scholarly journals Zinc Binding to Tau Influences Aggregation Kinetics and Oligomer Distribution

2019 ◽  
Vol 20 (23) ◽  
pp. 5979 ◽  
Author(s):  
Moreira ◽  
Cristóvão ◽  
Torres ◽  
Carapeto ◽  
Rodrigues ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Metal Ion ◽  
Molecular Mechanisms ◽  
Disease Onset ◽  
Native Mass Spectrometry ◽  
Mass Spectrometry Analysis ◽  
Zinc Binding ◽  
Lag Phase ◽  
Intrinsically Disordered ◽  
Tau Aggregation

Metal ions are well known modulators of protein aggregation and are key players in Alzheimer’s Disease, being found to be associated to pathologic protein deposits in diseased brains. Therefore, understanding how metals influence amyloid aggregation is critical in establishing molecular mechanisms that underlie disease onset and progression. Here, we report data on the interaction of full-length human Tau protein with calcium and zinc ions, evidencing that Tau self-assembly is differently regulated, depending on the type of bound metal ion. We established that Tau binds 4 Zn2+ and 1 Ca2+ per monomer while using native mass spectrometry analysis, without inducing order or substantial conformational changes in the intrinsically disordered Tau, as determined by structural analysis using circular dichroism and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopies. However, Tau aggregation is found to proceed differently in the calcium- and -zinc bound forms. While the rate of aggregation, as determined from thioflavin-T (ThT) fluorescence kinetics, is highly increased in both cases, the reaction proceeds via different mechanisms, as evidenced by the absence of the lag phase in the reaction of zinc-bound Tau. Monitoring Tau aggregation using native mass spectrometry indeed evidenced a distinct distribution of Tau conformers along the reaction, as confirmed by dynamic light scattering analysis. We propose that such differences arise from zinc binding at distinct locations within the Tau sequence that prompt both the rapid formation of seeding oligomers through interactions at high affinity sites within the repeat domains, as well as amorphous aggregation, through low affinity interactions with residues elsewhere in the sequence, including at the fuzzy coat domain.

scholarly journals Combinatorial native MS and LC-MS/MS approach reveals high intrinsic phosphorylation of human Tau but minimal levels of other key modifications

2020 ◽  
Author(s):  
Friedel Drepper ◽  
Jacek Biernat ◽  
Senthillvelrajan Kaniyappan ◽  
Helmut E. Meyer ◽  
Eva Maria Mandelkow ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Posttranslational Modifications ◽  
Native Mass Spectrometry ◽  
Eukaryotic Cell ◽  
Cell System ◽  
Mass Spectrometry Analysis ◽  
Phosphorylation Sites ◽  
Spectrometry Analysis ◽  
Phosphorylated Peptides ◽  
Tau Aggregation

AbstractAbnormal changes in the neuronal microtubule-associated protein Tau, such as hyperphosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer disease. Hyperphosphorylation is thought to take place before aggregation, and therefore it is often assumed that phosphorylation predisposes Tau towards aggregation. However, the nature and extent of phosphorylation has remained ill-defined. Tau protein contains up to 85 potential phosphorylation sites (80 Ser/Thr, and 5 Tyr P-sites), many of which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, limitations in methods (e.g. in mass spectrometry of phosphorylated peptides, or antibodies against phospho-epitopes) have led to conflicting results regarding the overall degree of phosphorylation of Tau in cells. Here we present results from a new approach, that is based on native mass spectrometry analysis of intact Tau expressed in a eukaryotic cell system (Sf9) which reveals Tau in different phosphorylation states. The extent of phosphorylation is remarkably heterogeneous with up to ∼20 phosphates (Pi) per molecule and distributed over 51 sites (including all P-sites published so far and additional 18 P-sites). The medium phosphorylated fraction Pm showed overall occupancies centered at 8 Pi (± 5 Pi) with a bell-shaped distribution, the highly phosphorylated fraction Ph had 14 Pi (± 6 Pi). The distribution of sites was remarkably asymmetric (with 71% of all P-sites located in the C-terminal half of Tau). All phosphorylation sites were on Ser or Thr residues, but none on Tyr. Other known posttranslational modifications of Tau were near or below our detection limit (e.g. acetylation, ubiquitination). None of the Tau fractions self-assemble readily, arguing that Tau aggregation is not promoted by phosphorylation per se but requires additional factors.

scholarly journals Mapping Interactome Networks of FOSL1 and FOSL2 in Human Th17 Cells

2021 ◽  
Author(s):  
Ankitha Shetty ◽  
Santosh D. Bhosale ◽  
Subhash Kumar Tripathi ◽  
Tanja Buchacher ◽  
Rahul Biradar ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cell Differentiation ◽  
Th17 Cell ◽  
Th17 Cells ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Binding Partners ◽  
Th17 Cell Differentiation

Dysregulated function of Th17 cells has implications in immunodeficiencies and autoimmune disorders. Th17 cell-differentiation is orchestrated by a complex network of transcription factors, including several members of the activator protein (AP-1) family. Among these, FOSL1 and FOSL2 influence the effector responses of Th17 cells. However, the molecular mechanisms underlying their functions are unclear, owing to the poorly characterized protein interaction networks of these factors. Here, we establish the first interactomes of FOSL1 and FOSL2 in human Th17 cells, using affinity purification–mass spectrometry analysis. In addition to the known JUN proteins, we identified several novel binding partners of FOSL1 and FOSL2. Gene ontology analysis found a major fraction of these interactors to be associated with RNA binding activity, which suggests new mechanistic links. Intriguingly, 29 proteins were found to share interactions with FOSL1 and FOSL2, and these included key regulators of Th17-fate. We further validated the binding partners identified in this study by using parallel reaction monitoring targeted mass spectrometry and other methods. Our study provides key insights into the interaction-based signaling mechanisms of FOSL1 and FOSL2 that potentially govern Th17 cell-differentiation and associated pathologies.

scholarly journals Depicting Conformational Ensembles of α-Synuclein by Single Molecule Force Spectroscopy and Native Mass Spectroscopy

2019 ◽  
Vol 20 (20) ◽  
pp. 5181 ◽  
Author(s):  
Roberta Corti ◽  
Claudia A. Marrano ◽  
Domenico Salerno ◽  
Stefania Brocca ◽  
Antonino Natalello ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Single Molecule ◽  
Conformational Changes ◽  
Intrinsically Disordered Proteins ◽  
Force Spectroscopy ◽  
Native Mass Spectrometry ◽  
Disordered Proteins ◽  
Unique Contribution ◽  
Single Molecule Force Spectroscopy ◽  
Intrinsically Disordered

Description of heterogeneous molecular ensembles, such as intrinsically disordered proteins, represents a challenge in structural biology and an urgent question posed by biochemistry to interpret many physiologically important, regulatory mechanisms. Single-molecule techniques can provide a unique contribution to this field. This work applies single molecule force spectroscopy to probe conformational properties of α-synuclein in solution and its conformational changes induced by ligand binding. The goal is to compare data from such an approach with those obtained by native mass spectrometry. These two orthogonal, biophysical methods are found to deliver a complex picture, in which monomeric α-synuclein in solution spontaneously populates compact and partially compacted states, which are differently stabilized by binding to aggregation inhibitors, such as dopamine and epigallocatechin-3-gallate. Analyses by circular dichroism and Fourier-transform infrared spectroscopy show that these transitions do not involve formation of secondary structure. This comparative analysis provides support to structural interpretation of charge-state distributions obtained by native mass spectrometry and helps, in turn, defining the conformational components detected by single molecule force spectroscopy.

scholarly journals The Cardiac Lipidome in Models of Cardiovascular Disease

Metabolites ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 254 ◽  
Author(s):  
Mateusz M. Tomczyk ◽  
Vernon W. Dolinsky
Keyword(s):  
Mass Spectrometry ◽  
Cardiovascular Disease ◽  
Molecular Mechanisms ◽  
Cell Structure ◽  
Myocardial Cell ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Cardiac Lipid ◽  
Lipid Species ◽  
Novel Biomarkers

Cardiovascular disease (CVD) is the leading cause of death worldwide. There are numerous factors involved in the development of CVD. Among these, lipids have an important role in maintaining the myocardial cell structure as well as cardiac function. Fatty acids (FA) are utilized for energy, but also contribute to the pathogenesis of CVD and heart failure. Advances in mass spectrometry methods have enabled the comprehensive analysis of a plethora of lipid species from a single sample comprised of a heterogeneous population of lipid molecules. Determining cardiac lipid alterations in different models of CVD identifies novel biomarkers as well as reveals molecular mechanisms that underlie disease development and progression. This information could inform the development of novel therapeutics in the treatment of CVD. Herein, we provide a review of recent studies of cardiac lipid profiles in myocardial infarction, obesity, and diabetic and dilated cardiomyopathy models of CVD by methods of mass spectrometry analysis.

scholarly journals Treatment of Microglia with Anti-PrP Antibodies Induces Neuronal Allergenicity

2020 ◽  
Author(s):  
Utpal Kumar Adhikari ◽  
Elif Sakiz ◽  
Umma Habiba ◽  
Sachin Kumar ◽  
Meena Mikhael ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Mechanisms ◽  
Neuroblastoma Cell ◽  
Mass Spectrometry Analysis ◽  
Cross Linking ◽  
Antibody Treatment ◽  
Spectrometry Analysis ◽  
N2a Cells ◽  
Related Proteins

Abstract Background: Previous reports identified proteins associated with ‘apoptosis’ following cross-linking PrPC with motif-specific anti-PrP antibodies in vivo and in vitro. The molecular mechanisms underlying this IgG-mediated neurotoxicity and the role of the activated proteins in the apoptotic pathways leading to neuronal death has not been properly defined. Previous reports implicated a number of proteins, including apolipoprotein E, cytoplasmic phospholipase A2, prostaglandin and calpain with anti-PrP antibody-mediated ‘apoptosis’, however, these proteins are also known to play an important role in allergy. In this study, we investigated whether cross-linking PrPC with anti-PrP antibodies stimulates a neuronal allergenic response.Methods: Initially, we predicted the allergenicity of the epitope sequences associated with ‘neurotoxic’ anti-PrP antibodies using allergenicity prediction servers. We then investigated whether anti-PrP antibody treatment of neuronal (N2a) and microglia (N11) cell lines leads to a neuronal allergenic response.Results: We found that both tail- and globular-epitopes were allergenic. Specifically, binding regions that contain epitopes for ‘neurotoxic’ antibodies such as ICSM18 (146-159), ICSM35 (91-110), POM 1 (138-147), POM 2 (57-88) and POM 3 (95-100) lead to activation of allergenic related proteins. Following direct application of anti-PrPC antibodies on N2a cells, mass spectrometry analysis identified 4 neuronal allergenic-related proteins when compared with untreated cells. Furthermore, mass spectrometry analysis identified 8 neuronal allergenic-related proteins following cross-linking N11 cells with anti-PrPC antibodies prior to co-culture with N2a cells, when compared with untreated cells. Of importance, we showed that the allergenic effects triggered by the anti-PrP antibodies were more potent when antibody-treated microglia were co-cultured with the neuroblastoma cell line. Furthermore, in both direct and co-culture with antibody-treated microglia, we demonstrate that the allergenic proteome was part of the PrPC-interactome. Conclusions: This study showed for the first time that anti-PrP antibody binding to PrPC triggers a neuronal allergenic response (we termed ‘IgG-Mediated Neuronal Allergenic Toxicity’) and highlights the important role of microglia in triggering IgG-mediated neuronal allergenic toxicity. Moreover, this study provides an important impetus for including allergenic assessment of therapeutic antibodies for neurodegenerative to derive safe and targeted biotherapeutics.

scholarly journals Native Mass Spectrometry Reveals the Conformational Diversity of the UVR8 Photoreceptor

2018 ◽  
Author(s):  
Inês S. Camacho ◽  
Alina Theisen ◽  
Linus O. Johannissen ◽  
L. Aranzazú Díaz-Ramos ◽  
John M. Christie ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Uv Light ◽  
Native Mass Spectrometry ◽  
Full Length ◽  
Structural Investigations ◽  
Core Domain ◽  
Intrinsically Disordered ◽  
The Core ◽  
C Terminus ◽  
Conformational Diversity

AbstractUVR8 is a plant photoreceptor protein that regulates photomorphogenic and protective responses to UV light. The inactive, homodimeric state absorbs UV-B light resulting in dissociation into monomers, which are considered to be the active state and comprise a β-propeller core domain and intrinsically disordered N- and C-terminal tails. The C-terminus is required for functional binding to signalling partner COP1. To date, however, structural studies have only been conducted with the core domain where the terminal tails have been truncated. Here, we report structural investigations of full-length UVR8 using native ion mobility mass spectrometry adapted for photo-activation. We show that, whilst truncated UVR8 photo-converts from a single conformation of dimers to a single monomer conformation, the full-length protein exist in numerous conformational families. The full-length dimer adopts both a compact state and an extended state where the C-terminus is primed for activation. In the monomer the extended C-terminus destabilises the core domain to produce highly extended yet stable conformations, which we propose are the fully active states that bind COP1. Our results reveal the conformational diversity of full-length UVR8. We also demonstrate the potential power of native mass spectrometry to probe functionally important structural dynamics of photoreceptor proteins throughout nature.TOC Graphic

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