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Modified Proteins (modified + protein)

Distribution by Scientific Domains

Life Sciences	37%
Chemistry	37%
Medical Sciences	25%

Selected Abstracts

Identification of Modified Proteins by Mass Spectrometry

IUBMB LIFE, Issue 2 2002
Albert Sickmann
Abstract Because it is obvious that high-throughput genomics do not lead to a molecular description or even a prediction of protein function, modern techniques for protein analysis become increasingly more important. Sequence analysis of proteins and peptides is not limited to the elucidation of the primary structure of a protein. The analysis of posttranslational modifications is an important task of protein chemistry in proteome research. Increased sensitivity in mass spectrometry as a result of more efficient ionization techniques and better detection systems has allowed the stepwise reduction of protein quantity for analysis. Protein spots of 2D-PAGE separated samples are now sufficient for an unequivocal identification of a protein by mass spectrometry. In addition to protein identification, a closer look at posttranslational modifications is now also possible. It is assumed that modifications such as phosphorylation or glycosylation exist on every second protein and that they are important for the protein function. [source]

Identification of domains on the extrinsic 23 kDa protein possibly involved in electrostatic interaction with the extrinsic 33 kDa protein in spinach photosystem II

FEBS JOURNAL, Issue 5 2004
Akihiko Tohri
To elucidate the domains on the extrinsic 23 kDa protein involved in electrostatic interaction with the extrinsic 33 kDa protein in spinach photosystem II, we modified amino or carboxyl groups of the 23 kDa protein to uncharged methyl ester groups with N -succinimidyl propionate or glycine methyl ester in the presence of a water-soluble carbodiimide, respectively. The N -succinimidyl propionate-modified 23 kDa protein did not bind to the 33 kDa protein associated with PSII membranes, whereas the glycine methyl ester-modified 23 kDa protein completely bound. This indicates that positive charges on the 23 kDa protein are important for electrostatic interaction with the 33 kDa protein associated with the PSII membranes. Mapping of the N -succinimidyl propionate-modified sites of the 23 kDa protein was performed using Staphylococcus V8 protease digestion of the modified protein followed by determination of the mass of the resultant peptide fragments with MALDI-TOF MS. The results showed that six domains (Lys11,Lys14, Lys27,Lys38, Lys40, Lys90,Lys96, Lys143,Lys152, Lys166,Lys174) were modified with N -succinimidyl propionate. In these domains, Lys11, Lys13, Lys33, Lys38, Lys143, Lys166, Lys170 and Lys174 were wholly conserved in the 23 kDa protein from 12 species of higher plants. These positively charged lysyl residues on the 23 kDa protein may be involved in electrostatic interactions with the negatively charged carboxyl groups on the 33 kDa protein, the latter has been suggested to be important for the 23 kDa binding [Bricker, T.M. & Frankel, L.K. (2003) Biochemistry42, 2056,2061]. [source]

Histidine and not tyrosine is required for the Peroxide-induced formation of haem to protein cross-linked myoglobin

IUBMB LIFE, Issue 8-9 2007
Brandon J. Reeder
Abstract Peroxide-induced oxidative modifications of haem proteins such as myoglobin and haemoglobin can lead to the formation of a covalent bond between the haem and globin. These haem to protein cross-linked forms of myoglobin and haemoglobin are cytotoxic and have been identified in pathological conditions in vivo. An understanding of the mechanism of haem to protein cross-link formation could provide important information on the mechanisms of the oxidative processes that lead to pathological complications associated with the formation of these altered myoglobins and haemoglobins. We have re-examined the mechanism of the formation of haem to protein cross-link to test the previously reported hypothesis that the haem forms a covalent bond to the protein via the tyrosine 103 residue (Catalano, C. E., Choe, Y. S., Ortiz de Montellano, P. R., J. Biol. Chem. 1989, 10534 - 10541). Comparison of native horse myoglobin, recombinant sperm whale myoglobin and Tyr103 , Phe sperm whale mutant shows that, contrary to the previously proposed mechanism of haem to protein cross-link formation, the absence of tyrosine 103 has no impact on the formation of haem to protein cross-links. In contrast, we have found that engineered myoglobins that lack the distal histidine residue either cannot generate haem to protein cross-links or show greatly suppressed levels of modified protein. Moreover, addition of a distal histidine to myoglobin from Aplysia limacina, that naturally lacks this histidine, restores the haem protein's capacity to generate haem to protein cross-links. The distal histidine is, therefore, vital for the formation of haem to protein cross-link and we explore this outcome. [source]

Astaxanthin, a dietary carotenoid, protects retinal cells against oxidative stress in-vitro and in mice in-vivo

JOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 10 2008
Yoshimi Nakajima
We have investigated whether astaxanthin exerted neuroprotective effects in retinal ganglion cells in-vitro and in-vivo. In-vitro, retinal damage was induced by 24-h hydrogen peroxide (H2O2) exposure or serum deprivation, and cell viability was measured using a WST assay. In cultured retinal ganglion cells (RGC-5, a rat ganglion cell-line transformed using E1A virus), astaxanthin inhibited the neurotoxicity induced by H2O2 or serum deprivation, and reduced the intracellular oxidation induced by various reactive oxygen species (ROS). Furthermore, astaxanthin decreased the radical generation induced by serum deprivation in RGC-5. In mice in-vivo, astaxanthin (100 mg kg,1, p.o., four times) reduced the retinal damage (a decrease in retinal ganglion cells and in thickness of inner plexiform layer) induced by intravitreal N -methyl- d -aspartate (NMDA) injection. Furthermore, astaxanthin reduced the expressions of 4-hydroxy-2-nonenal (4-HNE)-modified protein (indicator of lipid peroxidation) and 8-hydroxy-deoxyguanosine (8-OHdG; indicator of oxidative DNA damage). These findings indicated that astaxanthin had neuroprotective effects against retinal damage in-vitro and in-vivo, and that its protective effects may have been partly mediated via its antioxidant effects. [source]

Metabolism of Maillard reaction products by the human gut microbiota , implications for health

MOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 9 2006
Kieran M. Tuohy
Abstract The human colonic microbiota imparts metabolic versatility on the colon, interacts at many levels in healthy intestinal and systemic metabolism, and plays protective roles in chronic disease and acute infection. Colonic bacterial metabolism is largely dependant on dietary residues from the upper gut. Carbohydrates, resistant to digestion, drive colonic bacterial fermentation and the resulting end products are considered beneficial. Many colonic species ferment proteins but the end products are not always beneficial and include toxic compounds, such as amines and phenols. Most components of a typical Western diet are heat processed. The Maillard reaction, involving food protein and sugar, is a complex network of reactions occurring during thermal processing. The resultant modified protein resists digestion in the small intestine but is available for colonic bacterial fermentation. Little is known about the fate of the modified protein but some Maillard reaction products (MRP) are biologically active by, e. g. altering bacterial population levels within the colon or, upon absorption, interacting with human disease mechanisms by induction of inflammatory responses. This review presents current understanding of the interactions between MRP and intestinal bacteria. Recent scientific advances offering the possibility of elucidating the consequences of microbe-MRP interactions within the gut are discussed. [source]

Suppression of nNOS expression in rat enteric neurones by the receptor for advanced glycation end-products

NEUROGASTROENTEROLOGY & MOTILITY, Issue 5 2006
K. Korenaga
Abstract, Diabetes mellitus results in a loss of neuronal nitric oxide synthase (nNOS) expression in the myenteric plexus but the underlying mechanisms remain unknown. We hypothesized that this may be mediated by advanced glycation end-products (AGEs), a class of modified protein adducts formed by non-enzymatic glycation that interact with the receptor for AGE (RAGE) and which are important in the pathogenesis of other diabetic complications. Whole mount preparations of longitudinal muscles with adherent myenteric plexus (LM-MPs) from the duodenum of adult male rats were exposed to glycated bovines serum albumin (AGE-BSA) or BSA for 24 h. Western blotting, immunohistochemistry and real-time reverse transcriptase polymerase chain reaction (RT-PCR) for mRNA showed a significant reduction in nNOS expression in LM-MPs after exposure to AGE-BSA. NO release, as measured by the Griess reaction, was also significantly reduced by AGE-BSA. A neutralizing antibody against RAGE attenuated the reduction of nNOS protein caused by AGE-BSA. Immunohistochemistry revealed co-localization of RAGE expression with Hu, a marker for neuronal cells but not for S-100, a glial marker. Advanced glycation end-products reduce nNOS expression in the rat myenteric neurones acting via the receptor RAGE. Our results suggest novel pathways for disruption of the nitrergic phenotype in diabetes. [source]

High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids

BIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009
Aaron R. Goerke
Abstract We describe an E. coli -based cell-free system for the production of proteins with a non-natural amino acid (nnAA) incorporated site-specifically (modified protein). The mutant Methanococcus jannaschii tyrosyl-tRNA synthetase (mTyrRS) and tRNATyr pair were used as orthogonal elements. The mTyrRS experienced proteolysis and modified protein yields improved with higher synthetase addition (200,300 µg/mL). Product yields were also improved by increasing levels of total protein to 20 mg protein/mL and available vesicle surface area to 0.5 m2/mL. This new E. coli -based cell-free procedure produced up to 400 µg/mL of eCAT109pAz, 660 µg/mL of eDHFR10pAz, and 210 µg/mL of mDHFR31pAz with p -azido- L -phenylalanine (pAz) incorporated site-specifically at the amber nonsense codon. O -methyl- L -tyrosine and p -acetyl- L -phenylalanine were incorporated by similar protocols. The desired specificity for incorporation of the nnAA by the cell-free system was confirmed. Additionally, the modified proteins were enzymatically active and reactive for copper(I)-catalyzed (3,+,2) cycloadditions (click chemistry). Biotechnol. Bioeng. 2009;102: 400,416. © 2008 Wiley Periodicals, Inc. [source]

The use of transglutaminase in dairy products

INTERNATIONAL JOURNAL OF DAIRY TECHNOLOGY, Issue 1 2006
ELVAN ÖZRENK
Modification of proteins by enzymes such as transglutaminase (TG) has recently become of great interest to food scientists. TG (EC 2.3.2.13) catalyses the post-translational modification of proteins by transamidation of available glutamine residues by the formation of covalent cross-links between glutamine and lysine residues in proteins. It is suggested that TG is a useful tool for the production of dairy products. In this review, properties of TG and its possible use in the manufacture of dairy products are discussed. Aspects covered include reactions catalysed by the enzyme and properties of modified proteins of selected milk proteins and products. [source]

Solid-state glycation of ,-lactoglobulin by lactose and galactose: localization of the modified amino acids using mass spectrometric techniques

JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2004
François Fenaille
Abstract The Maillard reaction is commonly encountered during food processing or storage, and also in human nutrition, hence there is a need for analytical methodologies to identify and characterize the modified proteins. This paper reports specific methods using mass spectrometric techniques to localize protein modifications induced by lactose and galactose on ,-lactoglobulin (,-Lg) under solid-state glycation conditions. The extent of glycation was first determined by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The specific identification of lactose-modified amino acid residues was realized using both NanoESI-MS, NanoESI-MS/MS (neutral loss scanning modes) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) (with and without guanidination of lysine residues) on unfractionated digests. The results indicated that, after 8.25 h of incubation, the lysine residues were the main targets of lactose-induced modification. In addition to the 15 lysine residues, Leu1 (NH2 terminal) and the Arg124 were also found to be modified, thus leading to a total of 17 different modified amino acid residues (versus 15 found by LC/ESI-MS measurement). In a second set of experiments, different strategies consisting of constant neutral loss and precursor ion scanning were compared to characterize galactose-induced modifications. Owing to the high level of ,-Lg glycation, the combined use of these different strategies appeared to be necessary for determining the galactose-modified sites after 8.25 h of incubation. Thus, among the 22 galactose adducts deduced from the LC/ESI-MS measurement, apart from the N-terminal and classical lysine residues, we also observed a few arginine residues (Arg40, Arg124 and Arg148) that were modified, and also dialkylations on specific lysine residues (Lys47, Lys75). Copyright © 2003 John Wiley & Sons, Ltd. [source]

Mass spectrometry for the study of protein glycation in disease

MASS SPECTROMETRY REVIEWS, Issue 5 2006
Toshimitsu Niwa
Abstract The structural elucidation of advanced glycation end-product (AGE)-modified proteins and quantitative analysis of free AGEs have been successfully performed, by use of mass spectrometry (MS) in plasma and tissues of patients with AGE-related diseases, such as diabetes mellitus, uremia, cataract, and liver cirrhosis. Matrix-assisted laser desorption/ionization (MALDI)-MS made it possible to directly analyze the AGE-modified proteins such as albumin and IgG. However, because the direct structural analysis of intact AGE-modified proteins is often not easy due to the formation of broad and poorly resolved peaks, peptide mapping after enzymatic hydrolysis was introduced into the analysis of AGE-modified proteins and the site-specific analysis of defined AGEs by MALDI-MS. Liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) has been employed not only for the structural elucidation of enzymatically hydrolyzed AGEs-modified peptides but also for simultaneous quantification of free AGEs in plasma and tissues of patients. Based on many studies that use MS for the analysis of AGEs, there is no doubt as to the important role of protein-linked AGEs in several diseases. © 2006 Wiley-Liss, Inc. [source]

Intervention strategies to inhibit protein carbonylation by lipoxidation-derived reactive carbonyls

MEDICINAL RESEARCH REVIEWS, Issue 6 2007
Giancarlo Aldini
Abstract Protein carbonylation induced by reactive carbonyl species (RCS) generated by peroxidation of polyunsaturated fatty acids plays a significant role in the etiology and/or progression of several human diseases, such as cardiovascular (e.g., atherosclerosis, long-term complications of diabetes) and neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, and cerebral ischemia). Most of the biological effects of intermediate RCS, mainly ,,,-unsaturated aldehydes, di-aldehydes, and keto-aldehydes, are due to their capacity to react with the nucleophilic sites of proteins, forming advanced lipoxidation end-products (ALEs). Because of the emerging deleterious role of RCS/protein adducts in several human diseases, different potential therapeutic strategies have been developed in the last few years. This review sheds focus on fundamental studies on lipid-derived RCS generation, their biological effects, and their reactivity with proteins, with particular emphasis to 4-hydroxy- trans -2-nonenal (HNE)-, acrolein (ACR)-, malondialdehyde (MDA)-, and glyoxal (GO)-modified proteins. It also discusses the recently developed pharmacological approaches for the management of chronic diseases in which oxidative stress and RCS formation are massively involved. Inhibition of ALE formation, based on carbonyl-sequestering agents, seems to be the most promising pharmacological tool and is reviewed in detail. © 2006 Wiley Periodicals, Inc. Med Res Rev, 27, No. 6, 817,868, 2007 [source]

Renal carcinogenesis induced by ferric nitrilotriacetate in mice, and protection from it by Brazilian propolis and Artepillin C

PATHOLOGY INTERNATIONAL, Issue 9 2000
Tetsuo Kimoto
The protective effect of Brazilian propolis and its extract Artepillin C against ferric nitrilotriacetate (Fe-NTA)-induced renal lipid peroxidation and carcinogenesis was studied in male ddY mice. Fe-NTA-induced renal lipid peroxidation leads to a high incidence of renal cell carcinoma (RCC) in mice. Administration of propolis by gastric intubation 2 h before or Artepillin C at either the same time, 2 h, or 5 h before the intraperitoneal injection of Fe-NTA (7 mg Fe/kg) effectively inhibited renal lipid peroxidation. This was evaluated from the measurement of renal thiobarbituric acid-reactive substances (TBARS) or histochemical findings of 4-hydroxy-2-nonenal (4-HNE)-modified proteins and 8-hydroxy-2, -deoxyguanosine (8-OHdG). Repeated injection of Fe-NTA (10 mg Fe/kg per day, twice a week for a total of 16 times in 8 weeks) caused subacute nephrotoxicity as revealed by necrosis and pleomorphic large nuclear cells in the renal proximal tubules, and gave rise to RCC 12 months later. A protective effect from carcinogenicity was observed in mice given propolis or Artepillin C. Furthermore, the mice given Fe-NTA only developed multiple cysts composed of precancerous lesions with multilayered and proliferating large atypical cells. Mice treated with propolis and Artepillin C also had cysts, but these were dilated and composed of flat cells. These results suggest that propolis and Artepillin C prevent oxidative renal damage and the carcinogenesis induced by Fe-NTA in mice. [source]

Detection of Modified Tyrosines as an Inflammation Marker in a Photo-aged Skin Model

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2007
Yukiko Ishitsuka
Reactive nitrogen species, produced during the process of inflammation induced by various factors including UV radiation, modify amino acids in crucial proteins. It is assumed that skin tissue is more likely to be modified, as it is located at the outer layer of a body that is exposed to UV radiation on a daily basis. To investigate the influence of the modified tyrosine on UV-exposed skin, we detected the nitrotyrosine or halogenated tyrosine and dityrosine in photo-aged model mice. The back skin of mice was exposed to a dose of 10 J cm,2 day,1 every day for 15 weeks. Samples exhibiting typical symptoms of photo aging were provided to the immunofluorescence study. The quantification of modified proteins was accomplished through a chemical analytical method known as HPLC-tandem mass spectrometry. Analysis of the irradiated skin samples showed that all modified tyrosine except nitrotyrosine demonstrated statistically significant increases. The molecular weights of major modified proteins, confirmed as 25,50 kDa, were measured using Western blot analysis with an anti-nitrotyrosine antibody. Furthermore, the immunofluorescence study verified that the localization of myeloperoxidase conformed to that of nitrotyrosine. This result suggests that the modified tyrosine was produced during the process of inflammation by UV irradiation. In this study, we used a low dose of UV irradiation to which we are exposed in daily life. Our results suggest that UV exposure in daily life may induce the production of modified tyrosines and skin aging. [source]

Selective analysis of phosphopeptides within a protein mixture by chemical modification, reversible biotinylation and mass spectrometry

RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 16 2001
Maceij Adamczyk
A new method combining chemical modification and affinity purification is described for the characterization of serine and threonine phosphopeptides in proteins. The method is based on the conversion of phosphoserine and phosphothreonine residues to S -(2-mercaptoethyl)cysteinyl or ,-methyl- S -(2-mercaptoethyl)cysteinyl residues by ,-elimination/1,2-ethanedithiol addition, followed by reversible biotinylation of the modified proteins. After trypsin digestion, the biotinylated peptides were affinity-isolated and enriched, and subsequently subjected to structural characterization by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Database searching allowed for automated identification of modified residues that were originally phosphorylated. The applicability of the method is demonstrated by the identification of all known phosphorylation sites in a mixture of ,-casein, ,-casein, and ovalbumin. The technique has potential for adaptations to proteome-wide analysis of protein phosphorylation. Copyright © 2001 John Wiley & Sons, Ltd. [source]

High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids

Protein Targets of Oxidative Damage in Human Neurodegenerative Diseases with Abnormal Protein Aggregates

BRAIN PATHOLOGY, Issue 2 2010
Anna Martínez
Abstract Human neurodegenerative diseases with abnormal protein aggregates are associated with aberrant post-translational modifications, solubility, aggregation and fibril formation of selected proteins which cannot be degraded by cytosolic proteases, ubiquitin,protesome system and autophagy, and, therefore, accumulate in cells and extracellular compartments as residual debris. In addition to the accumulation of "primary" proteins, several other mechanisms are involved in the degenerative process and probably may explain crucial aspects such as the timing, selective cellular vulnerability and progression of the disease in particular individuals. One of these mechanisms is oxidative stress, which occurs in the vast majority of, if not all, degenerative diseases of the nervous system. The present review covers most of the protein targets that have been recognized as modified proteins mainly using bidimensional gel electrophoresis, Western blotting with oxidative and nitrosative markers, and identified by mass spectrometry in Alzheimer disease; certain tauopathies such as progressive supranuclear palsy, Pick disease, argyrophilic grain disease and frontotemporal lobar degeneration linked to mutations in tau protein, for example, FTLD-tau, Parkinson disease and related ,-synucleinopathies; Huntington disease; and amyotrophic lateral sclerosis, together with related animal and cellular models. Vulnerable proteins can be mostly grouped in defined metabolic pathways covering glycolysis and energy metabolism, cytoskeletal, chaperoning, cellular stress responses, and members of the ubiquitin,proteasome system. Available information points to the fact that vital metabolic pathways are hampered by protein oxidative damage in several human degenerative diseases and that oxidative damage occurs at very early stages of the disease. Yet parallel functional studies are limited and further work is needed to document whether protein oxidation results in loss of activity and impaired performance. A better understanding of proteins susceptible to oxidation and nitration may serve to define damaged metabolic networks at early stages of disease and to advance therapeutic interventions to attenuate disease progression. [source]