20316893
Structure of human dual specificity protein phosphatase 23, VHZ, enzyme-substrate/product complex.
Protein phosphorylation plays a crucial role in mitogenic signal transduction and regulation of cell growth and differentiation. Dual specificity protein phosphatase 23 (DUSP23) or VHZ mediates dephosphorylation of phospho-tyrosyl (pTyr) and phospho-seryl/threonyl (pSer/pThr) residues in specific proteins. In vitro, it can dephosphorylate p44ERK1 but not p54SAPK-beta and enhance activation of c-Jun N-terminal kinase (JNK) and p38. Human VHZ, the smallest of the catalytically active protein-tyrosine phosphatases (PTP) reported to date (150 residues), is a class I Cys-based PTP and bears the distinctive active site signature motif HCXXGXXRS(T). We present the crystal structure of VHZ determined at 1.93A resolution. The polypeptide chain adopts the typical alphabetaalpha PTP fold, giving rise to a shallow active site cleft that supports dual phosphorylated substrate specificity. Within our crystals, the Thr-135-Tyr-136 from a symmetry-related molecule bind in the active site with a malate ion, where they mimic the phosphorylated TY motif of the MAPK activation loop in an enzyme-substrate/product complex. Analyses of intermolecular interactions between the enzyme and this pseudo substrate/product along with functional analysis of Phe-66, Leu-97, and Phe-99 residues provide insights into the mechanism of substrate binding and catalysis in VHZ.
205373753
SNAP-25 substrate peptide (residues 180-183) binds to but bypasses cleavage by catalytically active Clostridium botulinum neurotoxin E.
Clostridium botulinum neurotoxins are the most potent toxins to humans. The recognition and cleavage of SNAREs are prime evente in exhibiting their toxicity. We report here the crystal structure of the catalytically active full-length botulinum serotype E catalytic domain (BoNT E) in complex with SNAP-25 (a SNARE protein) substrate peptide Arg(180)-Ile(181)-Met(182)-Glu(183) (P1-P3'). It is remarkable that the peptide spanning the scissile bond binds to but bypasses cleavage by the enzyme and inhibits the catalysis fairly with K(i) approximately 69 microm. The inhibitory peptide occupies the active site of BoNT E and shows well defined electron density. The catalytic zinc and the conserved key residue Tyr(350) of the enzyme facilitate the docking of Arg(180) (P1) by interacting with its carbonyl oxygen that displaces the nucleophilic water. The general base Glu(212) side chain interacts with the main chain amino group of P1 and P1'. Conserved Arg(347) of BoNT E stabilizes the proper docking of the Ile(181) (P1') main chain, whereas the hydrophobic pockets stabilize the side chains of Ile(181) (P1') and Met(182) (P2'), and the 250 loop stabilizes Glu(183) (P3'). Structural and functional analysis revealed an important role for the P1' residue and S1' pocket in driving substrate recognition and docking at the active site. This study is the first of its kind and rationalizes the substrate cleavage strategy of BoNT E. Also, our complex structure opens up an excellent opportunity of structure-based drug design for this fast acting and extremely toxic high priority BoNT E.
21302804
Structural analysis of a ternary complex of allantoate amidohydrolase from Escherichia coli reveals its mechanics.
Purine metabolism plays a major role in regulating the availability of purine nucleotides destined for nucleic acid synthesis. Allantoate amidohydrolase catalyzes the conversion of allantoate to (S)-ureidoglycolate, one of the crucial alternate steps in purine metabolism. The crystal structure of a ternary complex of allantoate amidohydrolase with its substrate allantoate and an allosteric effector, a sulfate ion, from Escherichia coli was determined to understand better the catalytic mechanism and substrate specificity. The 2.25 A resolution X-ray structure reveals an alpha/beta scaffold akin to zinc exopeptidases of the peptidase M20 family and lacks the (beta/alpha)(8)-barrel fold characteristic of the amidohydrolases. Arrangement of the substrate and the two co-catalytic zinc ions at the active site governs catalytic specificity for hydrolysis of N-carbamyl versus the peptide bond in exopeptidases. In its crystalline form, allantoate amidohydrolase adopts a relatively open conformation. However, structural analysis reveals the possibility of a significant movement of domains via rotation about two hinge regions upon allosteric effector and substrate binding resulting in a closed catalytically competent conformation by bringing the substrate allantoate closer to co-catalytic zinc ions. Two cis-prolyl peptide bonds found on either side of the dimerization domain in close proximity to the substrate and ligand-binding sites may be involved in protein folding and in preserving the integrity of the catalytic site.
19975781
Cloning, high level expression, purification, and crystallization of the full length Clostridium botulinum neurotoxin type E light chain.
The catalytic activity of the highly potent botulinum neurotoxins are confined to their N-terminal light chains ( approximately 50kDa). A full-length light chain for the type E neurotoxin with a C-terminal 6x His-tag, BoNT/E-LC, has been cloned in a pET-9c vector and over-expressed in BL21 (DE3) cells. BoNT/E-LC was purified to homogeneity by affinity chromatography on Ni-NTA agarose followed by exclusion chromatography using a Superdex-75 sizing column. The purified protein has very good solubility and can be stored stably at -20 degrees C; however, it seems to undergo auto-proteolysis when stored at temperature #10878;4-10 degrees C. BoNT/E-LC is active on its natural substrate, the synaptosomal associated 25kDa protein, SNAP-25, indicating that it retains a native-like conformation and therefore can be considered as a useful tool in studying the structure/function of the catalytic light chain. Recombinant BoNT/E-LC has been crystallized under five different conditions and at various pHs. Crystals diffract to better than 2.1A.
556786
A novel mode of dimerization via formation of a glutamate anhydride crosslink in a protein crystal structure.
Acid anhydrides are formed by removal of water, are very active, and can react with water to form the original binary components.
12841382
Mode of VAMP substrate recognition and inhibition of Clostridium botulinum neurotoxin F
Clostridium botulinum neurotoxins (BoNTs) cleave neuronal proteins responsible for neurotransmitter release, causing the neuroparalytic disease botulism. BoNT serotypes B, D, F and G cleave and inactivate vesicle-associated membrane protein (VAMP), each at a unique peptide bond. The specificity of BoNTs depends on the mode of substrate recognition. We have investigated the mechanism of substrate recognition of BoNT F by determining the crystal structures of its complex with two substrate-based inhibitors, VAMP 22-58/Gln58D-cysteine and 27-58/Gln58D-cysteine. The inhibitors bind to BoNT F in the canonical direction (as seen for BoNTs A and E substrates) but are positioned specifically via three major exosites away from the active site. The cysteine sulfur of the inhibitors interacts with the zinc and exists as sulfinic acid in the inhibitor VAMP 27-58/Gln58D-cysteine. Arg133 and Arg171, which form part of two separate exosites, are crucial for substrate binding and catalysis.
10815047
Metabolism of benzo[a]pyrene by human melanocytes in culture.
Benzo[a]pyrene (B[a]P) is a ubiquitous environmental pollutant and known skin carcinogen. In the present study, in vitro addition of [3H]B[a]P to normal human melanocytes in culture, isolated from adult human skin, resulted in the metabolism of [3H]B[a]P both intracellularly and extracellularly. HPLC analysis showed that [3H]B[a]P-9,10- and 7,8-diol were the major intracellular and extracellular metabolites followed by 3,6-quinone, 9-hydroxy and 3-hydroxy metabolites. Significant amounts of the [3H]B[a]P metabolites were found to be present in the sonicated cell suspension and culture medium as the glucuronide and sulfate conjugates. In total 37.3% of the [3H]B[a]P added in the culture medium was metabolized by melanocytes, of which 21.1% was quantified as the intracellular and 16.2% as the extracellular metabolites. Our data show that human melanocytes are capable of metabolizing B[a]P.
28746093
Reproducibility of Renal Function Measurements in Adult Men with Diabetic Nephropathy: Research and Clinical Implications
Background/Aim: Measurement of the renal function is critical to follow progression of kidney disease. Short-term and long-term variabilities in these measurements have significant impacts on clinical decision making and clinical trials. The goal of this study was to describe the variability in these measurements and to calculate minimum sample size estimates over varying time frames for clinical trials. Methods: We studied 44 elderly men with diabetic nephropathy who participated in a clinical trial. Glomerular filtration rate and renal plasma flow were measured by continuous infusion technique with five urine collection periods on two occasions 4 months apart. Protein and creatinine excretion rates were measured in the same specimens. In addition, two consecutive 24-hour specimens every month for 4 months were collected to analyze urine protein, creatinine, urea nitrogen, and electrolytes. A hierarchical random effects model was used to analyze the reproducibility from hour to hour, from day to day, and from month to month. Results: A total of 824 urine specimens were analyzed, of which 412 constituted specimens collected in the short term and 412 were 24-hour urine collections. Hour-to-hour variation accounted for 45% for urinary clearance of iothalamate, but for only 0.5% of the variability in plasma clearance of iothalamate. Day-to-day variability in 24-hour urinary excretion rates for creatinine was 46% and for protein 10%. Month-to-month variability in 24-hour excretion rates for creatinine was 11% and for protein 19%. The urine protein/creatinine ratio had a day-to-day variability of 2% and a month-to-month variability of 19%. Sample size requirements can be reduced by correcting for urine creatinine for some but not all urinary analytes. Conclusions: In nephrotic men with diabetic nephropathy, the coefficient of variation in the month-to-month protein excretion rate is 36%. Approximately 28 patients in each arm of two groups are needed to detect a difference in protein excretion rate of 28% (1 g/day in this study). The coefficient of variation in plasma iothalamate clearance over 4 months is 16%. To detect a 10% change in glomerular filtration rate between two groups, 44 patients per group are needed. To be deemed statistically significant, a change in daily protein excretion rate of at least 72% over month(s) is needed in individual patients.
30259650
Cell signaling and regulators of cell cycle as molecular targets for prostate cancer prevention by dietary agents.
Prostate cancer (PCA) is the most common invasive malignancy and leading cause (after lung) of cancer deaths in males. Since PCA is initially androgen-dependent, strategies are targeted toward androgen depletion for its control. However, tumor re-growth mostly occurs following this modality, and is androgen-independent. A loss of functional androgen receptor and an enhanced expression of growth factor receptors (e.g. erbB family members) and associated ligands have been shown to be the causal genetic events in PCA progression. These genetic alterations lead to an epigenetic mechanism where a feed-back autocrine loop between membrane receptor (e.g. epidermal growth factor receptor [erbB1] and associated ligand (e.g. transforming growth factor-alpha) results in an enhanced activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) as an essential component of the uncontrolled growth of PCA at an advanced and androgen-independent stage. Together, we rationalized that inhibiting these epigenetic events would be useful in controlling advanced PCA growth. Dietary polyphenolic flavonoids and isoflavones are being studied extensively as cancer-preventive and interventive agents. Therefore, we focused our attention on silymarin, genistein, and epigallocatechin 3-gallate (EGCG), present in milk thistle, soy beans, and green tea, respectively. The effect of these agents was assessed on the erbB1-Shc-ERK1/2 signal transduction pathway, cell cycle regulatory molecules, and cell growth and death. In androgen-independent human prostate carcinoma DU145 cells, silymarin, genistein, and EGCG resulted in a significant to complete inhibition of transforming growth factor-alpha-caused activation of membrane receptor erbB1 followed by inhibition of downstream cytoplasmic signaling target Shc activation and a decrease in its binding with erbB1, without an alteration in their protein expression. Silymarin and genistein also inhibited ERK1/2 activation, suggesting that these agents impair the activation of erbB1-Shc-ERK1/2 signaling in DU145 cells. In the case of EGCG, a further increase in ERK1/2 activation was observed that was related to its pro-oxidant and apoptotic activities. Silymarin, genistein, and EGCG also resulted in a significant induction of Cip1/p21 and Kip1/p27 and a decrease in cyclin-dependent kinase (CDK) 4, but a moderate inhibition of CDK2, cyclin D1, and cyclin E was observed. An enhanced level of Cip1/p21 and Kip1/27 also led to an increase in their binding to CDK4 and CDK2. Treatment of cells with silymarin, genistein, and EGCG also resulted in strong cell growth inhibition at lower doses, and complete inhibition at higher doses. In contrast to silymarin, higher doses of genistein also showed cell death. A more profound cytotoxic effect was observed in the case of EGCG, with strong cell death at lower doses and complete loss of viability at higher doses. Together, these results suggest that cell signaling and regulators of cell cycle are potential epigenetic molecular targets for prostate cancer prevention by dietary agents. More studies, therefore, are needed with these agents to explore their anticarcinogenic potential against human prostate cancer.
42508732
Three-dimensional computed tomographic analysis of the maxilla in unilateral cleft lip and palate: implications for rhinoplasty.
BACKGROUND
The cleft lip nose is a complex 3-dimensional (3D) midfacial soft tissue and bony deformity. The contribution of maxillary hypoplasia to the etiology of this deformity has often been implicated for the suboptimal results of surgical treatment. The dimensions of the maxilla in unilateral cleft lip and palate (UCLP) have not been studied especially in relation to the volumetric and other asymmetries on the either side in unilateral clefts. The purpose of this article is to assess the relevant maxillary parameters of length, width, height, depth, and volume in patients with UCLP and compare the parameters of the cleft and noncleft sides.


METHODS
Fifteen patients with UCLP were treated by a standard protocol by a single surgeon and orthodontist. The measurements of maxilla were taken using a combination of axial, coronal, lateral, and 3D reconstructed images. The volume of each maxilla was calculated on 3D reconstructions using the technique of manual segmentation, which allowed complete reconstruction of the right and left maxilla individually.


RESULTS
In general, the cleft maxillary length, width, height, depth, and volume have been found to be reduced when compared with the normal sides with significant P values (Wilcoxon signed rank test Z, P < 0.001).


CONCLUSIONS
The study demonstrates the anatomy of the maxillary asymmetry existing in UCLP and underlines the importance of correcting the bony deficiency by appropriate techniques to harmonize the results of rhinoplasty in unilateral cleft lip nose deformity. Augmentation of the deficient maxilla by specific contoured bone grafts allows restoration of the symmetry of the nasal platform in unilateral cleft lip nose deformity.
20137332
The unrecognized skeletal components of the cleft lip nose deformity.