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991.
Adrien W. Schmid Diego Chiappe V��r��ne Pignat Valerie Grimminger Ivan Hang Marc Moniatte Hilal A. Lashuel 《The Journal of biological chemistry》2009,284(19):13128-13142
Tissue transglutaminase (tTG) has been implicated in the pathogenesis of
Parkinson disease (PD). However, exactly how tTG modulates the structural and
functional properties of α-synuclein (α-syn) and contributes to
the pathogenesis of PD remains unknown. Using site-directed mutagenesis
combined with detailed biophysical and mass spectrometry analyses, we sought
to identify the exact residues involved in tTG-catalyzed cross-linking of
wild-type α-syn and α-syn mutants associated with PD. To better
understand the structural consequences of each cross-linking reaction, we
determined the effect of tTG-catalyzed cross-linking on the oligomerization,
fibrillization, and membrane binding of α-syn in vitro. Our
findings show that tTG-catalyzed cross-linking of monomeric α-syn
involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed
cross-linked monomeric α-syn composed of either wild-type or Gln →
Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping
by mass spectrometry. Using this approach, we identified the glutamine and
lysine residues involved in tTG-catalyzed intramolecular cross-linking of
α-syn. These studies demonstrate for the first time that
Gln79 and Gln109 serve as the primary tTG reactive
sites. Mutating both residues to asparagine abolishes tTG-catalyzed
cross-linking of α-syn and tTG-induced inhibition of α-syn
fibrillization in vitro. To further elucidate the sequence and
structural basis underlying these effects, we identified the lysine residues
that form isopeptide bonds with Gln79 and Gln109. This
study provides mechanistic insight into the sequence and structural basis of
the inhibitory effects of tTG on α-syn fibrillogenesis in vivo,
and it sheds light on the potential role of tTG cross-linking on modulating
the physiological and pathogenic properties of α-syn.Parkinson disease
(PD)2 is a progressive
movement disorder that is caused by the loss of dopaminergic neurons in the
substantia nigra, the part of the brain responsible for controlling movement.
Clinically, PD is manifested in symptoms that include tremors, rigidity, and
difficulty in initiating movement (bradykinesia). Pathologically, PD is
characterized by the presence of intraneuronal, cytoplasmic inclusions known
as Lewy bodies (LB), which are composed primarily of the protein
“α-synuclein” (α-syn)
(1) and are seen in the
post-mortem brains of PD patients with the sporadic or familial forms of the
disease (2). α-Syn is a
presynaptic protein of 140 residues with a “natively” unfolded
structure (3). Three missense
point mutations in α-syn (A30P, E46K, and A53T) are associated with the
early-onset, dominant, inherited form of PD
(4,
5). Moreover, duplication or
triplication of the α-syn gene has been linked to the familial
form of PD, suggesting that an increase in α-syn expression is
sufficient to cause PD. Together, these findings suggest that α-syn
plays a central role in the pathogenesis of PD.The molecular and cellular determinants that govern α-syn
oligomerization and fibrillogenesis in vivo remain poorly understood.
In vitro aggregation studies have shown that the mutations associated
with PD (A30P, E46K, and A53T) accelerate α-syn oligomerization, but
only E46K and A53T α-syn show higher propensity to fibrillize than
wild-type (WT) α-syn
(6-8).
This suggests that oligomerization, rather than fibrillization, is linked to
early-onset familial PD (9).
Our understanding of the molecular composition and biochemical state of
α-syn in LBs has provided important clues about protein-protein
interactions and post-translational modifications that may play a role in
modulating oligomerization, fibrillogenesis, and LB formation of the protein.
In addition to ubiquitination
(10), phosphorylation
(11,
12), nitration
(13,
14), and C-terminal truncation
(15,
16), analysis of post-mortem
brain tissues from PD and Lewy bodies in dementia patients has confirmed the
colocalization of tissue transglutaminase (tTG)-catalyzed cross-linked
α-syn monomers and higher molecular aggregates in LBs within
dopaminergic neurons (17,
18). Tissue transglutaminase
catalyzes a calcium-dependent transamidating reaction involving glutamine and
lysine residues, which results in the formation of a covalent cross-link via
ε-(γ-glutamyl) lysine bonds
(Fig. 2F). To date,
seven different isoforms of tTGs have been reported, of which only tTG2 seems
to be expressed in the human brain
(19), whereas tTG1 and tTG3
are more abundantly found in stratified squamous epithelia
(20). Subsequent
immuno-histochemical, colocalization, and immunoprecipitation studies have
shown that the levels of tTG and cross-linked α-syn species are
increased in the substantia nigra of PD brains
(17). These findings, combined
with the known role of tTG in cross-linking and stabilizing bimolecular
assemblies, led to the hypothesis that tTG plays an important role in the
initiation and propagation of α-syn fibril formation and that it
contributes to fibril stability in LBs. This hypothesis was initially
supported by in vitro studies demonstrating that tTG catalyzes the
polymerization of the α-syn-derived non-amyloid component (NAC) peptide
via intermolecular covalent cross-linking of residues Gln79 and
Lys80 (21) and by
other studies suggesting that tTG promotes the fibrillization of amyloidogenic
proteins implicated in the pathogenesis of other neurodegenerative diseases
such as Alzheimer disease, supranuclear palsy, Huntington disease, and other
polyglutamine diseases
(22-24).
However, recent in vitro studies with full-length α-syn have
shown that tTG catalyzes intramolecular cross-linking of monomeric α-syn
and inhibits, rather than promotes, its fibrillization in vitro
(25,
26). The structural basis of
this inhibitory effect and the exact residues involved in tTG-mediated
cross-linking of α-syn, as well as structural and functional
consequences of these modifications, remain poorly understood.Open in a separate windowFIGURE 2.tTG-catalyzed cross-linking of α-syn involves one to three
intramolecular cross-links. A-C, MALDI-TOF/TOF analysis of native
(—) and cross-linked (- - -) α-syn, showing that most
tTG-catalyzed cross-linking products of WT or disease-associated mutant forms
of α-syn are intramolecularly linked (predominant peak with two
cross-links), and up to three intramolecular cross-links can occur (left
shoulder). The abbreviations M and m/cl are
used to designate native and cross-linked α-synuclein, respectively.
D and E, kinetic analysis of α-syn (A30P)
cross-linking monitored by MALDI-TOF and SDS-PAGE. F, schematic
depiction of the tTG-catalyzed chemical reaction (isodipeptide formation)
between glutamine and lysine residues.In this study, we have identified the primary glutamine and lysine residues
involved in tTG-catalyzed, intramolecularly cross-linked monomeric α-syn
and investigated how cross-linking these residues affects the oligomerization,
fibrillization, and membrane binding of α-syn in vitro. Using
single-site mutagenesis and mass spectrometry applied to exhaustive
proteolytic digests of native and cross-linked monomeric α-syn, we
identified Gln109 and Gln79 as the major tTG substrates.
We demonstrate that the altered electrophoretic mobility of the
intramolecularly cross-linked α-syn in SDS-PAGE occurs as a result of
tTG-catalyzed cross-linking of Gln109 to lysine residues in the N
terminus of α-syn, which leads to the formation of more compact
monomers. Consistent with previous studies, we show that intramolecularly
cross-linked α-syn forms off-pathway oligomers that are distinct from
those formed by the wild-type protein and that do not convert to fibrils
within the time scale of our experiments (3-5 days). We also show that
membrane-bound α-syn is a substrate of tTG and that intramolecular
cross-linking does not interfere with the ability of monomeric α-syn to
adopt an α-helical conformation upon binding to synthetic membranes.
These studies provide novel mechanistic insight into the sequence and
structural basis of events that allow tTG to inhibit α-syn
fibrillogenesis, and they shed light on the potential role of tTG-catalyzed
cross-linking in modulating the physiological and pathogenic properties of
α-syn. 相似文献
992.
Martin Lee Miller Boumediene Soufi Carsten Jers Nikolaj Blom Boris Macek Ivan Mijakovic Dr. 《Proteomics》2009,9(1):116-125
There is ample evidence for the involvement of protein phosphorylation on serine/threonine/tyrosine in bacterial signaling and regulation, but very few exact phosphorylation sites have been experimentally determined. Recently, gel‐free high accuracy MS studies reported over 150 phosphorylation sites in two bacterial model organisms Bacillus subtilis and Escherichia coli. Interestingly, the analysis of these phosphorylation sites revealed that most of them are not characteristic for eukaryotic‐type protein kinases, which explains the poor performance of eukaryotic data‐trained phosphorylation predictors on bacterial systems. We used these large bacterial datasets and neural network algorithms to create the first bacteria‐specific protein phosphorylation predictor: NetPhosBac. With respect to predicting bacterial phosphorylation sites, NetPhosBac significantly outperformed all benchmark predictors. Moreover, NetPhosBac predictions of phosphorylation sites in E. coli proteins were experimentally verified on protein and site‐specific levels. In conclusion, NetPhosBac clearly illustrates the advantage of taxa‐specific predictors and we hope it will provide a useful asset to the microbiological community. 相似文献
993.
Mihály L. Kecskés Michael T. Rose Erwin Michel Benôit Lauby Malala Rakotondrainibe Andrea Casteriano Attila Palágyi Anaëlle Moutouvirin Sébastien Elter Romain Guillas Ganisan Krishnen Ivan R. Kennedy 《Engineering in Life Science》2009,9(6):431-436
Rapid, specific techniques are essential to monitor the quality of inoculant plant growth‐promoting strains at all stages of manufacture from starter culture to the final product in its carrier medium. In this study, colony immunoblotting was evaluated for the specific detection and enumeration of Citrobacter freundii, one component of a Vietnamese commercial inoculant plant growth‐promoting product used to improve the yield and nutrient efficiency of paddy rice. For quality control of either sterilised or unsterilised carrier media in commercial products colony immunoblotting proved to be a promising tool. Furthermore, it was possible using this technique to measure the survival of this strain in soil and the rhizosphere. 相似文献
994.
995.
Barbara Lazzari Andrea Caprera Alessandro Cestaro Ivan Merelli Marcello Del Corvo Paolo Fontana Luciano Milanesi Riccardo Velasco Alessandra Stella 《BMC plant biology》2009,9(1):82
Background
Two complete genome sequences are available for Vitis vinifera Pinot noir. Based on the sequence and gene predictions produced by the IASMA, we performed an in silico detection of putative microRNA genes and of their targets, and collected the most reliable microRNA predictions in a web database. The application is available at . 相似文献996.
Ivan Dimitrijevic Marie-Louise Edvinsson Qingwen Chen Malin Malmsjö Per-Ola Kimblad Lars Edvinsson 《BMC cardiovascular disorders》2009,9(1):1-11
Background
Endothelin-1 and angiotensin II are strong vasoconstrictors. Patients with ischemic heart disease have elevated plasma levels of endothelin-1 and angiotensin II and show increased vascular tone. The aim of the present study was to examine the endothelin and angiotensin II receptor expression in subcutaneous arteries from patients with different degrees of ischemic heart disease.Methods
Subcutaneous arteries were obtained, by biopsy from the abdomen, from patients undergoing coronary artery bypass graft (CABG) surgery because of ischemic heart disease (n = 15), patients with angina pectoris without established myocardial infarction (n = 15) and matched cardiovascular healthy controls (n = 15). Endothelin type A (ETA) and type B (ETB), and angiotensin type 1 (AT1) and type 2 (AT2) receptors expression and function were examined using immunohistochemistry, Western blot and in vitro pharmacology.Results
ETA and, to a lesser extent, ETB receptor staining was observed in the healthy vascular smooth muscle cells. The level of ETB receptor expression was higher in patients undergoing CABG surgery (250% ± 23%; P < 0.05) and in the patients with angina pectoris (199% ± 6%; P < 0.05), than in the healthy controls (100% ± 28%). The data was confirmed by Western blotting. Arteries from CABG patients showed increased vasoconstriction upon administration of the selective ETB receptor agonist sarafotoxin S6c, compared to healthy controls (P < 0.05). No such difference was found for the ETA receptors. AT1 and, to a lesser extent, AT2 receptor immunostaining was seen in the vascular smooth muscle cells. The level of AT1 receptor expression was higher in both the angina pectoris (128% ± 25%; P < 0.05) and in the CABG patients (203% ± 41%; P < 0.05), as compared to the healthy controls (100% ± 25%). The increased AT1 receptor expression was confirmed by Western blotting. Myograph experiment did however not show any change in vasoconstriction to angiotensin II in CABG patients compared to healthy controls (P = n.s).Conclusion
The results demonstrate, for the first time, upregulation of ETB and AT1 receptors in vascular smooth muscle cells in ischemic heart disease. These receptors may play a role in the pathophysiology of ischemic heart disease and could provide important targets for pharmaceutical interventions. 相似文献997.
Background
A salient purpose for studying gene regulatory networks is to derive intervention strategies, the goals being to identify potential drug targets and design gene-based therapeutic intervention. Optimal stochastic control based on the transition probability matrix of the underlying Markov chain has been studied extensively for probabilistic Boolean networks. Optimization is based on minimization of a cost function and a key goal of control is to reduce the steady-state probability mass of undesirable network states. Owing to computational complexity, it is difficult to apply optimal control for large networks. 相似文献998.
Tamara Smutná Vera L. Gon?alves Lígia M. Saraiva Jan Tachezy Miguel Teixeira Ivan Hrdy 《Eukaryotic cell》2009,8(1):47-55
Trichomonas vaginalis is one of a few eukaryotes that have been found to encode several homologues of flavodiiron proteins (FDPs). Widespread among anaerobic prokaryotes, these proteins are believed to function as oxygen and/or nitric oxide reductases to provide protection against oxidative/nitrosative stresses and host immune responses. One of the T. vaginalis FDP homologues is equipped with a hydrogenosomal targeting sequence and is expressed in the hydrogenosomes, oxygen-sensitive organelles that participate in carbohydrate metabolism and assemble iron-sulfur clusters. The bacterial homologues characterized thus far have been dimers or tetramers; the trichomonad protein is a dimer of identical 45-kDa subunits, each noncovalently binding one flavin mononucleotide. The protein reduces dioxygen to water but is unable to utilize nitric oxide as a substrate, similarly to its closest homologue from another human parasite Giardia intestinalis and related archaebacterial proteins. T. vaginalis FDP is able to accept electrons derived from pyruvate or NADH via ferredoxin and is proposed to play a role in the protection of hydrogenosomes against oxygen.Flavodiiron proteins (FDPs) constitute a recently established superfamily of soluble enzymes, thus far exclusively found in anaerobic and facultative aerobic organisms (2, 19, 54). Originally, the function ascribed to these proteins was the reduction of molecular oxygen to water as reported for Desulfovibrio gigas rubredoxin:oxygen oxidoreductase, the first thoroughly characterized protein of this type. This protein was found to utilize electrons derived from glycolysis for safe, four-electron reduction of dioxygen, thus protecting the anaerobic bacterium from the deleterious effects of oxidative stress (19). Later, some of these proteins were also shown to be involved in the reduction of nitric oxide in addition to their oxygen-reducing activity, thereby probably protecting the microbial organism against NO released during the immune response of the higher eukaryote host. The ratio of FDP activity toward oxygen and NO may differ substantially in various organisms; in some cases, FDP is almost exclusively reactive with oxygen, in others it is reactive with NO (20, 21, 43).FDPs are modular proteins, with flavodoxin-like and metallo-β-lactamase-like domains as their core modules. This two-domain structure is found in the simplest and most common members of the family, named class A FDPs. These proteins are the terminal elements of a multicomponent electron transporting chain that uses the reducing power of NAD(P)H to reduce and detoxify dioxygen and/or nitric oxide (41). Proximal electron donors to most class A FDPs are soluble electron transfer proteins. In the class A FDP rubredoxin:oxygen oxidoreductase from the sulfate-reducing bacterium Desulfovibrio gigas, the electron donor is a small protein, rubredoxin, that itself is reduced by an NADH:rubredoxin oxidoreductase (9, 10, 22). Besides rubredoxin, roles for other iron-sulfur flavoproteins in electron transport to FDPs have been suggested in several Archaea (41); coenzyme F420H2 is the electron donor for the FDP in the methanogenic archaeon Methanothermobacter marburgensis (44). The members of other FDP classes have additional domains fused to the C terminus that participate in electron transfer from the ultimate donor molecule [NAD(P)H] to the terminal electron acceptor (41).While originally believed to be restricted solely to prokaryotes, recent progress in genome sequencing projects have revealed homologous protein sequences in the genomes of several “amitochondriate” anaerobic protists, mostly with parasitic lifestyles, such as Trichomonas, Giardia, Entamoeba, Spironucleus, and a free-living Mastigamoeba (1, 2, 33, 42). Giardia intestinalis is the only eukaryotic organism to have had data on its FDP published recently. In line with what is known for the prokaryotic homologues, the giardial protein was shown to possess high oxygen (but not NO)-reducing activity and was therefore proposed to participate in protection against oxidative stress (13).Trichomonas vaginalis is an anaerobic (or microaerophilic) protozoan parasite causing human trichomoniasis, the most common nonviral sexually transmitted infection (38), for which oxygen concentrations higher than those encountered in situ in the vagina (i.e., concentrations above ∼60 μM) are toxic (17). The glucose metabolism of T. vaginalis is compartmentalized; while the reactions of classical glycolysis producing lactate, as well as the branch resulting in the formation of glycerol (8, 48) occur in the cytosol, a substantial portion of glycolytic carbon is diverted into the hydrogenosome, a mitochondrion-related organelle where the reactions of extended glycolysis produce additional ATP by oxidative decarboxylation of pyruvate (47, 48). Typical in the trichomonad hydrogenosome is the presence of the iron-sulfur (FeS) cluster-containing enzymes pyruvate:ferredoxin oxidoreductase (PFOR), hydrogenase, and the electron carrier ferredoxin, which are involved in the generation of molecular hydrogen using electrons released from pyruvate (36). PFOR and hydrogenase are highly oxygen-sensitive enzymes (29, 32), and it is likely that the sensitivity of trichomonads to oxygen could at least in part be due to the inactivation of these key hydrogenosomal proteins.T. vaginalis must cope with low oxygen concentrations in its natural environment and, accordingly, possesses defense mechanisms to combat oxidative damage caused by oxygen itself or by reactive oxygen species that arise either enzymatically or when the reduced prosthetic groups of enzymes such as flavins and FeS clusters come into contact with oxygen. Most eukaryotes utilize glutathione as a key redox buffer and antioxidant, but trichomonads lack this and similar thiols (17). Cysteine has been suggested as a major reducing buffer and antioxidant (17), and it is believed that the organism relies upon cytosolic NADH oxidase (reducing oxygen to water) and NADPH oxidase (reducing oxygen to hydrogen peroxide) to prevent the permeation of oxygen into the hydrogenosomes (31). Proteins of the peroxiredoxin cascade (11) are also important for cytosolic peroxide detoxification. The identified defense mechanisms of hydrogenosomes include superoxide dismutase activity (17, 30) and recently found putative peroxidases that might provide protection against peroxides (39), but the protein that was suggested long ago to be responsible for oxygen uptake and detoxification has never been identified (6).We describe here the properties of a class A FDP from T. vaginalis hydrogenosomes and suggest its role in the metabolism of oxygen and protection of the organelle. 相似文献
999.
1000.
Hiromi Hosako Gail S. Martin Marianne Barrier Yian A. Chen Ivan V. Ivanov Philip E. Mirkes 《Birth defects research. Part A, Clinical and molecular teratology》2009,85(6):546-555
BACKGROUND: Neural tube defects (NTDs) are one of the most common human birth defects, with a prevalence of approximately 1 in 1000 live births in the United States. In animal studies, deletion of p53 leads to a significant increase in embryos that exhibit exencephaly. Whereas several studies have closely investigated the morphologic changes of p53‐deficient embryos, no study has reported the molecular‐level alteration in p53‐deficient embryos. Here we attempt to identify genes and microRNAs (miRNAs) modified by deletion of p53 in day 8.5 mouse embryos. METHODS: Mouse embryos from p53 heterozygous crosses were collected, genotyped, and embryos of similar genotype (+/+; +/?; ?/?) were pooled. RNA from the pooled samples was isolated to determine mRNA and miRNA expression levels using Whole Genome Bioarrays and Low Density Arrays, respectively. RESULTS: In p53 ?/? embryos, 388 genes showed statistically significant alteration in gene expression of more than twofold compared to p53 +/+ embryos. Expression of p53 and well known p53 target genes, such as p21 and cyclin G1, were significantly down‐regulated in p53 ?/? embryos. In contrast, expression of other p53 target genes, such as Mdm2, Noxa, and Puma, were unchanged. We also identified six genes (Csk, Itga3, Jarid2, Prkaca, Rarg, and Sall4), known to cause NTDs when deleted, that are also down‐regulated in p53 ?/? embryos. Finally, five miRNAs (mir‐1, mir‐30e‐3p, mir‐142‐3p, mir‐301, and mir‐331) also showed statistically significant alterations in expression levels in p53 ?/? embryos compared to p53 +/+ embryos. Combined analysis of the experimental data using stepwise regression model and two publicly available algorithms identified putative target genes of these miRNAs. CONCLUSIONS: Our data have identified genes and miRNAs that may be involved in the mechanisms underlining NTDs and begin to define the developmental role of p53 in the etiology of NTDs. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc. 相似文献