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Comparison of strategies for non-perturbing labeling of ?-synuclein to study amyloidogenesis.

ABSTRACT: Characterization of the amyloidogenic Parkinson's disease protein ?-synuclein (?S) has proven difficult due to its structural plasticity. Here, we present a number of complementary methods to site-specifically introduce fluorescent probes to examine ?S fibril formation and cellular uptake. By using various combinations of conventional Cys modification, amber codon suppression, transferase mediated N-terminal modification, and native chemical ligation, several variants of singly- and doubly-labeled ?S were produced. We validated the nonperturbative nature of the label by a combination of in vitro aggregation kinetics measurements and imaging of the resulting fibrils. The labeled ?S can then be used to monitor conformational changes during fibril formation or cellular uptake of ?S fibrils in models of disease propagation.

SUBMITTER: Haney CM

PROVIDER: S-EPMC4733880 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Comparison of strategies for non-perturbing labeling of α-synuclein to study amyloidogenesis.

Haney Conor M CM Wissner Rebecca F RF Warner John B JB Wang Yanxin J YJ Ferrie John J JJ J Covell Dustin D Karpowicz Richard J RJ Lee Virginia M-Y VM Petersson E James EJ

Organic & biomolecular chemistry 20160201 5

Characterization of the amyloidogenic Parkinson's disease protein α-synuclein (αS) has proven difficult due to its structural plasticity. Here, we present a number of complementary methods to site-specifically introduce fluorescent probes to examine αS fibril formation and cellular uptake. By using various combinations of conventional Cys modification, amber codon suppression, transferase mediated N-terminal modification, and native chemical ligation, several variants of singly- and doubly-label ...[more]

PMID: 26695131

Similar Datasets

Correction: Comparison of strategies for non-perturbing labeling of α-synuclein to study amyloidogenesis.

Project description:Correction for 'Comparison of strategies for non-perturbing labeling of α-synuclein to study amyloidogenesis' by Conor M. Haney, et al., Org. Biomol. Chem., 2016, 14, 1584-1592.

| S-EPMC5553073 | biostudies-other

Modulating the Amyloidogenesis of ?-Synuclein.

Project description:Alpha-Synuclein is found in the neuronal cells but its native function is not well known. While ? -synuclein is an intrinsically disordered protein that adopts a helical conformation upon membrane binding, numerous studies have shown that oligomeric ?-forms of this protein are cytotoxic. This response to misfolded species contributes to Parkinson's Disease etiology and symptoms. The resulting amyloid fibrils are an established diagnostic in Parkinson's Disease. In this review, we focus on strategies that have been used to inhibit the amyloidogenesis of ? -synuclein either by stabilizing the native state, or by redirecting the pathway to less toxic aggregates. Small molecules such as polyphenols, peptides as well as large proteins have proven effective at protecting cells against the cytotoxicity of ?-synuclein. These strategies may lead to the development of therapeutic agents that could prove useful in combating this disease.

| S-EPMC4857621 | biostudies-literature

SARS-COV-2 Spike Protein Fragment eases Amyloidogenesis of α-Synuclein.

Project description:Parkinson's Disease is accompanied by presence of amyloids in the brain formed of α-synuclein chains. Correlation between COVID-19 and the onset of Parkinson's disease let to the idea that amyloidogenic segments in SARS-COV-2 proteins can induce aggregation of α-synuclein. Using molecular dynamic simulations, we show that the fragment FKNIDGYFKI of the spike protein, which is unique for SARS-COV-2, shifts preferentially the ensemble of α-synuclein monomer towards rod-like fibril seeding conformations, and at the same time stabilizes differentially this polymorph over the competing twister-like structure. Our results are compared with earlier work relying on a different protein fragment that is not specific for SARS-COV-2.

| S-EPMC10197603 | biostudies-literature

Natural biomolecules and protein aggregation: emerging strategies against amyloidogenesis.

Project description:Biomolecular self-assembly is a fundamental process in all organisms. As primary components of the life molecular machinery, proteins have a vast array of resources available to them for self-assembly in a functional structure. Protein self-assembly, however, can also occur in an aberrant way, giving rise to non-native aggregated structures responsible for severe, progressive human diseases that have a serious social impact. Different neurodegenerative disorders, like Huntington's, Alzheimer's, and spongiform encephalopathy diseases, have in common the presence of insoluble protein aggregates, generally termed "amyloid," that share several physicochemical features: a fibrillar morphology, a predominantly beta-sheet secondary structure, birefringence upon staining with the dye Congo red, insolubility in common solvents and detergents, and protease resistance. Conformational constrains, hydrophobic and stacking interactions can play a key role in the fibrillogenesis process and protein-protein and peptide-peptide interactions-resulting in self-assembly phenomena of peptides yielding fibrils-that can be modulated and influenced by natural biomolecules. Small organic molecules, which possess both hydrophilic and hydrophobic moieties able to bind to peptide/protein molecules through hydrogen bonds and hydrophobic and aromatic interactions, are potential candidates against amyloidogenesis. In this review some significant case examples will be critically discussed.

| S-EPMC3546742 | biostudies-literature

Labeling Strategies Matter for Super-Resolution Microscopy: A Comparison between HaloTags and SNAP-tags.

Project description:Super-resolution microscopy requires that subcellular structures are labeled with bright and photostable fluorophores, especially for live-cell imaging. Organic fluorophores may help here as they can yield more photons-by orders of magnitude-than fluorescent proteins. To achieve molecular specificity with organic fluorophores in live cells, self-labeling proteins are often used, with HaloTags and SNAP-tags being the most common. However, how these two different tagging systems compare with each other is unclear, especially for stimulated emission depletion (STED) microscopy, which is limited to a small repertoire of fluorophores in living cells. Herein, we compare the two labeling approaches in confocal and STED imaging using various proteins and two model systems. Strikingly, we find that the fluorescent signal can be up to 9-fold higher with HaloTags than with SNAP-tags when using far-red rhodamine derivatives. This result demonstrates that the labeling strategy matters and can greatly influence the duration of super-resolution imaging.

| S-EPMC6474801 | biostudies-literature

Comparison of arterial spin labeling registration strategies in the multi-center GENetic frontotemporal dementia initiative (GENFI).

Project description:PurposeTo compare registration strategies to align arterial spin labeling (ASL) with 3D T1-weighted (T1w) images, with the goal of reducing the between-subject variability of cerebral blood flow (CBF) images.Materials and methodsMulti-center 3T ASL data were collected at eight sites with four different sequences in the multi-center GENetic Frontotemporal dementia Initiative (GENFI) study. In a total of 48 healthy controls, we compared the following image registration options: (I) which images to use for registration (perfusion-weighted images [PWI] to the segmented gray matter (GM) probability map (pGM) (CBF-pGM) or M0 to T1w (M0-T1w); (II) which transformation to use (rigid-body or non-rigid); and (III) whether to mask or not (no masking, M0-based FMRIB software library Brain Extraction Tool [BET] masking). In addition to visual comparison, we quantified image similarity using the Pearson correlation coefficient (CC), and used the Mann-Whitney U rank sum test.ResultsCBF-pGM outperformed M0-T1w (CC improvement 47.2% ± 22.0%; P < 0.001), and the non-rigid transformation outperformed rigid-body (20.6% ± 5.3%; P < 0.001). Masking only improved the M0-T1w rigid-body registration (14.5% ± 15.5%; P = 0.007).ConclusionThe choice of image registration strategy impacts ASL group analyses. The non-rigid transformation is promising but requires validation. CBF-pGM rigid-body registration without masking can be used as a default strategy. In patients with expansive perfusion deficits, M0-T1w may outperform CBF-pGM in sequences with high effective spatial resolution. BET-masking only improves M0-T1w registration when the M0 image has sufficient contrast.Level of evidence1 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2018;47:131-140.

| S-EPMC6485386 | biostudies-literature

Multi-organ comparison of flow-based arterial spin labeling techniques: Spatially non-selective labeling for cerebral and renal perfusion imaging.

Project description:PurposeFlow-based arterial spin labeling (ASL) techniques provide a transit-time insensitive alternative to the more conventional spatially selective ASL techniques. However, it is not clear which flow-based ASL technique performs best and also, how these techniques perform outside the brain (taking into account eg, flow-dynamics, field-inhomogeneity, and organ motion). In the current study we aimed to compare 4 flow-based ASL techniques (ie, velocity selective ASL, acceleration selective ASL, multiple velocity selective saturation ASL, and velocity selective inversion prepared ASL [VSI-ASL]) to the current spatially selective reference techniques in brain (ie, pseudo-continuous ASL [pCASL]) and kidney (ie, pCASL and flow alternating inversion recovery [FAIR]).MethodsBrain (n = 5) and kidney (n = 6) scans were performed in healthy subjects at 3T. Perfusion-weighted signal (PWS) maps were generated and ASL techniques were compared based on temporal SNR (tSNR), sensitivity to perfusion changes using a visual stimulus (brain) and robustness to respiratory motion by comparing scans acquired in paced-breathing and free-breathing (kidney).ResultsIn brain, all flow-based ASL techniques showed similar tSNR as pCASL, but only VSI-ASL showed similar sensitivity to perfusion changes. In kidney, all flow-based ASL techniques had comparable tSNR, although all lower than FAIR. In addition, VSI-ASL showed a sensitivity to B1 -inhomogeneity. All ASL techniques were relatively robust to respiratory motion.ConclusionIn both brain and kidney, flow-based ASL techniques provide a planning-free and transit-time insensitive alternative to spatially selective ASL techniques. VSI-ASL shows the most potential overall, showing similar performance as the golden standard pCASL in brain. However, in kidney, a reduction of B1 -sensitivity of VSI-ASL is necessary to match the performance of FAIR.

| S-EPMC7898485 | biostudies-literature

Fluorine-18 radiochemistry, labeling strategies and synthetic routes.

Project description:Fluorine-18 is the most frequently used radioisotope in positron emission tomography (PET) radiopharmaceuticals in both clinical and preclinical research. Its physical and nuclear characteristics (97% β(+) decay, 109.7 min half-life, 635 keV positron energy), along with high specific activity and ease of large scale production, make it an attractive nuclide for radiochemical labeling and molecular imaging. Versatile chemistry including nucleophilic and electrophilic substitutions allows direct or indirect introduction of (18)F into molecules of interest. The significant increase in (18)F radiotracers for PET imaging accentuates the need for simple and efficient (18)F-labeling procedures. In this review, we will describe the current radiosynthesis routes and strategies for (18)F labeling of small molecules and biomolecules.

| S-EPMC4306521 | biostudies-other

Resin supported acyl carrier protein labeling strategies.

Project description:The post-translational modifying enzymes phophopantetheinyl transferase and acyl carrier protein hydrolase have shown utility in the functional modification of acyl carrier proteins. Here we develop these tools as immobilized biocatalysts on agarose supports. New utility is imparted through these methods, enabling rapid and label-independent protein purification. Immobilization of acyl carrier protein is also demonstrated for rapid activity assays of these 4'-phosophopantetheine modifying enzymes, displaying a particular advantage in the case of phosphopantetheine removal, where few orthogonal techniques have been demonstrated. These tools further enrich the suite of functional utility of 4'-phosophopantetheine chemistry, with applications to protein functionalization, materials, and natural product biosynthetic studies.

| S-EPMC4012645 | biostudies-literature

A simple non-perturbing cell migration assay insensitive to proliferation effects.

Project description:Migration is a fundamental cellular behavior that plays an indispensable role in development and homeostasis, but can also contribute to pathology such as cancer metastasis. Due to its relevance to many aspects of human health, the ability to accurately measure cell migration is of broad interest, and numerous approaches have been developed. One of the most commonly employed approaches, because of its simplicity and throughput, is the exclusion zone assay in which cells are allowed to migrate into an initially cell-free region. A major drawback of this assay is that it relies on simply counting cells in the exclusion zone and therefore cannot distinguish the effects of proliferation from migration. We report here a simple modification to the exclusion zone migration assay that exclusively measures cell migration and is not affected by proliferation. This approach makes use of a lineage-tracing vital stain that is retained through cell generations and effectively reads out migration relative to the original, parental cell population. This modification is simple, robust, non-perturbing, and inexpensive. We validate the method in a panel of cell lines under conditions that inhibit or promote migration and demonstrate its use in normal and cancer cell lines as well as primary cells.

| S-EPMC4989229 | biostudies-literature

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