Solution structure and function of a conserved protein SP14.3 encoded by an essential Streptococcus pneumoniae gene

Streptococcus pneumoniae is a major human pathogen that causes high mortality and morbidity rates and has developed resistance to many antibiotics. The genome of S. pneumoniae has recently been completely sequenced revealing many genes encoding hypothetical proteins of unknown function. We have found that the gene encoding one such conserved protein, SP14.3, is essential for growth of S. pneumonia. Since it is essential, SP14.3 represents a potential target for drug discovery. Here, we describe the three-dimensional solution structure of SP14.3 as determined by NMR spectroscopy. The structure consists of two domains each with an alpha/beta-fold. The N-terminal domain contains two alpha-helices and a three-stranded beta-sheet, while the C-terminal domain is composed of one alpha-helix and a five-stranded beta-sheet. The N-terminal domain of the protein contains a highly negatively charged surface and resembles the fold of the N-terminal domain of Thermus thermophilus ribosomal protein S3. The C-terminal domain has a protein fold similar to human small nuclear ribonucleoprotein Sm D3 and Haloarcula marismortui ribosomal protein L21E. The two domains of the protein tumble in solution overall as a whole with an overall molecular rotational correlation time (tau(m)) of 12.9 ns at 25 degrees C. The relative orientation of the two domains is not defined by the nuclear Overhauser effect data. Indeed, residual dipolar couplings and the structure calculations indicate that the relative orientation of the two domains is not rigidly oriented with respect to one another in solution.     

Structure of the N-terminal RNA-binding domain of the SARS CoV nucleocapsid protein

The severe acute respiratory syndrome (SARS) virus belongs to the Coronaviridea family of viruses. Its virion encodes several proteins including a replicase and four structural proteins. Here we describe the three-dimensional structure of the N-terminal domain of the SARS coronavirus (CoV) nucleocapsid protein. The protein consists of a five-stranded beta sheet with a folding topology distinct from other RNA-binding proteins. Single-stranded RNAs bind to the protein surface at the junction between a flexible, positively charged beta hairpin and the core structure. NMR-based screening was used to identify low molecular weight compounds that bind to this site.     

Fluorescence assay of SIRT protein deacetylases using an acetylated peptide substrate and a secondary trypsin reaction

A novel fluorescent substrate was devised for the sirtuin (SIRT) class of human protein deacetylases comprised of a peptide sequence containing a single acetyl-lysine residue, with a fluorescent group (tetramethylrhodamine-6-carboxylic acid, 6-TAMRA) near the carboxyl terminus and a nonfluorescent quenching group (QSY-7) near the amino terminus. The peptide sequence is modeled after the p53 acetylation site but is unreactive toward trypsin because all other lysine and arginine residues have been replaced by serine. However, the SIRT-deacetylated peptide is readily cleaved by trypsin, resulting in a maximal 30-fold enhancement of the 6-TAMRA fluorescence. Nicotinamide at millimolar concentrations stops the deacetylation but does not inhibit trypsin, and a microtiter plate assay of the SIRTs has been devised using the fluorescent substrate and these reagents. Using this method, the kinetics of the reaction of the cosubstrate nicotinamide adenine dinucleotide and the competitive inhibitor nicotinamide with SIRT1 and SIRT2 has been analyzed. Several nicotinamide analogs have also been tested as inhibitors and found to have much lower affinity for these enzymes than does the parent compound.     

Evolution of the allosteric ligand sites of mammalian phosphofructo-1-kinase

Mammalian phosphofructokinase (PFK) has evolved by a process of tandem gene duplication and fusion to yield a protein that is more than double the size of prokaryotic PFKs. On the basis of complete conservation of active site residues in the N-terminal half of the eukaryotic enzyme with those of the bacterial PFKs, one assumes that the active site of the eukaryotic PFK is located in the N-terminal half. Again using sequence comparisons, the four allosteric ligand sites of mammalian PFK have been thought to arise from the duplicated catalytic and regulatory sites of the ancestral PFK. Previous site-directed mutagenesis studies [Li et al. (1999) Biochemistry 38, 16407-16412; Chang and Kemp (2002) Biochem. Biophys. Res. Commun. 290, 670-675] have identified the origins of the citrate and fructose 2,6-bisphosphate sites. Here, site-directed mutagenesis of two arginine residues (Arg-433 and Arg-429) of mouse phosphofructokinase is used to identify the ATP inhibitory site, and, by inference, the AMP/ADP site. Mutation of the residues to alanine reduced ATP inhibition in the case of Arg-429 and eliminated ATP inhibition in the instance of Arg-433. The Arg-433 mutant could be inhibited by citrate, and that inhibition could be reversed by fructose 2,6-bisphosphate and cyclic AMP, a high-affinity ligand for the AMP/ADP binding site. It is concluded that the two inhibitors, ATP and citrate, of mammalian PFK interact with sites that have evolved from the duplicated phosphoenolpyruvate/ADP allosteric site of the ancestral PFK. The two sites for activators, fructose 2,6-bisphosphate and AMP or ADP, have evolved from the catalytic site of the ancestral precursor.     

Organization of fusicoccin receptor in higher plant plasma membrane: relationship between affinity and molecular mass

Higher plant plasma membranes carry receptors of different affinity for the phytotoxin fusicoccin. Reception of fusicoccin involves proteins belonging to the highly conserved 14-3-3 family, but the complete structure of the fusicoccin receptor (FCR) is unknown. Using radiation inactivation analysis, we estimated the molecular masses of low-affinity and high-affinity FCR at 63 +/- 7 and 130 +/- 15 kD, respectively. The dose dependences of receptor inactivation indicate that microsomal specimens contain "silent" FCRs of 420 +/- 90 kD in amounts commensurate with that of the active FCRs. Both low- and high-affinity FCRs are inactivated by hydrolytic enzymes from the outer surface of the plasma membrane, and impairment of protoplast integrity causes an irreversible transition of the low-affinity binding site into the high-affinity one. A scheme is proposed for the organization of different types of FCR in the plasma membrane, implying that the membrane affinity for fusicoccin reflects the interaction between proteins in the FCR complex.     

Heterogenous stomatal closure in response to leaf water deficits is not a universal phenomenon

The extent and occurrence of water stress-induced “patchy” CO₂ uptake across the surface of leaves was evaluated in a number of plant species. Leaves, while still attached to a plant, were illuminated and exposed to air containing [¹⁴C]CO₂ before autoradiographs were developed. Plant water deficits that caused leaf water potential depression to −1.1 megapascals during a 4-day period did result in heterogenous CO₂ assimilation patterns in bean (Phaseolus vulgaris). However, when the same level of stress was imposed more gradually (during 17 days), no patchy stomatal closure was evident. The patchy CO₂ assimilation pattern that occurs when bean plants are subjected to a rapidly imposed stress could induce artifacts in gas exchange studies such that an effect of stress on chloroplast metabolism is incorrectly deduced. This problem was characterized by examining the relationship between photosynthesis and internal [CO₂] in stressed bean leaves. When extent of heterogenous CO₂ uptake was estimated and accounted for, there appeared to be little difference in this relationship between control and stressed leaves. Subjecting spinach (Spinacea oleracea) plants to stress (leaf water potential depression to −1.5 megapascals) did not appear to cause patchy stomatal closure. Wheat (Triticum aestivum) plants also showed homogenous CO₂ assimilation patterns when stressed to a leaf water potential of −2.6 megapascals. It was concluded that water stress-induced patchy stomatal closure can occur to an extent that could influence the analysis of gas exchange studies. However, this phenomenon was not found to be a general response. Not all stress regimens will induce patchiness; nor will all plant species demonstrate this response to water deficits.     

Andrenocorticotropin and β-lipotropin in the hypothalamus

Adrenocorticotropin and β-lipotropin (β-LPH) have been localized by immunoperoxidase methods in nerve cells and fibers of the hypothalamus and brain stem of the ewe. 6-μm sections were immunostained first for either ACTH or β-LPH. The reaction products and the antibody complexes were then eluted completely from the tissue, and the same section was immunostained for the second peptide. Absorption of the primary antisera with a variety of peptide fragments of ACTH and β-LPH demonstrated, immunocytochemically as well as by radioimmunoassay, that the ACTH and β-LPH antisera were directed to the COOH- and NH(2)-termini of the peptides, respectively. Neither antiserum recognized any portion of the heterologous peptide. In the sequential staining procedure on the same tissue section, preincubation of the antisera with the homologous peptide abolished the staining, whereas preincubation with the heterologous peptide did not affect it, regardless of the order followed. Every nerve cell in the arcuate nucleus that contained ACTH also contained β-LPH, but β-LPH cells appeared, probably falsely, to be twice as numerous as ACTH cells. β-LPH-positive fibers in and beyond the hypothalamus were also more numerous and stained more intensively than ACTH fibers. The salient exception was fibers in the infundibular zona externa, where the opposite was true. Our observations establish that ACTH and β-LPH are contained in the same nerve cells They stongly favor biosynthesis in brain, probably from a common precursor molecule, as has been demonstrated in the pituitary gland. The complexity of the cytologic distribution pattern described suggests that the two peptides are not processed in the same manner by the nerve cell.     

Production via good manufacturing practice of exofucosylated human mesenchymal stromal cells for clinical applications

Background

The regenerative and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) have raised great hope for their use in cell therapy. However, when intravenously infused, hMSCs fail to reach sites of tissue injury. Fucose addition in α(1,3)-linkage to terminal sialyllactosamines on CD44 creates the molecule known as hematopoietic cell E-/L-selectin ligand (HCELL), programming hMSC binding to E-selectin that is expressed on microvascular endothelial cells of bone marrow (BM), skin and at all sites of inflammation. Here we describe how this modification on BM-derived hMSCs (BM-hMSCs) can be adapted to good manufacturing practice (GMP) standards.