Mass Spectrometry Analysis of Wild-Type and Knock-in Q140/Q140 Huntington’s Disease Mouse Brains Reveals Changes in Glycerophospholipids Including Alterations in Phosphatidic Acid and Lyso-Phosphatidic Acid

2015 ◽  
Vol 4 (2) ◽  
pp. 187-201 ◽  
Author(s):  
Petr Vodicka ◽  
Shunyan Mo ◽  
Adelaide Tousley ◽  
Karin M. Green ◽  
Ellen Sapp ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Phosphatidic Acid ◽  
Huntington's Disease ◽  
Huntington’S Disease ◽  
Mass Spectrometry Analysis ◽  
Wild Type ◽  
Spectrometry Analysis

scholarly journals PPP2R5D Promotes Hepatitis C Virus Infection Through Binding to NS5B Protein

2021 ◽  
Author(s):  
Muhammad Ikram Anwar ◽  
Ni Li ◽  
Qing Zhou ◽  
Mingxiao Chen ◽  
Chengguang Hu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Infection ◽  
Hcv Infection ◽  
Mass Spectrometry Analysis ◽  
Enhancement Effect ◽  
Wild Type ◽  
Spectrometry Analysis ◽  
Adaptive Mutations

Abstract Background: Hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis C, end-stage liver diseases, and hepatocellular carcinoma. The development of infectious HCV cell culture systems primarily relied on the replication enhancement effect of adaptive mutations. Although the mode of action may vary, those adaptive mutations could direct the study of virus-host interactions required for efficient virus infection. We previously identified a substitution D559G in NS5B (RNA dependent RNA polymerase) critical for the replication of HCV genomes. In this study, we set out to study whether D559G-NS5B specifically interacted with some host factors crucial for HCV infection.Methods: Through mass spectrometry analysis of immunoprecipitation mixture of ectopically expressed wild-type and D559G-mutated NS5B, we identified candidate factors showing potential interactions with NS5B and D559G-NS5B. The requirement of selected host factor in HCV infection in vitro was demonstrated by gene knockout, overexpression, virus infection, and co-immunoprecipitation approaches.Results: From the results of immunoprecipitation and mass spectrometry analysis, we selected protein phosphatase 2 regulatory subunit B’delta (PPP2R5D) for further characterization. Co-immunoprecipitation confirmed that both wild-type and D559G NS5B proteins interacted with PPP2R5D, but the interaction between D559G-NS5B and PPP2R5D was more efficient. Silencing of PPP2R5D decreased HCV infection, and knockout of PPP2R5D nearly eliminated HCV infection in Huh7.5 cells. Transient and stably overexpression of PPP2R5D in PPP2R5D-knockout cells restored HCV infection to a level close to that seen for wild-type Huh7.5 cells. Conclusions: PPP2R5D is required for HCV infection in cultured hepatoma cells, and PPP2R5D may function through binding to HCV NS5B. The underlying mechanism of PPP2R5D in the complete HCV life cycle requires further investigation.

scholarly journals Revisiting G3BP-S149 phosphorylation and its impact on stress granule assembly

2019 ◽  
Author(s):  
Marc D. Panas ◽  
Nancy Kedersha ◽  
Tim Schulte ◽  
Rui M. Branca ◽  
Pavel Ivanov ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Environmental Stress ◽  
Sodium Arsenite ◽  
Protein Structures ◽  
Stress Granules ◽  
Stress Granule ◽  
Mass Spectrometry Analysis ◽  
Wild Type ◽  
Spectrometry Analysis

AbstractStress granules (SGs) are cytoplasmic, non-membranous RNA/protein structures that assemble in response to environmental stress. G3BP is a critical SG-nucleating protein, and its ability to regulate SGs has been reported to be regulated by serine 149 phosphorylation. We now report that the constructs engineered to contain non-phosphorylatable and phosphomimetic (G3BP1-S149A and G3BP1-S149E, respectively) mutations used in many studies include additional unintended mutations (A54T/S149A and S99P/S149E) one of which (S99P) is responsible for the effects on SG assembly attributed to S149E. Specifically, the S99P mutation alone reduces SG nucleation and impairs the ability to rescue SG assembly in ΔΔG3BP1/2 U2OS KO cells, challenging the widely-stated conclusion that de-phosphorylation of serine 149 in G3BP1 promotes SG assembly. We used comparative mass spectrometry analysis of both (1) ectopically expressed GFP-G3BP1 in ΔΔG3BP1/2 U2OS KO and (2) endogenous G3BP1 in wild-type U2OS, with and without sodium arsenite treatment, in an attempt to reproduce earlier findings, but found no significant changes in S149 phosphorylation that correlate with arsenite-induced SG formation.

scholarly journals Effect of Ocular Hypertension on D-β-Aspartic Acid-Containing Proteins in the Retinas of Rats

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Takashi Kanamoto ◽  
Takashi Tachibana ◽  
Yasushi Kitaoka ◽  
Toshio Hisatomi ◽  
Yasuhiro Ikeda ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ocular Hypertension ◽  
Aspartic Acid ◽  
Atp Synthase ◽  
Wistar Rats ◽  
Protein Band ◽  
Mass Spectrometry Analysis ◽  
Liquid Chromatography Mass Spectrometry ◽  
Spectrometry Analysis ◽  
Sds Page

Purpose. To investigate the effect of ocular hypertension-induced isomerization of aspartic acid in retinal proteins. Methods. Adult Wistar rats with ocular hypertension were used as an experimental model. D-β-aspartic acid-containing proteins were isolated by SDS-PAGE and western blot with an anti-D-β-aspartic acid antibody and identified by liquid chromatography-mass spectrometry analysis. The concentration of ATP was measured by ELISA. Results. D-β-aspartic acid was expressed in a protein band at around 44.5 kDa at much higher quantities in the retinas of rats with ocular hypertension than in those of normotensive rats. The 44.5 kDa protein band was mainly composed of α-enolase, S-arrestin, and ATP synthase subunits α and β, in both the ocular hypertensive and normotensive retinas. Moreover, increasing intraocular pressure was correlated with increasing ATP concentrations in the retinas of rats. Conclusion. Ocular hypertension affected the expression of proteins containing D-β-aspartic acid, including ATP synthase subunits, and up-regulation of ATP in the retinas of rats.

scholarly journals N-Terminomics Strategies for Protease Substrates Profiling

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4699
Author(s):  
Mubashir Mintoo ◽  
Amritangshu Chakravarty ◽  
Ronak Tilvawala
Keyword(s):  
Mass Spectrometry ◽  
Protease Activity ◽  
Viral Infections ◽  
Mass Spectrometry Analysis ◽  
Post Translational Modification ◽  
Spectrometry Analysis ◽  
Physiological Conditions ◽  
Inflammatory Conditions ◽  
Biological Mixtures

Proteases play a central role in various biochemical pathways catalyzing and regulating key biological events. Proteases catalyze an irreversible post-translational modification called proteolysis by hydrolyzing peptide bonds in proteins. Given the destructive potential of proteolysis, protease activity is tightly regulated. Dysregulation of protease activity has been reported in numerous disease conditions, including cancers, neurodegenerative diseases, inflammatory conditions, cardiovascular diseases, and viral infections. The proteolytic profile of a cell, tissue, or organ is governed by protease activation, activity, and substrate specificity. Thus, identifying protease substrates and proteolytic events under physiological conditions can provide crucial information about how the change in protease regulation can alter the cellular proteolytic landscape. In recent years, mass spectrometry-based techniques called N-terminomics have become instrumental in identifying protease substrates from complex biological mixtures. N-terminomics employs the labeling and enrichment of native and neo-N-termini peptides, generated upon proteolysis followed by mass spectrometry analysis allowing protease substrate profiling directly from biological samples. In this review, we provide a brief overview of N-terminomics techniques, focusing on their strengths, weaknesses, limitations, and providing specific examples where they were successfully employed to identify protease substrates in vivo and under physiological conditions. In addition, we explore the current trends in the protease field and the potential for future developments.

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