Phytochemistry. 2010 Dec 4. [Epub ahead of print]
From protein catalogues towards targeted proteomics approaches in cereal grains.
Finnie C, Sultan A, Grasser KD.
Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Bldg 224, DK-2800 Kgs. Lyngby, Denmark.
Abstract
Due to their importance for human nutrition, the protein content of cereal grains has been a subject of intense study for over a century and cereal grains were not surprisingly one of the earliest subjects for 2D-gel-based proteome analysis. Over the last two decades, countless cereal grain proteomes, mostly derived using 2D-gel based technologies, have been described and hundreds of proteins identified. However, very little is still known about post-translational modifications, subcellular proteomes, and protein-protein interactions in cereal grains. Development of techniques for improved extraction, separation and identification of proteins and peptides is facilitating functional proteomics and analysis of sub-proteomes from small amounts of starting material, such as seed tissues. The combination of proteomics with structural and functional analysis is increasingly applied to target subsets of proteins. These "next-generation" proteomics studies will vastly increase our depth of knowledge about the processes controlling cereal grain development, nutritional and processing characteristics.
Copyright © 2010 Elsevier Ltd. All rights reserved.PMID: 21134685 [PubMed - as supplied by publisher]
J Inorg Biochem. 2011 Jan;105(1):111-117.
Cadmium(II)-stimulated enzyme activation of Arabidopsis thaliana phytochelatin synthase 1.
Ogawa S, Yoshidomi T, Yoshimura E.
Abstract
Phytochelatin (PC), a class of heavy metal-binding peptides, is synthesized from the tripeptide glutathione (GSH) and/or previously synthesized PC in a reaction mediated by PC synthase (PCS). In the present study, the PC production rate catalyzed by recombinant Arabidopsis PCS1 (rAtPCS1) in the presence of a constant free Cd(II) level increased steadily and the kinetic parameters were approximated using a substituted-enzyme mechanism in which GSH and bis(glutathionato)cadmium acted as co-substrates. In contrast, the PC production rate as a function of GSH concentration at a constant total Cd(II) concentration reached a maximum, which shifted toward higher GSH concentrations as the concentration of Cd(II) was increased. These observations are consistent with the suggestion that rAtPCS1 possesses a Cd(II) binding site where Cd(II) binds to activate the enzyme. The affinity constant, optimized using a one-site mathematical model, successfully simulated the experimental data for the assay system using lower concentrations of Cd(II) (5 or 10μM) but not for the assay using higher concentrations (50 or 500μM), where a sigmoidal increase in PCS activity was evident. Furthermore, the PCS activity determined at a constant GSH concentration as a function of Cd(II) concentration also reached a maximum. These findings demonstrate that rAtPCS1 also possesses a second Cd(II) binding site where Cd(II) binds to induce an inhibitory effect. A two-site mathematical model was applied successfully to account for the observed phenomena, supporting the suggestion that rAtPCS1 possesses two Cd(II) binding sites.
Copyright © 2010 Elsevier Inc. All rights reserved.PMID: 21134609 [PubMed - as supplied by publisher]
J Inorg Biochem. 2011 Jan;105(1):102-110. Epub 2010 Sep 29.
Copper effective binding with 32-62 and 94-125 peptide fragments of histone H2B.
Zavitsanos K, Nunes AM, Malandrinos G, Hadjiliadis N.
Abstract
In an attempt to investigate the role of histone H2B in Cu(II) induced toxicity and carcinogenesis, we synthesized the terminally blocked peptides H2B(32-62) (SRKESYSVYVYKVLKQVH(48)PDTGISSKAMGIM) and Η2Β(94-125) (IQTAVRLLLPGELAKH(110)AVSEGTKAVTKYTSS), mimicking the N-terminal histone-fold domain and C-terminal tail of histone H2B, respectively and studied their interaction with Cu(II) ions by means of potentiometric titrations and spectroscopic techniques (UV-visible, CD and EPR). Both peptides, H2B(32-62) and H2B(94-125), interacted efficiently with Cu(II) ions, forming several species from pH 4 to 11, with His(48) and His(110) serving as anchors for metal binding. In H2B(32-62), the effective Cu(II) binding is emphasized by the formation of a soluble Cu(II)-H2B(32-62) complex, unlike the unbound peptide that precipitated over pH 7.9. At physiological pH, both peptides form tetragonal 3N species with a {N(Im), 2N(-)} coordination mode. At this pH, H2B(32-62) presented the formation of coordination isomers, differentiated by the presence in one of them, of an axial coordination of the carboxylate group of Asp(50). Copper binding with both H2B(32-62) and H2B(94-125) may induce a conformational change in the peptides' original structure. At physiological conditions, this effect may interfere with nucleosome's structure and dynamics, including the ubiquitination of Lys(120) which is linked to gene silencing.
Copyright © 2010 Elsevier Inc. All rights reserved.PMID: 21134608 [PubMed - as supplied by publisher]
J Inorg Biochem. 2011 Jan;105(1):52-57. Epub 2010 Oct 8.
Ca(2+)-induced self-assembly in designed peptides with optimally spaced gamma-carboxyglutamic acid residues.
Dai Q, Dong M, Liu Z, Prorok M, Castellino FJ.
Institute of Biotechnology, Beijing 100071, China.
Abstract
We have previously elucidated a new paradigm for the metal ion-induced helix-helix assembly in the natural γ-carboxyglutamic acid (Gla)-containing class of conantokin (con) peptides, typified by con-G and a variant of con-T, con-T[K7Gla], independent of the hydrophobic effect. In these "metallo-zipper" structures, Gla residues spaced at i, i+4, i+7, i+11 intervals, which is similar to the arrangement of a and d residues in typical heptads of coiled-coils, coordinate with Ca(2+) and form specific antiparallel helical dimers. In order to evaluate the common role of Gla residues in peptide self-assembly, we extend herein the same Gla arrangement to designed peptides: NH(2)-(γLSγEAK)(3)-CONH(2) (peptide 1) and NH(2)-γLSγEAKγLSγQANγLSγKAE-CONH(2) (peptide 2). Peptide 1 and peptide 2 exhibit no helicity alone, but undergo structural transitions to helical conformations in the presence of a variety of divalent cations. Sedimentation equilibrium ultracentrifugation analyses showed that peptide 1 and peptide 2 form helical dimers in the presence of Ca(2+), but not Mg(2+). Folding and thiol-disulfide rearrangement assays with Cys-containing peptide variants indicated that the helical dimers are mixtures of antiparallel and parallel dimers, which is different from the strict antiparallel strand orientations of con-G and con-T[K7γGla] dimers. These findings suggest that the Gla arrangement, i, i+4, i+7, i+11, i+14, plays a key role in helix formation, without a strict adherence to strand orientation of the helical dimer.
Copyright © 2010 Elsevier Inc. All rights reserved.PMID: 21134602 [PubMed - as supplied by publisher]
J Inorg Biochem. 2011 Jan;105(1):10-16. Epub 2010 Sep 29.
Selective peptide bond hydrolysis of cysteine peptides in the presence of Ni(II) ions.
Protas AM, Bonna A, Kopera E, Bal W.
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland.
Abstract
Recently, we described a sequence-specific R1-(Ser/Thr) peptide bond hydrolysis reaction in peptides of a general sequence R1-(Ser/Thr)-Xaa-His-Zaa-R, which occurs in the presence of Ni(II) ions [A. Krężel, E. Kopera, A. M. Protas, A. Wysłouch-Cieszyńska, J. Poznański, W. Bal, J. Am. Chem. Soc. 132 (2010) 3355-3366]. In this study we explored the possibility of substituting the Ser/Thr and the His residues, necessary for the reaction to occur according to the Ni(II)-assisted acyl shift reaction mechanism, with Cys residues. We tested this concept by synthesizing three homologous peptides: R1-Ser-Arg-Cys-Trp-R2, R1-Cys-Arg-His-Trp-R2, and R1-Cys-Arg-Cys-Trp-R2, and the R1-Ser-Arg-His-Trp-R2 peptide as comparator (R1 and R2 were CH3CO-Gly-Ala and Lys-Phe-Leu-NH2, respectively). We studied their hydrolysis in the presence of Ni(II) ions, under anaerobic conditions and in the presence of TCEP as a thiol group antioxidant. We measured hydrolysis rates using HPLC and identified products of reaction using electrospray mass spectrometry. Potentiometry and UV-vis spectroscopy were used to assess Ni(II) complexation. We demonstrated that Ni(II) is not compatible with the Cys substitution of the Ser/Thr acyl acceptor residue, but the substitution of the Ni(II) binding His residue with a Cys yields a peptide susceptible to Ni(II)-related hydrolysis. The relatively high activity of the R1-Ser-Arg-Cys-Trp-R2 peptide at pH 7.0 suggests that this peptide and its Cys-containing analogs might be useful in practical applications of Ni(II)-dependent peptide bond hydrolysis.
Copyright © 2010 Elsevier Inc. All rights reserved.PMID: 21134597 [PubMed - as supplied by publisher]
Exp Cell Res. 2010 Dec 3. [Epub ahead of print]
The human cathelicidin, LL-37, induces granzyme-mediated apoptosis in cytotoxic T lymphocytes.
Mader JS, Marcet-Palacios M, Hancock RE, Bleackley RC.
Department of Biochemistry, School of Molecular and Systems Medicine, Faculty of Medicine, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
Abstract
LL-37 is a human cationic host defense peptide (antimicrobial peptide) belonging to the cathelicidin family of peptides. In this study, LL-37 was shown to kill stimulated CD8(+) T cells (Cytotoxic T lymphocytes; CTLs) via apoptosis, while having no cytotoxic effect on non-stimulated CD8(+) or CD4(+) T cells, or stimulated CD4(+) T cells. Of interest, the CD8(+) cells were much more sensitive to LL-37 than many other cell types. LL-37 exposure resulted in DNA fragmentation, chromatin condensation, and the release of both granzyme A and granzyme B from intracellular granules. The importance of granzyme family members in the apoptosis of CTLs following LL-37 treatment was analyzed by using C57Bl/6 lymphocytes obtained from mice that were homozygous for null mutations in the granzyme B gene, the granzyme A gene, or both granzyme A and B. Granzyme A and granzyme B were both shown to play an important role in LL-37-induced apoptosis of CTLs. Further analysis revealed that apoptosis occurred primarily through granzyme A-mediated caspase-independent apoptosis. However, caspase-dependent cell death was also observed. This suggests that LL-37 induces apoptosis in CTLs via multiple different mechanisms, initiated by the LL-37-induced leakage of granzymes from cytolytic granules. Our results imply the existence of a novel mechanism of crosstalk between the inflammatory and adaptive immune systems. Cells such as neutrophils, at the site of a tumor for example, could influence the effector activity of CTL through the secretion of LL-37.
Copyright © 2010. Published by Elsevier Inc.PMID: 21134367 [PubMed - as supplied by publisher]
BMC Evol Biol. 2010 Dec 6;10(1):379. [Epub ahead of print]
Evolution of plant phage-type RNA polymerases: The genome of the basal angiosperm Nuphar advena encodes two mitochondrial and one plastid phage-type RNA polymerases.
Yin C, Richter U, Borner T, Weihe A.
Abstract
ABSTRACT:
BACKGROUND: In mono- and eudicotyledonous plants, a small nuclear gene family (RpoT, RNA polymerase of the T3/T7 type) encodes mitochondrial as well as chloroplast RNA polymerases homologous to the T-odd bacteriophage enzymes. RpoT genes from angiosperms are well characterized, whereas data from deeper branching plant species are limited to the moss Physcomitrella and the spikemoss Selaginella. To further elucidate the molecular evolution of the RpoT polymerases in the plant kingdom and to get more insight into the potential importance of having more than one phage-type RNA polymerase (RNAP) available, we searched for the respective genes in the basal angiosperm Nuphar advena.
RESULTS: By screening a set of BAC library filters, three RpoT genes were identified. Both genomic gene sequences and full-length cDNAs were determined. The NaRpoT mRNAs specify putative polypeptides of 996, 990 and 985 amino acids, respectively. All three genes comprise 19 exons and 18 introns, conserved in their positions with those known from RpoT genes of other land plants. The encoded proteins show a high degree of conservation at the amino acid sequence level, including all functional crucial regions and residues known from the phage T7 RNAP. The N-terminal transit peptides of two of the encoded polymerases, NaRpoTm1 and NaRpoTm2, conferred targeting of green fluorescent protein (GFP) exclusively to mitochondria, whereas the third polymerase, NaRpoTp, was targeted to chloroplasts. Remarkably, translation of NaRpoTp mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis and the Nicotiana RpoTp polymerase, N. advena RpoTp provides another example for a plant mRNA that is exclusively translated from a non-AUG codon. In contrast to the RpoT of the lycophyte Selaginella and those of the moss Physcomitrella, which are according to phylogenetic analyses in sister positions to all other phage-type polymerases of angiosperms, the Nuphar RpoTs clustered with the well separated clades of mitochondrial (NaRpoTm1 and NaRpoTm2) and plastid (NaRpoTp) polymerases.
CONCLUSIONS: Nuphar advena encodes two mitochondrial and one plastid phage-type RNAP. Identification of a plastid-localized phage-type RNAP in this basal angiosperm, orthologous to all other RpoTp enzymes of flowering plants, suggests that the duplication event giving rise to a nuclear gene-encoded plastid RNA polymerase, not present in lycopods, took place after the split of lycopods from all other tracheophytes. A dual-targeted mitochondrial and plastididal RNA polymerase (RpoTmp), as present in eudicots but not monocots, was not detected in Nuphar suggesting that its occurrence is an evolutionary novelty of eudicotyledonous plants like Arabidopsis.
PMID: 21134269 [PubMed - as supplied by publisher]
Wound Repair Regen. 2010 Dec 6. doi: 10.1111/j.1524-475X.2010.00642.x. [Epub ahead of print]
Bioactive peptides derived from vascular endothelial cell extracellular matrices promote microvascular morphogenesis and wound healing in vitro.
Demidova-Rice TN, Geevarghese A, Herman IM.
Graduate Program in Cellular and Molecular Physiology, Center for Innovations in Wound Healing Research, Sackler School of Graduate Biomedical Sciences, School of Medicine, Tufts University, Boston, Massachusetts.
Abstract
Studies in our laboratory indicate that collagenase from Clostridium histolyticum promotes endothelial cell and keratinocyte responses to injury in vitro and wound healing in vivo. We postulate that matrix degradation by Clostridial collagenase creates bioactive fragments that can stimulate cellular responses to injury and angiogenesis. To test this hypothesis, we performed limited digestion of defined capillary-endothelial-derived extracellular matrices using purified human or bacterial collagenases. Immunoprecipitation with antibodies recognizing collagens I, II, III, IV, and V, followed by mass spectrometry reveals the presence of unique fragments in bacterial, but not human-enzyme-digested matrix. Results show that there are several bioactive peptides liberated from Clostridial collagenase-treated matrices, which facilitate endothelial responses to injury, and accelerate microvascular remodeling in vitro. Fragments of collagen IV, fibrillin-1, tenascin X, and a novel peptide created by combining specific amino acids contained within fibrillin 1 and tenascin X each have profound proangiogenic properties. The peptides used at 10-100 nM increase rates of microvascular endothelial cell proliferation by up to 47% and in vitro angiogenesis by 200% when compared with serum-stimulated controls. Current studies are aimed at revealing the molecular mechanisms regulating peptide-induced wound healing while extending these in vitro observations using animal modeling.
© 2010 by the Wound Healing Society.PMID: 21134032 [PubMed - as supplied by publisher]
Anat Histol Embryol. 2010 Dec 6. doi: 10.1111/j.1439-0264.2010.01055.x. [Epub ahead of print]
The Small Intestine of the Adult New Hampshire Chicken: an Immunohistochemical Study.
Pirone A, Ding BA, Lenzi C, Baglini A, Giannessi E, Romboli I.
Addresses of authors: Department of Animal Productions, Section of Anatomy, University of Pisa, Pisa, Italy Department of Animal Science, Qing Hai University, 810016 Xining, China Department of Pathological Anatomy, Prophylaxis and Food Hygiene, University of Pisa, Pisa, Italy.
Abstract
With 3 figures SUMMARY: The presence and distribution of glucose-dependent insulinotropic polypeptide or gastric inhibitory polypeptide (GIP), gastric-releasing peptide (GRP) and glucagon immunoreactivity were studied in the small intestine of the New Hampshire chicken using immunohistochemistry. This is the first report of the presence of GIP-immunoreactive (ir) cells in avian small intestine. GIP, GRP and glucagon immunoreactivity was localized in the epithelium of the villi and crypts of the duodenum, jejunum and ileum. In particular, both in the duodenum and in the jejunum immunoreactive endocrine cells to GIP, GRP and glucagon were observed. In the ileum, we noticed GIP-ir and glucagon-ir cells. GRP-ir was found in nerve fibres of all three segments of the small intestine. The distribution of these bioactive agents in the intestinal tract of the chicken suggests that GIP and glucagon may play a role in the enteropancreatic axis in which intestinal peptides modulate pancreas secretion.
© 2010 Blackwell Verlag GmbH.PMID: 21133986 [PubMed - as supplied by publisher]
Br J Pharmacol. 2010 Dec 6. doi: 10.1111/j.1476-5381.2010.01146.x. [Epub ahead of print]
Prolyl oligopeptidase induces angiogenesis both in vitro and in vivo in a novel regulatory manner.
Myöhänen T, Tenorio-Laranga J, Jokinen B, Vázquez-Sánchez R, Moreno-Baylach M, García-Horsman J, Männistö P.
Division of Pharmacology and Toxicology, Faculty of Pharmacy, University of Helsinki, P.O. Box 56 (Viikinkaari 5E), FIN-00014 University of Helsinki, Finland.
Abstract
Background and purpose: A serine protease, prolyl oligopeptidase (POP) has been reported to be involved in the release of the pro-angiogenic tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (Ac-SDKP) from its precursor, 43-mer thymosin β4 (Tß4). Recently, it was shown that both POP activity and the levels of Ac-SDKP are increased in malignant tumours. The aim of this study was to clarify the release of Ac-SDKP, and test if POP and a POP inhibitor, KYP-2047, can affect angiogenesis. Experimental approach: We used HPLC for bioanalytical and an enzyme immunoassay for pharmacological analysis. Angiogenesis of HUVEC was assessed in vitro using a "tube formation" assay and in vivo using a Matrigel plug assay in adult male rats. Moreover, colocalization of POP and blood vessels was studied. Key results: We showed the sequential hydrolysis of Tβ4: the first step hydrolysis by proteases to <30-mer>© 2010 The Authors. British Journal of Pharmacology © 2010 The British Pharmacological Society.
PMID: 21133893 [PubMed - as supplied by publisher]
J Interferon Cytokine Res. 2010 Dec 6. [Epub ahead of print]
Cytokine and Chemokine Responses to Selected Early Secreted Antigenic Target-6 and Culture Filtrate Protein-10 Peptides in Tuberculosis.
Murthy MK, Kaliappan T, Raja A.
Department of Immunology, Tuberculosis Research Centre (ICMR) , Chetput, Chennai, India .
Abstract
Cytokine [tumor necrosis factor-α, interleukin-2 (IL-2), IL-4] and chemokine [regulated upon activation normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein-1] responses to selected early secreted antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) peptides were studied in healthy household contacts and patients with pulmonary tuberculosis (PTB). It was observed that Th1 cytokines and chemokine RANTES positive T cells were elevated in response to the peptides Esp1, Esp6, Cfp6, and Cfp8 in healthy household contacts. IL-4 positive T cells were enhanced by Esp1 and Esp6 in PTB. Monocyte chemoattractant protein-1 positive monocytes increased in response to the peptides Esp1, Esp6, Cfp8, and Cfp9 in PTB. These peptides deserve attention for further immune studies.
PMID: 21133811 [PubMed - as supplied by publisher]
J Aerosol Med Pulm Drug Deliv. 2010 Dec;23(S2):S71-S87.
The Particle has Landed-Characterizing the Fate of Inhaled Pharmaceuticals.
Patton JS, Brain JD, Davies LA, Fiegel J, Gumbleton M, Kim KJ, Sakagami M, Vanbever R, Ehrhardt C.
1 Dance Pharmaceuticals , San Francisco, California.
Abstract
Abstract Although there is a modest body of literature on the absorption of inhaled pharmaceuticals by normal lungs and some limited information from diseased lungs, there is still a surprising lack of mechanistic knowledge about the details of the processes involved. Where are molecules absorbed, what mechanisms are involved, how well are different lung regions penetrated, what are the determinants of metabolism and dissolution, and how best can one retard the clearance of molecules deposited in the lung or induce intracellular uptake by lung cells? Some general principles are evident: (1) small hydrophobic molecules are absorbed very fast (within tens of seconds) usually with little metabolism; (2) small hydrophilic molecules are absorbed fast (within tens of minutes), again with minimal metabolism; (3) very low water solubility of the drug can retard absorption; (4) peptides are rapidly absorbed but are significantly metabolized unless chemically protected against peptidases; (5) larger proteins are more slowly absorbed with variable bioavailabilities; and 6) insulin seems to be best absorbed distally in the lungs while certain antibodies appear to be preferentially absorbed in the upper airways. For local lung disease applications, and some systemic applications as well, many small molecules are absorbed much too fast for convenient and effective therapies. For systemic delivery of peptides and proteins, absorption may sometimes be too fast. Bioavailabilities are often too low for cost-effective and reliable treatments. A better understanding of the determinants of pulmonary drug dissolution, absorption, metabolism, and how to target specific regions and/or cells in the lung will enable safer and more effective inhaled medicines in the future.
PMID: 21133802 [PubMed - as supplied by publisher]
Leuk Lymphoma. 2010 Dec 6. [Epub ahead of print]
Identification of new possible targets for leukemia treatment by kinase activity profiling.
Ter Elst A, Diks SH, Kampen KR, Hoogerbrugge PM, Ruijtenbeek R, Boender PJ, Sikkema AH, Scherpen FJ, Kamps WA, Peppelenbosch MP, de Bont ES.
Department of Pediatric Oncology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
Abstract
To date, the biology of acute leukemia has been unclear, and defining new therapeutic targets without prior knowledge remains complicated. The use of high-throughput techniques would enable us to learn more about the biology of the disease, and make it possible to directly assess a broader range of therapeutic targets. In this study we have identified comprehensive tyrosine kinase activity profiles in leukemia samples using the PamChip® kinase activity profiling system. Strikingly, 31% (44/120) of the detected peptides were active in all three groups of leukemia samples. The recently reported activity of platelet-derived growth factor receptor (PDGFR) and neurotrophic tyrosine kinase receptors (NTRK1 and NTRK2) in leukemia could be appreciated in our array results. In addition, high levels of peptide phosphorylation were demonstrated for peptides related to macrophage stimulating 1 receptor (MST1R). A provisional signal transduction scheme of the common active peptides was constructed and used to specifically select an inhibitor for leukemic blast cell survival assays. As expected, a dose-dependent decrease in leukemic blast cell survival was achieved for all leukemia samples. Our data demonstrate that kinase activity profiling in leukemic samples is feasible and provides novel insights into the pathogenesis of leukemia. This approach can be used for the rapid discovery of potential drug targets.
PMID: 21133721 [PubMed - as supplied by publisher]
Expert Rev Anti Infect Ther. 2010 Dec;8(12):1359-1369.
Vitamin D: emerging roles in infection and immunity.
Department of Otolaryngology-Head and Neck Surgery, Counties-Manukau District Health Board, Auckland, New Zealand and Auckland Audiology, 10 Owens Road, Epsom, Auckland 1023, New Zealand. jbartley@ihug.co.nz.
Abstract
In the preantibiotic era, TB of the skin was treated successfully with UV light. By the 1920s, pulmonary TB was being treated with regular sun exposure. During the last decade, basic laboratory research into the antimicrobial actions of vitamin D has provided new insights into these historical observations. Vitamin D has a critical role in the innate immune system through the production of antimicrobial peptides - particularly cathelicidin. Vitamin D would appear to have an important role in respiratory tract, skin and potentially gut health. A number of autoimmune diseases, including multiple sclerosis, Type I diabetes, systemic lupus erythematosus and rheumatoid arthritis, are associated with vitamin D deficiency. Vitamin D could have an important role in the prevention and possible treatment of these conditions; however, much of the current evidence relates to basic science and epidemiological research. In many situations, appropriate double-blind, randomized controlled trial data to guide clinicians treating infectious and autoimmune disease is still lacking.
PMID: 21133662 [PubMed - as supplied by publisher]
J Proteome Res. 2010 Dec 6. [Epub ahead of print]
Identification of novel proteins from the venom of a cryptic snake Drysdalia coronoides by a combined transcriptomics and proteomics approach.
Chatrath ST, Chapeaurouge A, Lin Q, Lim TK, Dunstan N, Mirtschin P, Kumar P, Kini MR.
Abstract
We have investigated the transcriptome and proteome of the venom of a cryptic Australian elapid snake Drysdalia coronoides. To probe into the transcriptome, we constructed a partial cDNA library from the venom gland of D. coronoides. The proteome of the venom of D. coronoides was explored by tryptic digestion of the crude venom followed by HPLC separation of the resulting peptides and MALDI-TOF/TOF mass spectrometric analysis. Importantly, the tandem MS data of the tryptic peptides of the venom not only confirmed the predicted protein sequences deduced from the transcriptome but also added to our knowledge about the venom composition through identification of two more toxin families. Using both the approaches, we were able to identify proteins belonging to eight different snake venom protein super-families, namely, three-finger toxins, serine protease inhibitors, cysteine rich secretory proteins, phospholipases A2, venom nerve growth factors, snake venom metalloproteases, vespryns and a new family phospholipase B. We also identified three novel proteins belonging to 3FTx super-family.
PMID: 21133350 [PubMed - as supplied by publisher]
Proteomics. 2010 Nov 25. [Epub ahead of print]
A proteome-scale study on in vivo protein N(α)-acetylation using an optimized method.
Zhang X, Ye J, Engholm-Keller K, Højrup P.
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.
Abstract
Protein N-terminal acetylation (N(α)-acetylation) is among the most common modifications in eukaryotes. We previously described a simple method to enrich N(α)-modified peptides using CNBr-activated Sepharose resin. A limitation of this method is that an optimal ratio of sample to resin had to be determined prior to the analysis since Lys-containing N(α)-modified peptides may be lost. To address this problem, we hereby present an optimized method by the introduction of double incubation at pH 6.0. We demonstrate with the optimized method that the N(α)-modified peptides can be enriched regardless of whether ε-NH(2) is present or not, and the sample to resin ratio optimization is no longer necessary. Another improvement was accomplished by the inclusion of the singly charged precursor for MS/MS fragmentation to alleviate the shortcoming of the reduced charge state of N(α)-modified peptides. We employed a duplicate experiment using 80 μg samples each and identified 922 IPI annotated and 103 IPI unannotated acetylated N-termini from 989 proteins, so far the largest acetylated N-termini data set acquired from a tryptic digest. Furthermore, the reproducibility of the N(α)-acetyl proteome approach was evaluated and its complementarity to the regular proteome approach was analyzed. The unexpected coupling of CNBr-activated Sepharose to His-containing peptides via the imidazole group was discovered.
PMID: 21132856 [PubMed - as supplied by publisher]
Proteomics. 2010 Nov 25. [Epub ahead of print]
Combining peptide recognition specificity and context information for the prediction of the 14-3-3-mediated interactome in S. cerevisiae and H. sapiens.
Panni S, Montecchi-Palazzi L, Kiemer L, Cabibbo A, Paoluzi S, Santonico E, Landgraf C, Volkmer-Engert R, Bachi A, Castagnoli L, Cesareni G.
Department of Biology, University of Rome "Tor Vergata", Rome, Italy.
Abstract
Large-scale interaction studies contribute the largest fraction of protein interactions information in databases. However, co-purification of non-specific or indirect ligands, often results in data sets that are affected by a considerable number of false positives. For the fraction of interactions mediated by short linear peptides, we present here a combined experimental and computational strategy for ranking the reliability of the inferred partners. We apply this strategy to the family of 14-3-3 domains. We have first characterized the recognition specificity of this domain family, largely confirming the results of previous analyses, while revealing new features of the preferred sequence context of 14-3-3 phospho-peptide partners. Notably, a proline next to the carboxy side of the phospho-amino acid functions as a potent inhibitor of 14-3-3 binding. The position-specific information about residue preference was encoded in a scoring matrix and two regular expressions. The integration of these three features in a single predictive model outperforms publicly available prediction tools. Next we have combined, by a naïve Bayesian approach, these "peptide features" with "protein features", such as protein co-expression and co-localization. Our approach provides an orthogonal reliability assessment and maps with high confidence the 14-3-3 peptide target on the partner proteins.
PMID: 21132852 [PubMed - as supplied by publisher]
Proteomics. 2010 Nov 25. [Epub ahead of print]
Fragmentation-free LC-MS can identify hundreds of proteins.
Bochet P, Rügheimer F, Guina T, Brooks P, Goodlett D, Clote P, Schwikowski B.
Laboratoire de Biologie Systémique, Département Génomes et Génétique, Institut Pasteur, Paris, France.
Abstract
One of the most common approaches for large-scale protein identification is LC, followed by MS. If more than a few proteins are to be identified, the additional fragmentation of individual peptides has so far been considered as indispensable, and thus, the associated costs, in terms of instrument time and infrastructure, as unavoidable. Here, we present evidence to the contrary. Using a combination of (i) highly accurate and precise mass measurements, (ii) modern retention time prediction, and (iii) a robust scoring algorithm, we were able to identify 257 proteins of Francisella tularensis from a single LC-MS experiment in a fragmentation-free approach (i.e. without experimental fragmentation spectra). This number amounts to 59% of the number of proteins identified in a standard fragmentation-based approach, when executed with the same false discovery rate. Independent evidence supports at least 27 of a set of 31 proteins that were identified only in the fragmentation-free approach. Our results suggest that additional developments in retention time prediction, measurement technology, and scoring algorithms may render fragmentation-free approaches an interesting complement or an alternative to fragmentation-based approaches.
PMID: 21132849 [PubMed - as supplied by publisher]
Eur J Immunol. 2010 Oct 27. [Epub ahead of print]
TLR5 functions as an endocytic receptor to enhance flagellin-specific adaptive immunity.
Letran SE, Lee SJ, Atif SM, Uematsu S, Akira S, McSorley SJ.
Center for Infectious Diseases and Microbiology Translational Research, Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, McGuire Translational Research Facility, University of Minnesota Medical School, Minneapolis, MN, USA.
Abstract
Innate immune activation via TLR induces dendritic cell maturation and secretion of inflammatory mediators, generating favorable conditions for naïve T-cell activation. Here, we demonstrate a previously unknown function for TLR5, namely that it enhances MHC class-II presentation of flagellin epitopes to CD4(+) T cells and is required for induction of robust flagellin-specific adaptive immune responses. Flagellin-specific CD4(+) T cells expanded poorly in TLR5-deficient mice immunized with flagellin, a deficiency that persisted even when additional TLR agonists were provided. Flagellin-specific IgG responses were similarly depressed in the absence of TLR5. In marked contrast, TLR5-deficient mice developed robust flagellin-specific T-cell responses when immunized with processed flagellin peptide. Surprisingly, the adaptor molecule Myd88 was not required for robust CD4(+) T-cell responses to flagellin, indicating that TLR5 enhances flagellin-specific CD4(+) T-cell responses in the absence of conventional TLR signaling. A requirement for TLR5 in generating flagellin-specific CD4(+) T-cell activation was also observed when using an in vitro dendritic cell culture system. Together, these data uncover an Myd88-independent function for dendritic cell TLR5 in enhancing the presentation of peptides to flagellin-specific CD4(+) T cells.
PMID: 21132712 [PubMed - as supplied by publisher]
Ann Biomed Eng. 2010 Dec 4. [Epub ahead of print]
Multifunctional FePt Nanoparticles for Radiation-Guided Targeting and Imaging of Cancer.
Hariri G, Wellons MS, Morris WH 3rd, Lukehart CM, Hallahan DE.
Department of Chemistry, Vanderbilt University, Nashville, TN, 37235, USA.
Abstract
A multifunctional FePt nanoparticle was developed that targets tumor microvasculature via "radiation-guided" peptides, and is detected by both near-infrared (NIR) fluorescence imaging and analytical mass spectrometry methods. Tumor specific binding was first measured by biotinylated peptide linked to fluorophore-conjugated streptavidin. This showed tumor selective binding to tumors using the HVGGSSV peptide. FePt nanoparticles were synthesized sequentially by surface modification with poly(L: )lysine, poly(ethylene) glycol conjugation, and functionalized with HVGGSSV peptide and fluorescent probe Alexa fluor 750. NIR fluorescence imaging and ICP-MS analysis showed significant HVGGSSV-FePt nanoparticle binding to irradiated tumors as compared to unirradiated tumors and controls. Results indicate that multifunctional FePt nanoparticles have potential application for radiation-guided targeting and imaging of cancer.
PMID: 21132370 [PubMed - as supplied by publisher]
1 comment:
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