Celkový extrakt z kôry nadzemnej časti Mahonia aquifolium sa študoval z hľadiska chemickéhozloženia a účinku na komplementový systém. Výsledky ukázali, že celkový extrakt vykazujeantikomplementovú aktivitu na aktiváciu komplementu klasickou cestou, ktorá môže súvisieťs prítomnosťou alkaloidov. Frakcia BBI alkaloidov a izolovaný hlavný protoberberínový alkaloid –berberín sa vyznačujú vyššou antikomplementovou aktivitou v porovnaní s celkovým extraktom.Výsledky ukazujú, že berberín a frakcia so zmesou BBI alkaloidov sa zúčastňujú na imunomodulačnom účinku celkového extraktu M. aquifolium.
Crude extracts obtained from the stem bark of Mahonia aquifolium have been investigated as tothe chemical composition and anticomplementary activity. The results show that their anticomplementary activity is mainly due to the alkaloid components. Especially the BBI alkaloid fraction andberberine showed a strong inhibitory effect on CH50 total hemolytic complement assay. The crudeextract of M. aquifolium was less active than berberine or the fraction BBI alkaloids. The resultsindicate that the fraction of BBI alkaloids and berberine largely account for the immunomodulatoryactivity of the crude extract of M. aquifolium.
Protein phosphorylation was repeatedly shown to be the most dynamic post-translational modification mediated by a huge orchestra of protein kinases and phosphatases. Upon landing on a stigma, pollen grain dehydration and activation are accompanied by changes in protein phosphorylation together with the translation activation of stored mRNAs. To enable studies of the total phosphoproteome, it is usually necessary to apply various enrichment techniques. In this chapter, one of these protocols that worked previously well on tobacco mature pollen is presented in more detail. The method comprises of three basic steps: (1) picking flowers from the flowering tobacco plants (Nicotiana tabacum cv. Samsun), and collection of the shed pollen grains; (2) extraction of total proteins by TCA/acetone; (3) phosphoprotein enrichment by MOAC with aluminum hydroxide matrix. Taken together this protocol describes how to isolate phosphoproteins out of tobacco mature pollen.
- MeSH
- Chromatography, Affinity MeSH
- Phosphoproteins chemistry isolation & purification metabolism MeSH
- Aluminum Hydroxide chemistry MeSH
- Protein Processing, Post-Translational MeSH
- Pollen metabolism MeSH
- Plant Proteins chemistry isolation & purification metabolism MeSH
- Nicotiana metabolism MeSH
- Publication type
- Journal Article MeSH
Protein misfolding has been proposed to be a common pathogenic mechanism in many inborn errors of metabolism including cystathionine β-synthase (CBS) deficiency. In this work, we describe the structural properties of nine CBS mutants that represent a common molecular pathology in the CBS gene. Using thermolysin in two proteolytic techniques, we examined conformation of these mutants directly in crude cell extracts after expression in E. coli. Proteolysis with thermolysin under native conditions appeared to be a useful technique even for very unstable mutant proteins, whereas pulse proteolysis in a urea gradient had limited values for the study of the majority of CBS mutants due to their instability. Mutants in the active core had either slightly increased unfolding (p.A114V, p.E302K and p.G307S) or extensive unfolding with decreased stability (p.H65R, p.T191M, p.I278T and p.R369C). The extent of the unfolding inversely correlated with the previously determined degree of tetrameric assembly and with the catalytic activity. In contrast, mutants bearing aminoacid substitutions in the C-terminal regulatory domain (p.R439Q and p.D444N) had increased global stability with decreased flexibility. This study shows that proteolytic techniques can reveal conformational abnormalities even for CBS mutants that have activity and/or a degree of assembly similar to the wild-type enzyme. We present here a methodological strategy that may be used in cell lysates to evaluate properties of proteins that tend to misfold and aggregate and that may be important for conformational studies of disease-causing mutations in the field of inborn errors of metabolism.
- MeSH
- Time Factors MeSH
- Cystathionine beta-Synthase genetics MeSH
- Protein Denaturation MeSH
- Dimerization MeSH
- Escherichia coli metabolism MeSH
- Kinetics MeSH
- Protein Conformation MeSH
- Humans MeSH
- Urea chemistry MeSH
- Mutation MeSH
- Solvents MeSH
- Protein Folding MeSH
- Protein Structure, Tertiary MeSH
- Thermolysin chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Endogenous opioid peptides serve as potent analgesics through the opioid receptor (OR) activation. However, they often suffer from poor metabolic stability, low lipophilicity, and low blood-brain barrier permeability. Researchers have developed many strategies to overcome the drawbacks of current pain medications and unwanted biological effects produced by the interaction with opioid receptors. Here, we tested multifunctional enkephalin analogs LYS739 (MOR/DOR agonist and KOR partial antagonist) and LYS744 (MOR/DOR agonist and KOR full antagonist) under in vivo conditions in comparison with MOR agonist, morphine. We applied 2D electrophoretic resolution to investigate differences in proteome profiles of crude membrane (CM) fractions isolated from the rat brain cortex and hippocampus exposed to the drugs (10 mg/kg, seven days). Our results have shown that treatment with analog LYS739 induced the most protein changes in cortical and hippocampal samples. The identified proteins were mainly associated with energy metabolism, cell shape and movement, apoptosis, protein folding, regulation of redox homeostasis, and signal transduction. Among these, the isoform of mitochondrial ATP synthase subunit beta (ATP5F1B) was the only protein upregulation in the hippocampus but not in the brain cortex. Contrarily, the administration of analog LYS744 caused a small number of protein alterations in both brain parts. Our results indicate that the KOR full antagonism, together with MOR/DOR agonism of multifunctional opioid ligands, can be beneficial in treating chronic pain states by reducing changes in protein expression levels but retaining analgesic efficacy.
- MeSH
- Analgesics MeSH
- Enkephalins metabolism MeSH
- Hippocampus metabolism MeSH
- Rats MeSH
- Morphine * pharmacology MeSH
- Brain metabolism MeSH
- Analgesics, Opioid pharmacology MeSH
- Receptors, Opioid, mu * metabolism MeSH
- Receptors, Opioid metabolism MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
The fungus Geotrichum candidum 4013 produces two types of lipases (extracellular and cell-bound). Both enzymes were tested for their hydrolytic ability to p-nitrophenyl esters and compounds having a structure similar to the original substrate (triacylglycerols). Higher lipolytic activity of extracellular lipase was observed when triacylglycerols of medium- (C12) and long- (C18) chain fatty acids were used as substrates. Cell-bound lipase preferentially hydrolysed trimyristate (C14). The differences in the abilities of these two enzymes to hydrolyse p-nitrophenyl esters were observed as well. The order of extracellular lipase hydrolysis relation velocity was as follows: p-nitrophenyl decanoate > p-nitrophenyl caprylate > p-nitrophenyl laurate > p-nitrophenyl palmitate > p-nitrophenyl stearate. The cell-bound lipase indicates preference for p-nitrophenyl palmitate. The most striking differences in the ratios between the activity of both lipases (extracellular : cell-bound) towards different fatty acid methyl esters were 2.2 towards methyl hexanoate and 0.46 towards methyl stearate (C18). The Michaelis constant (K(m) ) and maximum reaction rate (V(max) ) for p-nitrophenyl palmitate hydrolysis of cell-bound lipase were significantly higher (K(m) 2.462 mM and V(max) 0.210 U/g/min) than those of extracellular lipase (K(m) 0.406 mM and V(max) 0.006 U/g/min).
- MeSH
- Fungal Proteins chemistry genetics metabolism MeSH
- Geotrichum chemistry enzymology genetics MeSH
- Hydrolysis MeSH
- Kinetics MeSH
- Lipase chemistry genetics metabolism MeSH
- Substrate Specificity MeSH
- Triglycerides chemistry metabolism MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
AGR2 is an oncogenic endoplasmic reticulum (ER)-resident protein disulfide isomerase. AGR2 protein has a relatively unique property for a chaperone in that it can bind sequence-specifically to a specific peptide motif (TTIYY). A synthetic TTIYY-containing peptide column was used to affinity-purify AGR2 from crude lysates highlighting peptide selectivity in complex mixtures. Hydrogen-deuterium exchange mass spectrometry localized the dominant region in AGR2 that interacts with the TTIYY peptide to within a structural loop from amino acids 131-135 (VDPSL). A peptide binding site consensus of Tx[IL][YF][YF] was developed for AGR2 by measuring its activity against a mutant peptide library. Screening the human proteome for proteins harboring this motif revealed an enrichment in transmembrane proteins and we focused on validating EpCAM as a potential AGR2-interacting protein. AGR2 and EpCAM proteins formed a dose-dependent protein-protein interaction in vitro Proximity ligation assays demonstrated that endogenous AGR2 and EpCAM protein associate in cells. Introducing a single alanine mutation in EpCAM at Tyr251 attenuated its binding to AGR2 in vitro and in cells. Hydrogen-deuterium exchange mass spectrometry was used to identify a stable binding site for AGR2 on EpCAM, adjacent to the TLIYY motif and surrounding EpCAM's detergent binding site. These data define a dominant site on AGR2 that mediates its specific peptide-binding function. EpCAM forms a model client protein for AGR2 to study how an ER-resident chaperone can dock specifically to a peptide motif and regulate the trafficking a protein destined for the secretory pathway.
- MeSH
- Epithelial Cell Adhesion Molecule genetics metabolism MeSH
- Humans MeSH
- MCF-7 Cells MeSH
- Peptides metabolism MeSH
- Proteins genetics metabolism MeSH
- Proto-Oncogene Proteins c-mdm2 metabolism MeSH
- Recombinant Proteins metabolism MeSH
- Protein Binding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
INTRODUCTION: Hemojuvelin (Hjv) is a key component of the signaling cascade that regulates liver hepcidin (Hamp) expression. The purpose of this study was to determine Hjv protein levels in mice and rats subjected to iron overload and iron deficiency. METHODS: C57BL/6 mice were injected with iron (200 mg/kg); iron deficiency was induced by feeding of an iron-deficient diet, or by repeated phlebotomies. Erythropoietin (EPO)-treated mice were administered recombinant EPO at 50 U/mouse. Wistar rats were injected with iron (1200 mg/kg), or fed an iron-deficient diet. Hjv protein was determined by immunoblotting, liver samples from Hjv-/- mice were used as negative controls. Mouse plasma Hjv content was determined by a commercial ELISA kit. RESULTS: Liver crude membrane fraction from both mice and rats displayed a major Hjv-specific band at 35 kDa, and a weaker band of 20 kDa. In mice, the intensity of these bands was not changed following iron injection, repeated bleeding, low iron diet or EPO administration. No change in liver crude membrane Hjv protein was observed in iron-treated or iron-deficient rats. ELISA assay for mouse plasma Hjv did not show significant difference between Hjv+/+ and Hjv-/- mice. Liver Hamp mRNA, Bmp6 mRNA and Id1 mRNA displayed the expected response to iron overload and iron deficiency. EPO treatment decreased Id1 mRNA, suggesting possible participation of the bone morphogenetic protein pathway in EPO-mediated downregulation of Hamp mRNA. DISCUSSION: Since no differences between Hjv protein levels were found following various experimental manipulations of body iron status, the results indicate that, in vivo, substantial changes in Hamp mRNA can occur without noticeable changes of membrane hemojuvelin content. Therefore, modulation of hemojuvelin protein content apparently does not represent the limiting step in the control of Hamp gene expression.
- MeSH
- Iron Deficiencies MeSH
- Iron, Dietary metabolism MeSH
- Erythropoietin pharmacology MeSH
- Liver drug effects metabolism MeSH
- Bone Morphogenetic Proteins genetics metabolism MeSH
- Rats MeSH
- Membrane Proteins genetics metabolism MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Rats, Wistar MeSH
- Iron Overload genetics metabolism MeSH
- Signal Transduction drug effects genetics MeSH
- Iron metabolism MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
