| 17081983 |
Global, in vivo, and site-specific phosphorylation dynamics in signaling networks |
10.1016/j.cell.2006.09.026 |
Cell |
Global, in vivo, and site-specific phosphorylation dynamics in signaling networks
Abstract
- Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.
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| 17087724 |
Human cationic trypsinogen is sulfated on Tyr154 |
10.1111/j.1742-4658.2006.05501.x. |
FEBS J |
Human cationic trypsinogen is sulfated on Tyr154
Abstract
- The crystal structure of human pancreatic cationic trypsin showed the chemical modification of Tyr154, which was originally described as phosphorylation [Gaboriaud C, Serre L, Guy-Crotte O, Forest E & Fontecilla-Camps JC (1996) J Mol Biol259, 995-1010]. Here we report that Tyr154 is sulfated, not phosphorylated. Cationic and anionic trypsinogens were purified from human pancreatic juice and subjected to alkaline hydrolysis. Modified tyrosine amino acids were separated on a Dowex cation-exchange column and analyzed by thin layer chromatography. Both human cationic and anionic trypsinogens contained tyrosine sulfate, but no tyrosine phosphate, whereas bovine trypsinogen contained neither. Furthermore, incorporation of [(35)S]SO(4) into human cationic trypsinogen transiently expressed by human embryonic kidney 239T cells was demonstrated. Mutation of Tyr154 to Phe abolished radioactive sulfate incorporation, confirming that Tyr154 is the site of sulfation in cationic trypsinogen. Sulfated pancreatic cationic trypsinogen exhibited faster autoactivation than a nonsulfated recombinant form, suggesting that tyrosine sulfation of trypsinogens might enhance intestinal digestive zymogen activation in humans. Finally, sequence alignment revealed that the sulfation motif is only conserved in primate trypsinogens, suggesting that typsinogen sulfation is absent in other vertebrates.
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| 17088326 |
Crystal structures of human alpha-defensins HNP4, HD5, and HD6 |
10.1110/ps.062336606. |
Protein Sci |
Crystal structures of human alpha-defensins HNP4, HD5, and HD6
Abstract
- Six alpha-defensins have been found in humans. These small arginine-rich peptides play important roles in various processes related to host defense, being the effectors and regulators of innate immunity as well as enhancers of adoptive immune responses. Four defensins, called neutrophil peptides 1 through 4, are stored primarily in polymorphonuclear leukocytes. Major sites of expression of defensins 5 and 6 are Paneth cells of human small intestine. So far, only one structure of human alpha-defensin (HNP3) has been reported, and the properties of the intestine defensins 5 and 6 are particularly poorly understood. In this report, we present the high-resolution X-ray structures of three human defensins, 4 through 6, supplemented with studies of their antimicrobial and chemotactic properties. Despite only modest amino acid sequence identity, all three defensins share their tertiary structures with other known alpha- and beta-defensins. Like HNP3 but in contrast to murine or rabbit alpha-defensins, human defensins 4-6 form characteristic dimers. Whereas antimicrobial and chemotactic activity of HNP4 is somewhat comparable to that of other human neutrophil defensins, neither of the intestinal defensins appears to be chemotactic, and for HD6 also an antimicrobial activity has yet to be observed. The unusual biological inactivity of HD6 may be associated with its structural properties, somewhat standing out when compared with other human alpha-defensins. The strongest cationic properties and unique distribution of charged residues on the molecular surface of HD5 may be associated with its highest bactericidal activity among human alpha-defensins.
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| 17090546 |
A novel neutrophil elastase inhibitor prevents elastase activation and surface cleavage of the epithelial sodium channel expressed in Xenopus laevis oocytes |
10.1074/jbc.M605125200. |
J Biol Chem |
A novel neutrophil elastase inhibitor prevents elastase activation and surface cleavage of the epithelial sodium channel expressed in Xenopus laevis oocytes
Abstract
- The amiloride-sensitive epithelial sodium channel (ENaC) constitutes a limiting step in sodium reabsorption across distal airway epithelium and controlling mucociliary clearance. ENaC is activated by serine proteases secreted in the extracellular milieu. In cystic fibrosis lungs, high concentrations of secreted neutrophil elastase (NE) are observed. hNE could activate ENaC and contribute to further decreased mucociliary clearance. The aims of this study were (i) to test the ability of an engineered human neutrophil elastase inhibitor (EPI-hNE4) to specifically inhibit the elastase activation of ENaC-mediated amiloride-sensitive currents (I(Na)) and (ii) to examine the effect of elastase on cell surface expression of ENaC and its cleavage pattern (exogenous proteolysis). Oocytes were exposed to hNE (10-100 microg/ml) and/or trypsin (10 microg/ml) for 2-5 min in the presence or absence of EPI-hNE4 (0.7 microm). hNE activated I(Na) 3.6-fold (p < 0.001) relative to non-treated hENaC-injected oocytes. EPI-hNE4 fully inhibited hNE-activated I(Na) but had no effect on trypsin- or prostasin-activated I(Na). The co-activation of I(Na) by hNE and trypsin was not additive. Biotinylation experiments revealed that cell surface gamma ENaC (but not alpha or beta ENaC) exposed to hNE for 2 min was cleaved (as a 67-kDa fragment) and correlated with increased I(Na). The elastase-induced exogenous proteolysis pattern is distinct from the endogenous proteolysis pattern induced upon preferential assembly, suggesting a causal relationship between gamma ENaC cleavage and ENaC activation, taking place at the plasma membrane.
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| 17102824 |
Wilms' tumor: the experience of the pediatric unit of Kasr El-Aini center of radiation oncology and nuclear medicine (NEMROCK) |
None |
J Egypt Natl Canc Inst |
Wilms' tumor: the experience of the pediatric unit of Kasr El-Aini center of radiation oncology and nuclear medicine (NEMROCK)
Abstract
- The aim of the present work is to study the treatment results of Wilms' tumor patients who had attended the pediatric unit of Kasr El-Aini center of radiation oncology and nuclear Medicine (NEMROCK) from January 1994 to January 2001.
Sixty-two new Wilms' tumor patients attended the clinic (NEMROCK) from January 1994 until January 2001. The diagnosis was confirmed pathologically. Stage I included 22 cases, stage II 17 cases, stage III 16 cases, and stage IV included 4 cases, whereas stage V included only 3 cases. Stage I cases received 6 months of vincristine and dactinomycin. Stage II with favorable histology (FH) received 1 year of vincristine and dactinomycin. Stage III and IV received 1 year of vincristine, dactinomycin and doxorubicin. Abdominal radiation therapy, 1080cGY, was given in case of tumor spillage during surgery either to the involved flank or the whole abdomen depending on whether contamination was limited to the flank only or the whole abdomen. In addition, radiation was given to metastatic sites in stage IV. Stages II, III, IV with unfavorable histology (UH) received 1 year of dactinomycin, vincristine, doxorubicin and cyclophosphamide in addition to radiation therapy. Stage V cases were diagnosed by surgical biopsy and were managed according to stage and pathology.
Forty patients (64.5%) had favorable histology while twenty-two patients (35.5%) had unfavorable histology. The 4-year overall survival rate was 70.1%. Stage I, II, and stages III+IV+V with favorable histology had a 4-year overall survival of 82.3%, 56% and 41%, respectively. Stages I to IV with unfavorable histology had a 4- year survival of 65.7%.
Multivariate analysis revealed that stage and residual disease after surgery significantly affected overall survival; while histopathology and stage affected significantly disease-free survival. Moreover, our study revealed that residual disease after surgery affected significantly the incidence of local recurrence and distant metastases.
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| 17106905 |
Diversity of the O-superfamily conotoxins from Conus miles |
10.1002/psc.802. |
J Pept Sci |
Diversity of the O-superfamily conotoxins from Conus miles
Abstract
- Conopeptides display prominent features of hypervariability and high selectivity of large gene families that mediate interactions between organisms. Remarkable sequence diversity of O-superfamily conotoxins was found in a worm-hunting cone snail Conus miles. Five novel cDNA sequences encoding O-superfamily precursor peptides were identified in C. miles native to Hainan by RT-PCR and 3'-RACE. They share the common cysteine pattern of the O-superfamily conotoxin (C-C-CC-C-C, with three disulfide bridges). The predicted peptides consist of 27-33 amino acids. We then performed a phylogenetic analysis of the new and published homologue sequences from C. miles and the other Conus species. Sequence divergence (%) and residue substitutions to view evolutionary relationships of the precursors' signal, propeptide, and mature toxin regions were analyzed. Percentage divergence of the amino acid sequences of the prepro region exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical with to highly divergent from inter- and intra-species. Despite the O-superfamily being a large and diverse group of peptides, widely distributed in the venom ducts of all major feeding types of Conus and discovered in several Conus species, it was for the first time that the newly found five O-superfamily peptides in this research came from the vermivorous C. miles. So far, conotoxins of the O-superfamily whose properties have been characterized are from piscivorous and molluscivorous Conus species, and their amino acid sequences and mode of action have been discussed in detail. The elucidated cDNAs of the five toxins are new and of importance and should attract the interest of researchers in the field, which would pave the way for a better understanding of the relationship of their structure and function.
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| 17107879 |
ANP and urodilatin: who is who in the kidney |
None |
Eur J Med Res |
ANP and urodilatin: who is who in the kidney
Abstract
- Mounting evidence suggests that urodilatin, not atrial natriuretic peptide (ANP) is the responsible peptide in regulation of renal Na superset+- and water homeostasis. Following the discovery of ANP this peptide was thought to be responsible for the induction of natriuresis and diuresis in the mammalian kidney. However, the isolation of urodilatin from human urine and substantial work contributed to a better understanding of the renal physiology of these two natriuretic peptides. Indeed, subsequent elucidation supported that urodilatin rather than ANP seems to be the natriuretic peptide responsible for the regulation of Na superset+- and water homeostasis in the kidney. Urodilatin - synthesized and secreted from the distal tubules of the kidney - may act as a paracrine mediator when secreted into the lumen. In contrast, while the role of ANP as regulator of the cardiovascular system is established, its physiological regulatory role on transport processes in the nephron is questionable. This review attempts to analyze the roles of both ANP and urodilatin and to discuss new potential candidates which may also play a role in electrolyte and water handling in the kidney.
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| 17115032 |
Tyrosine phosphorylation controls PCNA function through protein stability |
10.1038/ncb1501. |
Nat Cell Biol |
Tyrosine phosphorylation controls PCNA function through protein stability
Abstract
- The proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication and damage repair. How its function is controlled remains an important question. Here, we show that the chromatin-bound PCNA protein is phosphorylated on Tyr 211, which is required for maintaining its function on chromatin and is dependent on the tyrosine kinase activity of EGF receptor (EGFR) in the nucleus. Phosphorylation on Tyr 211 by EGFR stabilizes chromatin-bound PCNA protein and associated functions. Consistently, increased PCNA Tyr 211 phosphorylation coincides with pronounced cell proliferation, and is better correlated with poor survival of breast cancer patients, as well as nuclear EGFR in tumours, than is the total PCNA level. These results identify a novel nuclear mechanism linking tyrosine kinase receptor function with the regulation of the PCNA sliding clamp.
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| 17125217 |
Trungapeptins A-C, cyclodepsipeptides from the marine cyanobacterium Lyngbya majuscula |
10.1021/np050485a. |
J Nat Prod |
Trungapeptins A-C, cyclodepsipeptides from the marine cyanobacterium Lyngbya majuscula
Abstract
- Trungapeptins A-C (1-3) were isolated from the marine cyanobacterium Lyngbya majuscula collected from Trung Province, Thailand. Their gross structures were elucidated by interpretation of spectroscopic data. The absolute configurations of the amino acids and phenyllactic acid were determined by Marfey's and chiral HPLC analyses, respectively. The relative stereochemistry of 3-hydroxy-2-methyl-7-octynoic acid (Hmoya) of trungapeptin A was elucidated by application of the J-based configuration analysis, and its absolute stereochemistry was established to be 2S, 3R by Mosher's method. The structures of compounds 1-3 are closely related to the antanapeptins, a series of depsipeptides isolated from a Madagascan collection of L. majuscula.
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| 17125219 |
Kahalalide derivatives from the Indian sacoglossan mollusk Elysia grandifolia |
10.1021/np060172v. |
J Nat Prod |
Kahalalide derivatives from the Indian sacoglossan mollusk Elysia grandifolia
Abstract
- Two new cyclic depsipeptide derivatives, kahalalides R (1) and S (2), together with two known congeners, kahalalides F (3) and D (4), were isolated from the Indian sacoglossan mollusk Elysia grandifolia. The structures of the new compounds were unambiguously established on the basis of NMR spectroscopic (1H, 13C, COSY, HMBC) and mass spectrometric (FABMS, ESIMS, MALDI-TOF/PSD) data, which also included Marfey amino acid analyses. The new derivative kahalalide R was found to exert comparable or even higher cytotoxicity than the potential drug candidate kahalalide F toward the MCF7 human mammary carcinoma cell line.
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| 17125221 |
A chemical study of cyclic depsipeptides produced by a sponge-derived fungus |
10.1021/np060178k. |
J Nat Prod |
A chemical study of cyclic depsipeptides produced by a sponge-derived fungus
Abstract
- Two novel cyclic depsipeptides, guangomides A (1) and B (2), together with a new destruxin derivative (3) were isolated from the cytotoxic extract obtained from the saltwater culture of an unidentifiable sponge-derived fungus. The new structures were elucidated on the basis of analysis of extensive 1D and 2D NMR data sets, and the absolute configurations of 2S, 9S, 13S, 19S, 24R, 28R of 1 were determined on the basis of the combined X-ray and Marfey's method structure analysis. Identical absolute configurations were assumed for 2. The cytotoxicity of the extract was found to be due to brefeldin A, while 1 and 2 showed weak antibacterial activity against Staphylococcus epidermidis and Enterococcus durans.
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| 17129757 |
NMR structure determination of a synthetic analogue of bacillomycin Lc reveals the strategic role of L-Asn1 in the natural iturinic antibiotics |
10.1016/j.saa.2006.10.027. |
Spectrochim Acta A Mol Biomol Spectrosc |
NMR structure determination of a synthetic analogue of bacillomycin Lc reveals the strategic role of L-Asn1 in the natural iturinic antibiotics
Abstract
- Iturins are a group of antifungal produced by Bacillus subtilis. All are cyclic lipopeptides with seven alpha-amino acids of configuration LDDLLDL and one beta-amino fatty acid. The bacillomycin L is a member of this family and its NMR structure was previously resolved using the sequence Asp-Tyr-Asn-Ser-Gln-Ser-Thr. In this work, we carefully examined the NMR spectra of this compound and detected an error in the sequence. In fact, Asp1 and Gln5 need to be changed into Asn1 and Glu5, which therefore makes it identical to bacillomycin Lc. As a consequence, it now appears that all iturinic peptides with antibiotic activity share the common beta-amino fatty acid 8-L-Asn1-D-Tyr2-D-Asn3 sequence. To better understand the conformational influence of the acidic residue L-Asp1, present, for example in the inactive iturin C, the NMR structure of the synthetic analogue SCP [cyclo (L-Asp1-D-Tyr2-D-Asn3-L-Ser4-L-Gln5-D-Ser6-L-Thr7-beta-Ala8)] was determined and compared with bacillomycin Lc recalculated with the corrected sequence. In both cases, the conformers obtained were separated into two families of similar energy which essentially differ in the number and type of turns. A detailed analysis of both cyclopeptide structures is presented here. In addition, CD and FTIR spectra were performed and confirmed the conformational differences observed by NMR between both cyclopeptides.
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| 17139284 |
How many drug targets are there? |
10.1038/nrd2199. |
Nat Rev Drug Discov |
How many drug targets are there?
Abstract
- For the past decade, the number of molecular targets for approved drugs has been debated. Here, we reconcile apparently contradictory previous reports into a comprehensive survey, and propose a consensus number of current drug targets for all classes of approved therapeutic drugs. One striking feature is the relatively constant historical rate of target innovation (the rate at which drugs against new targets are launched); however, the rate of developing drugs against new families is significantly lower. The recent approval of drugs that target protein kinases highlights two additional trends: an emerging realization of the importance of polypharmacology, and also the power of a gene-family-led approach in generating novel and important therapies.
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| 17141187 |
Solution structure and membrane interaction mode of an antimicrobial peptide gaegurin 4 |
10.1016/j.bbrc.2006.11.064. |
Biochem Biophys Res Commun |
Solution structure and membrane interaction mode of an antimicrobial peptide gaegurin 4
Abstract
- We have applied NMR spectroscopy to determine the high-resolution structure of gaegurin 4, a 37-residue antimicrobial peptide from Rana rugosa, under varying hydrophobic conditions. Even in 100% H2O, gaegurin 4 contains a nascent turn near its C-terminal Rana box. Under a more hydrophobic condition it forms two amphipathic helices, one long encompassing residues 2-23 and the other consisting of residues 25-34, similar to what has been observed in cecropin A. Functional implication of the helix-breaking kink at Gly24 in gaegurin 4 was investigated by preparing several analogs. Based upon the current and previous results, we propose a novel seaanemone-like ion pore-forming model for gaegurin 4.
|
| 17142296 |
Isolation and structure-activity of mu-conotoxin TIIIA, a potent inhibitor of tetrodotoxin-sensitive voltage-gated sodium channels |
10.1124/mol.106.028225. |
Mol Pharmacol |
Isolation and structure-activity of mu-conotoxin TIIIA, a potent inhibitor of tetrodotoxin-sensitive voltage-gated sodium channels
Abstract
- Mu-conotoxins are three-loop peptides produced by cone snails to inhibit voltage-gated sodium channels during prey capture. Using polymerase chain reaction techniques, we identified a gene sequence from the venom duct of Conus tulipa encoding a new mu-conotoxin-TIIIA (TIIIA). A 125I-TIIIA binding assay was established to isolate native TIIIA from the crude venom of Conus striatus. The isolated peptide had three post-translational modifications, including two hydroxyproline residues and C-terminal amidation, and <35% homology to other mu-conotoxins. TIIIA potently displaced [3H]saxitoxin and 125I-TIIIA from rat brain (Nav1.2) and skeletal muscle (Nav1.4) membranes. Alanine and glutamine scans of TIIIA revealed several residues, including Arg14, that were critical for high-affinity binding to tetrodotoxin (TTX)-sensitive Na+ channels. We were surprised to find that [E15A]TIIIA had a 10-fold higher affinity than TIIIA for TTX-sensitive sodium channels (IC50, 15 vs. 148 pM at rat brain membrane). TIIIA was selective for Nav1.2 and -1.4 over Nav1.3, -1.5, -1.7, and -1.8 expressed in Xenopus laevis oocytes and had no effect on rat dorsal root ganglion neuron Na+ current. 1H NMR studies revealed that TIIIA adopted a single conformation in solution that was similar to the major conformation described previously for mu-conotoxin PIIIA. TIIIA and analogs provide new biochemical probes as well as insights into the structure-activity of mu-conotoxins.
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