2 without interacting with the enzyme active site. This assumption is based on the observations that catalase effectively inhibits tyrosine nitration by scavenging H2O2, which is one of the substrates for the nitration. In contrast, superoxide dismutase (SOD) does not alter the nitration reactions, indicating the absence of superoxide radical anion (O-2 center dot(-)) during the peroxidase-catalyzed nitration reactions. (C) 2010 Elsevier B.V. All rights reserved.

Empirical torsional potential functions from protein structure data. Phi- and psi-potentials for non-glycyl amino acid residues.

The torsional potential functions Vt(phi) and Vt(psi) around single bonds N--C alpha and C alpha--C, which can be used in conformational studies of oligopeptides, polypeptides and proteins, have been derived, using crystal structure data of 22 globular proteins, fitting the observed distribution in the (phi, psi)-plane with the value of Vtot(phi, psi), using the Boltzmann distribution. The averaged torsional potential functions, obtained from various amino acid residues in L-configuration, are Vt(phi) = 1.0 cos (phi + 60 degrees); Vt(psi) = 0.5 cos (psi + 60 degrees) - 1.0 cos (2 psi + 30 degrees) - 0.5 cos (3 psi + 30 degrees). The dipeptide energy maps Vtot(phi, psi) obtained using these functions, instead of the normally accepted torsional functions, were found to explain various observations, such as the absence of the left-handed alpha helix and the C7 conformation, and the relatively high density of points near the line psi = 0 degrees. These functions derived from observational data on protein structures, will, it is hoped, explain various previously unexplained facts in polypeptide conformation.

Over-expression and purification strategies for recombinant multi-protein oligomers: A case study of Mycobacterium tuberculosis sigma/anti-sigma factor protein complexes

The function of a protein in a cell often involves coordinated interactions with one or several regulatory partners. It is thus imperative to characterize a protein both in isolation as well as in the context of its complex with an interacting partner. High resolution structural information determined by X-ray crystallography and Nuclear Magnetic Resonance offer the best route to characterize protein complexes. These techniques, however, require highly purified and homogenous protein samples at high concentration. This requirement often presents a major hurdle for structural studies. Here we present a strategy based on co-expression and co-purification to obtain recombinant multi-protein complexes in the quantity and concentration range that can enable hitherto intractable structural projects. The feasibility of this strategy was examined using the sigma factor/anti-sigma factor protein complexes from Mycobacterium tuberculosis. The approach was successful across a wide range of sigma factors and their cognate interacting partners. It thus appears likely that the analysis of these complexes based on variations in expression constructs and procedures for the purification and characterization of these recombinant protein samples would be widely applicable for other multi-protein systems. (C) 2010 Elsevier Inc. All rights reserved.

High-resolution methyl edited GFT NMR experiments for protein resonance assignments and structure determination

Three-dimensional (3D) structure determination of proteins is benefitted by long-range distance constraints comprising the methyl groups, which constitute the hydrophobic core of proteins. However, in methyl groups (of Ala, Ile, Leu, Met, Thr and Val) there is a significant overlap of C-13 and H-1 chemical shifts. Such overlap can be resolved using the recently proposed (3,2)D HCCH-COSY, a G-matrix Fourier transform (GFT) NMR based experiment, which facilitates editing of methyl groups into distinct spectral regions by combining their C-13 chemical shifts with that of the neighboring, directly attached, C-13 nucleus. Using this principle, we present three GFT experiments: (a) (4,3)D NOESY-HCCH, (b) (4,3)D H-1-TOCSY-HCCH and (c) (4,3)D C-13-TOCSY-HCCH. These experiments provide unique 4D spectral information rapidly with high sensitivity and resolution for side-chain resonance assignments and NOE analysis of methyl groups. This is exemplified by (4,3)D NOESY-HCCH data acquired for 17.9 kDa non-deuterated cytosolic human J-protein co-chaperone, which provided crucial long-range distance constraints for its 3D structure determination.

X-ray and molecular-dynamics studies on Mycobacterium leprae single-stranded DNA-binding protein and comparison with other eubacterial SSB structures

The crystal structures of two forms of Mycobacterium leprae single-stranded DNA-binding protein (SSB) have been determined at 2.05 and 2.8 A resolution. Comparison of these structures with the structures of other eubacterial SSBs indicates considerable variation in their quaternary association, although the DNA-binding domains in all of them exhibit the same OB-fold. This variation has no linear correlation with sequence variation, but could be related to variation in protein stability. Molecular-dynamics simulations have been carried out on tetrameric molecules derived from the two forms and the prototype Escherichia coli SSB and the individual subunits of both proteins. Together, the X-ray studies and molecular-dynamics simulations yield information on the relatively rigid and flexible regions of the molecule and on the effect of oligomerization on flexibility. The simulations provide insight into the changes in subunit structure on oligomerization. They also provide insight into the stability and time evolution of the hydrogen bonds/water bridges that connect the two pairs of monomers in the tetramer.

Chloroquine Inhibits Heme-Dependent Protein Synthesis in Plasmodium falciparum

A cell-free protein-synthesizing system has been reconstituted using the S-30 fraction or ribosomes and the S-100 fraction from Plasmodium falciparum. Addition of heme in vitro stimulates cell-free protein synthesis strikingly. Chloroquine inhibits the heme-dependent protein synthesis in the parasite lysate. The drug has also been found to inhibit parasite protein synthesis in situ at therapeutic concentrations soon after addition to parasite cultures. Ribosomes as well as the S-100 fraction isolated from such chloroquine-treated cultures are defective in protein synthesis. Addition of hemin plus glucose 6-phosphate or high concentrations of GTP, cAMP, and an active preparation of eIF-2 to the parasite cell-free system restores protein synthesis to a significant extent in chloroquine-treated cultures. Under conditions of inhibition of protein synthesis in situ by chloroquine in the culture, the parasite eukaryotic initiation factor 2-alpha- (eIF-2-alpha) is phosphorylated in the parasite lysate to a greater extent than that observed in the control culture. Addition of hemin in vitro suppresses this phosphorylation. eIF-2-alpha kinase activity is present in the parasite lysate and is not a contaminant derived from the human erythrocytes used to culture the parasite. The heme-chloroquine interactive effects can also be demonstrated with purified eIF-2-alpha kinase from rabbit reticulocyte lysate. It is proposed that chloroquine inhibits heme-dependent protein synthesis in the parasite and this is an early event mediating the growth-inhibitory effects of the drug.

Effect of amino acid substitutions at the subunit interface on the stabiity and aggregation properties of a dimeric protein: role of Arg 178 and Arg 218 at the dimer interface of thymidylate synthase

The significance of two interface arginine residues on the structural integrity of an obligatory dimeric enzyme thymidylate synthase (TS) from Lactobacillus casei was investigated by thermal and chemical denaturation. While the R178F mutant showed apparent stability to thermal denaturation by its decreased tendency to aggregate, the Tm of the R218K mutant was lowered by 5 degrees C. Equilibrium denaturation studies in guanidinium chloride (GdmCl) and urea indicate that in both the mutants, replacement of Arg residues results in more labile quaternary and tertiary interactions. Circular dichroism studies in aqueous buffer suggest that the protein interior in R218K may be less well-packed as compared to the wild type protein. The results emphasize that quaternary interactions may influence the stability of the tertiary fold of TS. The amino acid replacements also lead to notable alteration in the ability of the unfolding intermediate of TS to aggregate. The aggregated state of partially unfolded intermediate in the R178F mutant is stable over a narrower range of denaturant concentrations. In contrast, there is an exaggerated tendency on the part of R218K to aggregate in intermediate concentrations of the denaturant. The 3 A crystal structure of the R178F mutant reveals no major structural change as a consequence of amino acid substitution. The results may be rationalized in terms of mutational effects on both the folded and unfolded state of the protein. Site specific amino acid substitutions are useful in identifying specific regions of TS involved in association of non-native protein structures.

Isolation of a phosphoryl choline-binding protein from the hemolymph of the snail, Achatina fulica

A phosphorylcholine-binding protein from the hemolymph of the snail Achatina fulica was purified to near homogeneity using a Sepharose phenylphosphorylcholine affinity column. The protein bound to the affinity column was eluted with 5 mM phosphorylcholine as a single symmetrical peak. The purified protein (400 Kda) contained 35–40% carbohydrate. On SDS-PAGE the protein separated into two bands of 20 and 24 Kda, and had a pI of 5.9. On immunodiffusion, antiserum to the snail phosphorylcholine binding protein did not cross-react against other phosphorylcholine binding proteins, like rat serum phosphorylcholine-binding protein (PCBP), limulus C-reactive protein (CRP), or human CRP. On pretreatment of the snail hemolymph with this antiserum, the hemagglutination titer of the hemolymph was markedly decreased. The purified snail phosphorylcholine binding protein agglutinated rabbit erythrocytes in the absence of divalent cation (Ca+2) but trace amount of Ca+2 increased its binding. The strongest inhibitor of the agglutination reaction was lactose, followed by melibiose and 2-deoxygalactose. The relationships of the snail phosphorylcholine binding protein to other hemolymph agglutinins and to CRPs are discussed in light of common phylogeny.

A simplified method for the purification of riboflavin-binding protein from hen's egg yolk

A simple and efficient procedure for the purification of the riboflavin-binding protein from hen's egg yolk is described. This method involves the removal by exclusion of lipoproteins and subsequent fractionation of soluble yolk proteins held on a DEAE-cellulose column by a salt gradient which is followed by purification by gel filtration on Sephadex G-100. The protein thus isolated is homogeneous by various physicoehemical, immunological, and functional criteria.

Quantitative and compositional changes in myelin of undernourished and protein malnourished rat brains

Effects of undernutrition and protein malnutrition on the quantitative and qualitative changes in myelin isolated from rat brain at 3 and 8 weeks of age were investigated. Undernutrition during suckling period was induced by increasing the litter size, and continued from the 3rd to the 8th week by limited food intake, or the rats were rehabilitated with adequate food. Protein malnutrition was induced by feeding the lactating dams 5% protein diet as against 25% protein diet in controls. The protein malnourished rats were rehabilitated from the 3rd to the 8th week with the normal 25% protein diet. Undernutrition produced 16% and 35% reductions in the myelin content at 3 and 8 weeks of age, respectively, and was only partially restored on rehabilitation. Protein malnutrition caused more drastic reduction of 27% in the myelin content at 3 weeks, which was also partially restored on rehabilitation. The specific activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase was not affected by undernutrition, whereas protein malnutrition caused a 25% reduction at 3 weeks, which was totally reversed by rehabilitation. Undernutrition had not altered the relative composition of myelin proteins, but protein malnutrition resulted in a significant reduction in the proteolipid protein at 3 weeks of age, which could be reversed by rehabilitation.

Proteomic Identification of Haptoglobin alpha 2 as a Glioblastoma Serum Biomarker: Implications in Cancer Cell Migration and Tumor Growth

Glioblastoma (GBM; grade IV astrocytoma) is the most malignant and common primary brain tumor in adults. Using combination of 2-DE and MALDI-TOF MS, we analyzed 14 GBM and 6 normal control sera and identified haptoglobin alpha 2 chain as an up-regulated serum protein in GBM patients. GBM-specific up-regulation was confirmed by ELISA based quantitation of haptoglobin (Hp) in the serum of 99 GBM patients as against lower grades (49 grade III/AA; 26 grade II/DA) and 26 normal individuals (p = 0.0001). Further validation using RT-qPCR on an independent set (n = 78) of tumor and normal brain (n = 4) samples and immunohistochemcial staining on a subset (n = 42) of above samples showed increasing levels of transcript and protein with tumor grade and were highest in GBM (p = < 0.0001 and < 0.0001, respectively). Overexpression of Hp either by stable integration of Hp cDNA or exogenous addition of purified Hp to immortalized astrocytes resulted in increased cell migration. RNAi-mediated silencing of Hp in glioma cells decreased cell migration. Further, we demonstrate that both human glioma and mouse melanoma cells overexpressing Hp showed increased tumor growth. Thus, we have identified haptoglobin as a GBM-specific serum marker with a role on glioma tumor growth and migration.

Isolation and characterization of thiamin-binding protein from chicken egg white

A thiamin-binding protein was isolated and characterized from chicken egg white by affinity chromatography on thiamin pyrophosphate coupled to aminoethyl-Sepharose. The high specificity of interaction between the thiamin-binding protein and the riboflavin-binding protein of the egg white, with a protein/protein molar ratio of 1.0, led to the development of an alternative procedure that used the riboflavin-binding protein immobilized on CNBr-activated Sepharose as the affinity matrix. The thiamin-binding protein thus isolated was homogeneous by the criteria of polyacrylamide-gel disc electrophoresis, double immunodiffusion and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, had a mol.wt. of 38,000 +/- 2000 and was not a glycoprotein. The protein bound [14C]thiamin was a molar ratio of 1.0, with dissociation constant (Kd) 0.3 micrometer.