Browse
Submit Data
Databases
API
Help

Dataset Information

25 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Effects of Glutamate Arginylation on α-Synuclein: Studying an Unusual Post-Translational Modification through Semisynthesis.

ABSTRACT: A variety of post-translational modifications (PTMs) are believed to regulate the behavior and function of α-synuclein (αS), an intrinsically disordered protein that mediates synaptic vesicle trafficking. Fibrils of αS are implicated in neurodegenerative disorders such as Parkinson's disease. In this study, we used chemical synthesis and biophysical techniques to characterize the neuroprotective effects of glutamate arginylation, a hitherto little characterized PTM in αS. We developed semisynthetic routes combining peptide synthesis, unnatural amino acid mutagenesis, and native chemical ligation (NCL) to site-specifically introduce the PTM of interest along with fluorescent probes into αS. We synthesized the arginylated glutamate as a protected amino acid, as well as a novel ligation handle for NCL, in order to generate full-length αS modified at various individual sites or a combination of sites. We assayed the lipid-vesicle binding affinities of arginylated αS using fluorescence correlation spectroscopy (FCS) and found that arginylated αS has the same vesicle affinity compared to control protein, suggesting that this PTM does not alter the native function of αS. On the other hand, we studied the aggregation kinetics of modified αS and found that arginylation at E83, but not E46, slows aggregation and decreases the percentage incorporation of monomer into fibrils in a dose-dependent manner. Arginylation at both sites also resulted in deceleration of fibril formation. Our study represents the first synthetic strategy for incorporating glutamate arginylation into proteins and provides insight into the neuroprotective effect of this unusual PTM.

SUBMITTER: Pan B

PROVIDER: S-EPMC8042788 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Elucidating the role of C-terminal post-translational modifications using protein semisynthesis strategies: α-synuclein phosphorylation at tyrosine 125.

Project description:Despite increasing evidence that supports the role of different post-translational modifications (PTMs) in modulating α-synuclein (α-syn) aggregation and toxicity, relatively little is known about the functional consequences of each modification and whether or not these modifications are regulated by each other. This lack of knowledge arises primarily from the current lack of tools and methodologies for the site-specific introduction of PTMs in α-syn. More specifically, the kinases that mediate selective and efficient phosphorylation of C-terminal tyrosine residues of α-syn remain to be identified. Unlike phospho-serine and phospho-threonine residues, which in some cases can be mimicked by serine/threonine → glutamate or aspartate substitutions, there are no natural amino acids that can mimic phospho-tyrosine. To address these challenges, we developed a general and efficient semisynthetic strategy that enables the site-specific introduction of single or multiple PTMs and the preparation of homogeneously C-terminal modified forms of α-syn in milligram quantities. These advances have allowed us to investigate, for the first time, the effects of selective phosphorylation at Y125 on the structure, aggregation, membrane binding, and subcellular localization of α-syn. The development of semisynthetic methods for the site-specific introduction of single or PTMs represents an important advance toward determining the roles of such modifications in α-syn structure, aggregation, and functions in heath and disease.

| S-EPMC3592575 | biostudies-literature

Post-translational modifications of soluble α-synuclein regulate the amplification of pathological α-synuclein.

Project description:Cell-to-cell transmission and subsequent amplification of pathological proteins promote neurodegenerative disease progression. Most research on this has focused on pathological protein seeds, but how their normal counterparts, which are converted to pathological forms during transmission, regulate transmission is less understood. Here we show in cultured cells that phosphorylation of soluble, nonpathological α-synuclein (α-Syn) at previously identified sites dramatically affects the amplification of pathological α-Syn, which underlies Parkinson's disease and other α-synucleinopathies, in a conformation- and phosphorylation site-specific manner. We performed LC-MS/MS analyses on soluble α-Syn purified from Parkinson's disease and other α-synucleinopathies, identifying many new α-Syn post-translational modifications (PTMs). In addition to phosphorylation, acetylation of soluble α-Syn also modified pathological α-Syn transmission in a site- and conformation-specific manner. Moreover, phosphorylation of soluble α-Syn could modulate the seeding properties of pathological α-Syn. Our study represents the first systematic analysis how of soluble α-Syn PTMs affect the spreading and amplification of pathological α-Syn, which may affect disease progression.

| S-EPMC10103650 | biostudies-literature

Iron-sulfur clusters are involved in post-translational arginylation.

Project description:Eukaryotic arginylation is an essential post-translational modification that modulates protein stability and regulates protein half-life. Arginylation is catalyzed by a family of enzymes known as the arginyl-tRNA transferases (ATE1s), which are conserved across the eukaryotic domain. Despite their conservation and importance, little is known regarding the structure, mechanism, and regulation of ATE1s. In this work, we show that ATE1s bind a previously undiscovered [Fe-S] cluster that is conserved across evolution. We characterize the nature of this [Fe-S] cluster and find that the presence of the [Fe-S] cluster in ATE1 is linked to its arginylation activity, both in vitro and in vivo, and the initiation of the yeast stress response. Importantly, the ATE1 [Fe-S] cluster is oxygen-sensitive, which could be a molecular mechanism of the N-degron pathway to sense oxidative stress. Taken together, our data provide the framework of a cluster-based paradigm of ATE1 regulatory control.

| S-EPMC9884297 | biostudies-literature

Post-translational arginylation of ornithine decarboxylase from rat hepatocytes.

Project description:Ornithine decarboxylase (ODC) was purified 6500-fold from NMRI mouse kidneys under conditions designed to inhibit degradation by proteinases. The enzyme was homogeneous by SDS/polyacrylamide-gel electrophoresis, and the specific activity was among the highest reported. The yield was 70%. A monoclonal antibody against this preparation was generated and used in studies to investigate the half-life of ODC in cultured rat hepatocytes labelled with [35S]methionine. This value was 39 +/- 4 min and was unchanged when either NH4Cl (as a lysosomotropic agent) or leupeptin (as a lysosomal proteinase inhibitor) was added to the culture medium. Thus the intracellular turnover of ODC in cultured hepatocytes occurs mainly in extra-lysosomal compartments. Arginylation of rat ODC was investigated in vitro by incubation with L-[3H]arginyl-tRNA, and the incorporation of the label was compared with that of total cytosolic proteins. Arginylated ODC had a specific radioactivity 8600 times that of the bulk of cytosolic protein. Edman degradation of this ODC showed that the post-translational arginylation occurred only at the alpha-amino end of the enzyme. The inhibitor of arginyl-tRNA:protein arginyltransferase (EC 2.3.2.8), L-glutamyl-L-valyl-L-phenylalanine, increased the half-life of ODC in cultured hepatocytes from 39 min to more than 90 min. The possible significance of the preferential post-translational arginylation of ornithine decarboxylase to its rapid turnover is discussed.

| S-EPMC1131293 | biostudies-other

Post-translational modifications of MSA alpha-synuclein

Project description:LC-MS/MS analysis of post-translational modifications in alpha-synuclein of five MSA cases.

2020-04-09 | PXD018434 | JPOST Repository

Protein arginylation targets alpha synuclein, facilitates normal brain health, and prevents neurodegeneration.

Project description:Alpha synuclein (?-syn) is a central player in neurodegeneration, but the mechanisms triggering its pathology are not fully understood. Here we found that ?-syn is a highly efficient substrate for arginyltransferase ATE1 and is arginylated in vivo by a novel mid-chain mechanism that targets the acidic side chains of E46 and E83. Lack of arginylation leads to increased ?-syn aggregation and causes the formation of larger pathological aggregates in neurons, accompanied by impairments in its ability to be cleared via normal degradation pathways. In the mouse brain, lack of arginylation leads to an increase in ?-syn's insoluble fraction, accompanied by behavioral changes characteristic for neurodegenerative pathology. Our data show that lack of arginylation in the brain leads to neurodegeneration, and suggests that ?-syn arginylation can be a previously unknown factor that facilitates normal ?-syn folding and function in vivo.

| S-EPMC5595787 | biostudies-literature

Global Post-Translational Modification Discovery.

Project description:A new global post-translational modification (PTM) discovery strategy, G-PTM-D, is described. A proteomics database containing UniProt-curated PTM information is supplemented with potential new modification types and sites discovered from a first-round search of mass spectrometry data with ultrawide precursor mass tolerance. A second-round search employing the supplemented database conducted with standard narrow mass tolerances yields deep coverage and a rich variety of peptide modifications with high confidence in complex unenriched samples. The G-PTM-D strategy represents a major advance to the previously reported G-PTM strategy and provides a powerful new capability to the proteomics research community.

| S-EPMC5387672 | biostudies-literature

ATE1-Mediated Post-Translational Arginylation Is an Essential Regulator of Eukaryotic Cellular Homeostasis.

Project description:Arginylation is a protein post-translational modification catalyzed by arginyl-tRNA transferases (ATE1s), which are critical enzymes conserved across all eukaryotes. Arginylation is a key step in the Arg N-degron pathway, a hierarchical cellular signaling pathway that links the ubiquitin-dependent degradation of a protein to the identity of its N-terminal amino acid side chain. The fidelity of ATE1-catalyzed arginylation is imperative, as this post-translational modification regulates several essential biological processes such as cardiovascular maturation, chromosomal segregation, and even the stress response. While the process of ATE1-catalyzed arginylation has been studied in detail at the cellular level, much remains unknown about the structure of this important enzyme, its mechanism of action, and its regulation. In this work, we detail the current state of knowledge on ATE1-catalyzed arginylation, and we discuss both ongoing and future directions that will reveal the structural and mechanistic details of this essential eukaryotic cellular regulator.

| S-EPMC7749041 | biostudies-literature

Exploring the role of post-translational modifications in regulating ?-synuclein interactions by studying the effects of phosphorylation on nanobody binding.

Project description:Intracellular deposits of ?-synuclein in the form of Lewy bodies are major hallmarks of Parkinson's disease (PD) and a range of related neurodegenerative disorders. Post-translational modifications (PTMs) of ?-synuclein are increasingly thought to be major modulators of its structure, function, degradation and toxicity. Among these PTMs, phosphorylation near the C-terminus at S129 has emerged as a dominant pathogenic modification as it is consistently observed to occur within the brain and cerebrospinal fluid (CSF) of post-mortem PD patients, and its level appears to correlate with disease progression. Phosphorylation at the neighboring tyrosine residue Y125 has also been shown to protect against ?-synuclein toxicity in a Drosophila model of PD. In the present study we address the potential roles of C-terminal phosphorylation in modulating the interaction of ?-synuclein with other protein partners, using a single domain antibody fragment (NbSyn87) that binds to the C-terminal region of ?-synuclein with nanomolar affinity. The results reveal that phosphorylation at S129 has negligible effect on the binding affinity of NbSyn87 to ?-synuclein while phosphorylation at Y125, only four residues away, decreases the binding affinity by a factor of 400. These findings show that, despite the fact that ?-synuclein is intrinsically disordered in solution, selective phosphorylation can modulate significantly its interactions with other molecules and suggest how this particular form of modification could play a key role in regulating the normal and aberrant function of ?-synuclein.

| S-EPMC6032363 | biostudies-literature

The alpha subunit of nitrile hydratase is sufficient for catalytic activity and post-translational modification.

Project description:Nitrile hydratases (NHases) possess a mononuclear iron or cobalt cofactor whose coordination environment includes rare post-translationally oxidized cysteine sulfenic and sulfinic acid ligands. This cofactor is located in the ?-subunit at the interfacial active site of the heterodimeric enzyme. Unlike canonical NHases, toyocamycin nitrile hydratase (TNHase) from Streptomyces rimosus is a unique three-subunit member of this family involved in the biosynthesis of pyrrolopyrimidine antibiotics. The subunits of TNHase are homologous to the ?- and ?-subunits of prototypical NHases. Herein we report the expression, purification, and characterization of the ?-subunit of TNHase. The UV-visible, EPR, and mass spectra of the ?-subunit TNHase provide evidence that this subunit alone is capable of synthesizing the active site complex with full post-translational modifications. Remarkably, the isolated post-translationally modified ?-subunit is also catalytically active with the natural substrate, toyocamycin, as well as the niacin precursor 3-cyanopyridine. Comparisons of the steady state kinetic parameters of the single subunit variant to the heterotrimeric protein clearly show that the additional subunits impart substrate specificity and catalytic efficiency. We conclude that the ?-subunit is the minimal sequence needed for nitrile hydration providing a simplified scaffold to study the mechanism and post-translational modification of this important class of catalysts.

| S-EPMC4075990 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data