scholarly journals Association of brain γ-tubulins with αβ-tubulin dimers

2002 ◽  
Vol 365 (3) ◽  
pp. 889-895 ◽  
Author(s):  
Vadym SULIMENKO ◽  
Tetyana SULIMENKO ◽  
Slobodan POZNANOVIC ◽  
Volodymyr NECHIPORUK-ZLOY ◽  
Konrad J. BÖHM ◽  
...  
Keyword(s):  
Post Translational Modification ◽  
Microtubule Nucleation ◽  
Two Dimensional Electrophoresis ◽  
Microtubule Protein ◽  
Brain Extracts ◽  
Polar Orientation ◽  
The Brain ◽  
Dimensional Electrophoresis ◽  
Multiple Charge

γ-Tubulin is necessary for nucleation and polar orientation of microtubules in vivo. The molecular mechanism of microtubule nucleation by γ-tubulin and the regulation of this process are not fully understood. Here we show that there are two γ-tubulin forms in the brain that are present in complexes of various sizes. Large complexes tend to dissociate in the presence of a high salt concentration. Both γ-tubulins co-polymerized with tubulin dimers, and multiple γ-tubulin bands were identified in microtubule protein preparations under conditions of non-denaturing electrophoresis. Immunoprecipitation experiments with monoclonal antibodies against γ-tubulin and α-tubulin revealed interactions of both γ-tubulin forms with tubulin dimers, irrespective of the size of complexes. We suggest that, besides small and large γ-tubulin complexes, other molecular γ-tubulin form(s) exist in brain extracts. Two-dimensional electrophoresis revealed multiple charge variants of γ-tubulin in both brain extracts and microtubule protein preparations. Post-translational modification(s) of γ-tubulins might therefore have an important role in the regulation of microtubule nucleation in neuronal cells.

Polyribosome fractions and protein synthesis in stem rust infected wheat

1986 ◽  
Vol 64 (9) ◽  
pp. 1916-1927 ◽  
Author(s):  
Andrew Greenland ◽  
Michael Shaw
Keyword(s):  
Protein Synthesis ◽  
Stem Rust ◽  
Rust Fungus ◽  
High Molecular Mass ◽  
In Vitro Translation ◽  
Ribosomal Subunits ◽  
Two Dimensional Electrophoresis ◽  
Dimensional Electrophoresis

The effects of infection by stem-rust fungus on polyribosomal RNA fractions and protein synthesis in vitro and in vivo in near-isogenic resistant (Sr6) and susceptible (sr6) lines of wheat were determined. In infected resistant leaves the proportion of ribosomes present as polyribosomes was greater than that in healthy (uninfected) leaves at 1, 3, and 6 days and that in susceptible leaves at 1 and 3 days after inoculation. In the latter there were large increases in the pelletable RNA content (ribosomes, ribosomal subunits, and polyribosomes) and proportion of ribosomes present as polyribosomes from day 6. In vitro translation failed to detect any marked differences in polyribosomal translation products in resistant and susceptible leaves in response to infection. Labelling of polypeptides in vivo and separation by one- and two-dimensional electrophoresis showed that at 1 day after inoculation, two groups of high molecular mass polypeptides (80–96 and 100–110 kDa) were more heavily labelled and two novel polypeptides were present in resistant and susceptible leaves in response to infection. Synthesis of the high molecular weight and two novel polypeptides was maintained in infected resistant leaves up to 6 days after inoculation. In susceptible leaves the amount of radiolabel incorporated into these polypeptides and several proteins prominently labelled in uninfected controls declined rapidly from 3 days after inoculation.

scholarly journals A method for detection of overoxidation of cysteines: peroxiredoxins are oxidized in vivo at the active-site cysteine during oxidative stress

2002 ◽  
Vol 366 (3) ◽  
pp. 777-785 ◽  
Author(s):  
Elsa WAGNER ◽  
Sylvie LUCHE ◽  
Lucia PENNA ◽  
Mireille CHEVALLET ◽  
Alain van DORSSELAER ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Active Site ◽  
Model System ◽  
Enzyme Digestion ◽  
Protein Isoforms ◽  
Two Dimensional Electrophoresis ◽  
Physiological Conditions ◽  
Active Site Cysteine ◽  
Dimensional Electrophoresis

Peroxiredoxins are often encountered as double spots when analysed by two-dimensional electrophoresis. The quantitative balance between these two spots depends on the physiological conditions, and is altered in favour of the acidic variant by oxidative stress for all the peroxiredoxins we could analyse. Using HeLa cells as a model system, we have further analysed the two protein isoforms represented by the two spots for each peroxiredoxin. The use of selected enzyme digestion and MS demonstrated that the acidic variant of all the peroxiredoxins analysed is irreversibly oxidized at the active-site cysteine into cysteine sulphinic or sulphonic acid. Thus, this acidic variant represents an inactivation form of the peroxiredoxins, and provides a useful marker of oxidative damage to the cells.

scholarly journals Protein lactylation induced by neural excitation

2021 ◽  
Author(s):  
Hideo Hagihara ◽  
Hirotaka Shoji ◽  
Hikari Otabi ◽  
Atsushi Toyoda ◽  
Kaoru Katoh ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Monocarboxylate Transporter ◽  
Brain Cells ◽  
Post Translational Modification ◽  
High Potassium ◽  
Neural Excitation ◽  
Lactate Levels ◽  
The Brain ◽  
Prefrontal Cortices

AbstractLactate is known to have diverse roles in the brain at the molecular and behavioral levels under both physiological and pathophysiological conditions, such as learning and memory and regulation of mood. Recently, a novel post-translational modification called lysine lactylation has been found in histone H3 of mouse macrophages, and the lactylation levels paralleled the intracellular lactate levels1. However, it is unknown whether lysine lactylation occurs in brain cells, and if it does, whether lactylation is induced by the stimuli that accompany changes in lactate levels. Herein, we reveal that lysine lactylation in brain cells is regulated by systemic changes in lactate levels, neural excitation, and behaviorally relevant stimuli. Lysine lactylation levels were increased by lactate treatment and by high-potassium-induced depolarization in cultured primary neurons; these increases were attenuated by pharmacological inhibition of monocarboxylate transporter 2 and lactate dehydrogenase, respectively, suggesting that both cell-autonomous and non-cell-autonomous neuronal mechanisms are involved in overall lysine lactylation. In vivo, electroconvulsive stimulation increased lysine lactylation levels in the prefrontal cortices of mice, and its levels were positively correlated with the expression levels of the neuronal activity marker c-Fos on an individual cell basis. In the social defeat stress model of depression in which brain lactate levels increase, lactylation levels were increased in the prefrontal cortices of the defeated mice, which was accompanied by increased c-Fos expression, decreased social behaviors, and increased anxiety-like behaviors, suggesting that stress-induced neuronal excitation may induce lysine lactylation, thereby affecting mood-related behaviors. Further, we identified 63 candidate lysine-lactylated proteins in the mouse cortex and found that lactylation levels in histone H1 increased in response to defeat stress. This study may open up an avenue for exploration of a novel role of neuronal activity-induced lactate mediated by protein lactylation in the brain.

scholarly journals In Vivo Regulation of Rat Epididymal Proteins by Retinoids: Analysis by Two—Dimensional Electrophoresis

1995 ◽  
Vol 35 (3) ◽  
pp. 247-259 ◽  
Author(s):  
C. Astraudo ◽  
A. Lefevre ◽  
F. Boue ◽  
F. Dürr ◽  
C. Finaz
Keyword(s):  
Two Dimensional ◽  
Two Dimensional Electrophoresis ◽  
Dimensional Electrophoresis

scholarly journals Regulation of Galactose Metabolism through the HisK:GalR Two-Component System in Thermoanaerobacter tengcongensis

2010 ◽  
Vol 192 (17) ◽  
pp. 4311-4316 ◽  
Author(s):  
Zhong Qian ◽  
Quanhui Wang ◽  
Wei Tong ◽  
Chuanqi Zhou ◽  
Qian Wang ◽  
...  
Keyword(s):  
Phosphoryl Group ◽  
Regulation Mechanism ◽  
Two Component System ◽  
Galactose Metabolism ◽  
Component System ◽  
Two Dimensional Electrophoresis ◽  
Thermoanaerobacter Tengcongensis ◽  
Two Component ◽  
Dimensional Electrophoresis

ABSTRACT Thermoanaerobacter tengcongensis could utilize galactose as a carbon source via the enzymes encoded by a novel gal operon, whose regulation mechanism has yet to be elucidated. We propose here that the gal operon in T. tengcongensis is regulated through a HisK:GalR two-component system. By using radioactive isotope assay and genetic analysis, we found that the kinase of this system, HisK, is phosphorylated by ATP, and the regulator, GalR, accepts a phosphoryl group during phosphorelay, in which the phosphoryl group at HisK-His-259 is transferred to GalR-Asp-56. Two-dimensional electrophoresis, followed by Western blotting, revealed that phosphorylation status of GalR is uniquely dependent on the galactose stimulus in vivo. Furthermore, DNA pulldown assays demonstrated that the phosphorylated GalR prefers binding to the operator DNA O2 , whereas the unphosphorylated GalR to O1 . A model of HisK:GalR is proposed to explain how galactose mediates the expression of the gal operon in T. tengcongensis.

scholarly journals Synapsin I: an actin-bundling protein under phosphorylation control.

1987 ◽  
Vol 105 (3) ◽  
pp. 1355-1363 ◽  
Author(s):  
T C Petrucci ◽  
J S Morrow
Keyword(s):  
Actin Filaments ◽  
Binding Activity ◽  
Brain Extract ◽  
Synapsin I ◽  
Peptide Fragments ◽  
Molecular Weights ◽  
Brain Extracts ◽  
The Brain ◽  
Precise Role

Synapsin I is a neuronal phosphoprotein comprised of two closely related polypeptides with apparent molecular weights of 78,000 and 76,000. It is found in association with the small vesicles clustered at the presynaptic junction. Its precise role is unknown, although it probably participates in vesicle clustering and/or release. Synapsin I is known to associate with vesicle membranes, microtubules, and neurofilaments. We have examined the interaction of purified phosphorylated and unphosphorylated bovine and human synapsin I with tubulin and actin filaments, using cosedimentation, viscometric, electrophoretic, and morphologic assays. As purified from brain homogenates, synapsin I decreases the steady-state viscosity of solutions containing F-actin, enhances the sedimentation of actin, and bundles actin filaments. Phosphorylation by cAMP-dependent kinase has minimal effect on this interaction, while phosphorylation by brain extracts or by purified calcium- and calmodulin-dependent kinase II reduces its actin-bundling and -binding activity. Synapsin's microtubule-binding activity, conversely, is stimulated after phosphorylation by the brain extract. Two complementary peptide fragments of synapsin generated by 2-nitro-5-thiocyanobenzoic cleavage and which map to opposite ends of the molecule participate in the bundling process, either by binding directly to actin or by binding to other synapsin I molecules. 2-Nitro-5-thiocyanobenzoic peptides arising from the central portion of the molecule demonstrate neither activity. In vivo, synapsin I may link small synaptic vesicles to the actin-based cortical cytoskeleton, and coordinate their availability for release in a Ca++-dependent fashion.

scholarly journals Ribosomes in RNA granules are stalled on mRNA sequences that are consensus sites for FMRP association

2021 ◽  
Author(s):  
Mina N. Anadolu ◽  
Senthilkumar Kailasam ◽  
Konstanze Simbriger ◽  
Jingyu Sun ◽  
Teodora Markova ◽  
...  
Keyword(s):  
Neuronal Development ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Fragile X ◽  
Ribosome Profiling ◽  
Cytoskeletal Proteins ◽  
Rna Granules ◽  
Brain Extracts ◽  
The Brain

AbstractLocal translation in neurons is mediated in part by the reactivation of stalled polysomes. However, the mechanism for stalling of the polysomes is not understood. Stalled polysomes may be enriched within neuronal RNA granules defined by dense collections of compacted ribosomes found in the pellet of sucrose gradients used to separate polysomes from monosomes. We find that this fraction, isolated from P5 rat brains of both sexes, is enriched in proteins implicated in stalled polysome function, such as the fragile X mental retardation protein (FMRP) and Up-frameshift mutation 1 homolog (UPF1). Ribosome profiling of this fraction showed an abundance of footprint reads derived from mRNAs of cytoskeletal proteins implicated in neuronal development and an enrichment of footprint reads on RNA binding proteins. Compared to those usually found in ribosome profiling studies, the footprint reads were more extended on their 3’end and were found in reproducible peaks in the mRNAs. These footprint reads were enriched in motifs previously associated with mRNAs cross-linked to FMRP in vivo, independently linking the ribosomes in the sedimented pellet to the ribosomes associated with FMRP in the cell. The data supports a model in which specific sequences in mRNAs act to stall translation elongation in neurons, attracting FMRP and beginning a process where stalled ribosomes are packaged and transported in RNA granules.Significance StatementThis work finds that neuronal ribosomes in RNA granules are concentrated at consensus sites previously identified through cross-linking FMRP to mRNAs in the brain. This strongly links the compacted ribosomes found in the pellet of sucrose gradients from brain extracts to stalled ribosomes regulated by FMRP and provides important insights into how stalling is accomplished. Many mRNAs important for neurodevelopment are enriched in these ribosomes. These results suggest that many studies on translation in the brain may need to be revised. The larger size of the ribosomal footprints on stalled polysomes and their sedimentation in the pellet of sucrose gradients suggests mRNAs found in these structures have not been assessed in many studies of neuronal translation.

scholarly journals Proteomic Identification of Novel Substrates of a Protein Isoaspartyl Methyltransferase Repair Enzyme

2006 ◽  
Vol 281 (43) ◽  
pp. 32619-32629 ◽  
Author(s):  
Vasanthy Vigneswara ◽  
Jonathan D. Lowenson ◽  
Claire D. Powell ◽  
Matthew Thakur ◽  
Kevin Bailey ◽  
...  
Keyword(s):  
Repair Enzyme ◽  
Knock Out ◽  
Methyl Donor ◽  
Brain Extracts ◽  
Knock Out Mice ◽  
First Time ◽  
The Brain ◽  
Phosphatidylethanolamine Binding Protein ◽  
Mammalian Protein

We report the use of a proteomic strategy to identify hitherto unknown substrates for mammalian protein l-isoaspartate O-methyltransferase. This methyltransferase initiates the repair of isoaspartyl residues in aged or stress-damaged proteins in vivo. Tissues from mice lacking the methyltransferase (Pcmt1-/-) accumulate more isoaspartyl residues than their wild-type littermates, with the most “damaged” residues arising in the brain. To identify the proteins containing these residues, brain homogenates from Pcmt1-/- mice were methylated by exogenous repair enzyme and the radiolabeled methyl donor S-adenosyl-[methyl-3H]methionine. Methylated proteins in the homogenates were resolved by both one-dimensional and two-dimensional electrophoresis, and methyltransferase substrates were identified by their increased radiolabeling when isolated from Pcmt1-/- animals compared with Pcmt1+/+ littermates. Mass spectrometric analyses of these isolated brain proteins reveal for the first time that microtubule-associated protein-2, calreticulin, clathrin light chains a and b, ubiquitin carboxyl-terminal hydrolase L1, phosphatidylethanolamine-binding protein, stathmin, β-synuclein, and α-synuclein, are all substrates for the l-isoaspartate methyltransferase in vivo. Our methodology for methyltransferase substrate identification was further supplemented by demonstrating that one of these methyltransferase targets, microtubule-associated protein-2, could be radiolabeled within Pcmt1-/- brain extracts using radioactive methyl donor and exogenous methyltransferase enzyme and then specifically immunoprecipitated with microtubule-associated protein-2 antibodies to recover co-localized protein with radioactivity. We comment on the functional significance of accumulation of relatively high levels of isoaspartate within these methyltransferase targets in the context of the histological and phenotypical changes associated with the methyltransferase knock-out mice.

Protein synthesis in flax following inoculation with flax rust

1986 ◽  
Vol 64 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Ben C. S. Sutton ◽  
Michael Shaw
Keyword(s):  
Protein Synthesis ◽  
Large Subunit ◽  
Two Dimensional ◽  
Bisphosphate Carboxylase ◽  
Two Dimensional Electrophoresis ◽  
Host Pathogen Interaction ◽  
Melampsora Lini ◽  
In Vivo Labelling ◽  
Dimensional Electrophoresis

Resistance to flax rust Melampsora lini (Ehrenb.) Lév. in flax carrying the N resistance gene is determined by 24 h postinoculation, at which time hypersensitivity is observed. We have examined protein synthesis in cotyledons inoculated with both virulent and avirulent races of rust by in vivo labelling with [35S]methionine. The pattern of protein synthesis was assessed by one- and two-dimensional electrophoresis 8, 13, and 18 h after inoculation. No changes in protein synthesis were observed in the first 14 h following inoculation; however, by 18 h after inoculation the susceptible combination showed a marked decrease in protein synthesis (22%; P = 0.01). This could be largely accounted for by the reduced synthesis of the ribulose 1,5-bisphosphate carboxylase large subunit, which was readily quantified on electrophoresis gels. In addition, a 30-kDa polypeptide also declined in the susceptible combination. Two-dimensional electrophoresis enabled changes to be detected in the synthesis of other minor polypeptides. None of these changes were observed in the resistant combination in which a small increase in the synthesis of the ribulose 1,5-bisphosphate carboxylase large subunit and the 30-kDa polypeptide was found. These results indicate that the outcome of the host–pathogen interaction has already been determined by 18 h after inoculation.

scholarly journals N368-Tau fragments generated by legumain are detected only in trace amount in the insoluble Tau aggregates isolated from AD brain

2019 ◽  
Vol 7 (1) ◽  
Author(s):  
Kerstin Schlegel ◽  
Khader Awwad ◽  
Roland G. Heym ◽  
David Holzinger ◽  
Annika Doell ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Minor Component ◽  
Cleavage Sites ◽  
Brain Extracts ◽  
And Control ◽  
Progressive Accumulation ◽  
The Brain ◽  
Tissue Fractionation

Abstract Intraneuronal insoluble inclusions made of Tau protein are neuropathological hallmarks of Alzheimer Disease (AD). Cleavage of Tau by legumain (LGMN) has been proposed to be crucial for aggregation of Tau into fibrils. However, it remains unclear if LGMN-cleaved Tau fragments accumulate in AD Tau inclusions. Using an in vitro enzymatic assay and non-targeted mass spectrometry, we identified four putative LGMN cleavage sites at Tau residues N167-, N255-, N296- and N368. Cleavage at N368 generates variously sized N368-Tau fragments that are aggregation prone in the Thioflavin T assay in vitro. N368-cleaved Tau is not detected in the brain of legumain knockout mice, indicating that LGMN is required for Tau cleavage in the mouse brain in vivo. Using a targeted mass spectrometry method in combination with tissue fractionation and biochemical analysis, we investigated whether N368-cleaved Tau is differentially produced and aggregated in brain of AD patients and control subjects. In brain soluble extracts, despite reduced uncleaved Tau in AD, levels of N368-cleaved Tau are comparable in AD and control hippocampus, suggesting that LGMN-mediated cleavage of Tau is not altered in AD. Consistently, levels of activated, cleaved LGMN are also similar in AD and control brain extracts. To assess the potential accumulation of N368-cleaved Tau in insoluble Tau aggregates, we analyzed sarkosyl-insoluble extracts from AD and control hippocampus. Both N368-cleaved Tau and uncleaved Tau were significantly increased in AD as a consequence of pathological Tau inclusions accumulation. However, the amount of N368-cleaved Tau represented only a very minor component (< 0.1%) of insoluble Tau. Our data indicate that LGMN physiologically cleaves Tau in the mouse and human brain generating N368-cleaved Tau fragments, which remain largely soluble and are present only in low proportion in Tau insoluble aggregates compared to uncleaved Tau. This suggests that LGMN-cleaved Tau has limited role in the progressive accumulation of Tau inclusions in AD.

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