RasGRP1 (CalDAG-GEF-II) Mediates L-DOPA-induced Dyskinesia in a Mouse Model of Parkinson Disease

ABSTRACTThe therapeutic benefits of L–3,4–dihydroxyphenylalanine (L-DOPA) in Parkinson disease (PD) patients diminishes with the onset of abnormal involuntary movements (L-DOPA induced dyskinesia), a debilitating motor side effect. L-DOPA induced dyskinesia are due to altered dopaminergic signaling in the striatum, a brain region that controls motor and cognitive functions. However, the molecular mechanisms that promote L-DOPA-induced dyskinesia remain unclear. Here, we have reported that RasGRP1 (also known as CalDAG-GEF-II) physiologically mediated L-DOPA induced dyskinesia in a 6-hydroxy dopamine (6-OHDA) lesioned mouse model of PD. In this study, L-DOPA treatment rapidly upregulated RasGRP1 in the striatum. Our findings showed that RasGRP1 deleted mice (RasGRP1−/−) had drastically diminished L-DOPA-induced dyskinesia, andRasGRP1−/−mice did not interfere with the therapeutic benefits of L-DOPA. In terms of its mechanism, RasGRP1 mediates L-DOPA-induced extracellular regulated kinase (ERK), the mammalian target of rapamycin kinase (mTOR) and the cAMP/PKA pathway and binds directly with Ras-homolog-enriched in the brain (Rheb), which is a potent activator of mTOR, both in vitro and in the intact striatum. High-resolution tandem mass tag mass spectrometry analysis of striatal tissue revealed significant targets, such as phosphodiesterase (Pde1c), Pde2a, catechol-o-methyltransferase (comt), and glutamate decarboxylase 1 and 2 (Gad1 and Gad2), which are downstream regulators of RasGRP1 and are linked to L-DOPA-induced dyskinesia vulnerability. Collectively, the findings of this study demonstrated that RasGRP1 is a major regulator of L-DOPA-induced dyskinesia in the striatum. Drugs or gene-depletion strategies targeting RasGRP1 may offer novel therapeutic opportunities for preventing L-DOPA-induced dyskinesia in PD patients.

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Functional Characterization of the Cleavage Specificity of the Sapovirus Chymotrypsin-Like Protease

Journal of Virology ◽

10.1128/jvi.00693-08 ◽

2008 ◽

Vol 82 (16) ◽

pp. 8085-8093 ◽

Cited By ~ 7

Author(s):

Ivonne Robel ◽

Julia Gebhardt ◽

Jeroen R. Mesters ◽

Alexander Gorbalenya ◽

Bruno Coutard ◽

...

Keyword(s):

High Performance ◽

Molecular Mechanisms ◽

Rna Virus ◽

Functional Characterization ◽

Mass Spectrometry Analysis ◽

Viral Protease ◽

Spectrometry Analysis ◽

Cleavage Specificity ◽

Scissile Bond

ABSTRACT Sapovirus is a positive-stranded RNA virus with a translational strategy based on processing of a polyprotein precursor by a chymotrypsin-like protease. So far, the molecular mechanisms regulating cleavage specificity of the viral protease are poorly understood. In this study, the catalytic activities and substrate specificities of the predicted forms of the viral protease, the 3C-like protease (NS6) and the 3CD-like protease-polymerase (NS6-7), were examined in vitro. The purified NS6 and NS6-7 were able to cleave synthetic peptides (15 to 17 residues) displaying the cleavage sites of the sapovirus polyprotein, both NS6 and NS6-7 proteins being active forms of the viral protease. High-performance liquid chromatography and subsequent mass spectrometry analysis of digested products showed a specific trans cleavage of peptides bearing Gln-Gly, Gln-Ala, Glu-Gly, Glu-Pro, or Glu-Lys at the scissile bond. In contrast, peptides bearing Glu-Ala or Gln-Asp at the scissile bond (NS4-NS5 and NS5-NS6, or NS6-NS7 junctions, respectively) were resistant to trans cleavage by NS6 or NS6-7 proteins, whereas cis cleavage of the Glu-Ala scissile bond of the NS5-NS6 junction was evidenced. Interestingly, the presence of a Phe at position P4 overruled the resistance to trans cleavage of the Glu-Ala junction (NS5-NS6), whereas substitutions at the P1 and P2′ positions altered the cleavage efficiency. The differential cleavage observed is supported by a model of the substrate-binding site of the sapovirus protease, indicating that the P4, P1, and P2′ positions in the substrate modulate the cleavage specificity and efficiency of the sapovirus chymotrypsin-like protease.

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DEPDC1/ EEF1A1 Complex Promotes the Progression of Human Osteosarcoma via Downregulation of FOXO3a

10.21203/rs.3.rs-94034/v1 ◽

2020 ◽

Author(s):

Lin Shen ◽

Han Lin ◽

Aijun Zhang ◽

Ronghan Liu ◽

Chendan Zhou ◽

...

Keyword(s):

Poor Prognosis ◽

Molecular Mechanisms ◽

Mass Spectrometry Analysis ◽

Osteosarcoma Cells ◽

Critical Pathway ◽

Spectrometry Analysis ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cells

Abstract BackgroundOsteosarcoma is a common primary malignant bone tumor with poor prognosis. Currently there is no effective therapeutic strategies primarily due to the insufficient understanding its underlying mechanisms. Here we aimed to decipher the molecular mechanisms underlying the osteosarcoma progression.MethodsGEO data analysis, immunohistochemistry, qRT-PCR and western blotting were performed to evaluate the expression of differentially genes in human osteosarcoma tissues. Stably transfected human osteosarcoma cells were injected in mouse model to assess the effect of DEPDC1 in vivo. The function of DEPDC1–EEF1A1–FOXO3a axis was detected by mass spectrometry analysis, co-immunoprecipitation (co-IP) experiments and RNA sequencing in vitro. ResultsBy exploring differentially expressed genes, we found DEPDC1 is highly expressed in human osteosarcoma cells and tissues. Mechanistically, we found the protein expressed by DEPDC1 can directly bind to EEF1A1 through three binding regions, thus forming a complex. Importantly, DEPDC1/EEF1A1 complex can directly inhibit the transcription and expression of FOXO3a in vitro and in vivo, thus promoting the metastasis and proliferation of osteosarcoma. The clinical relevance study showed that overexpression of DEPDC1/EEF1A1 complex is correlated with reduced survival rate of osteosarcoma patients.ConclusionsCollectively, this study demonstrated the DEPDC1/EEF1A1–FOXO3a axis as a critical pathway that promotes the progression of osteosarcoma and leads to poor prognosis. Genetically targeting or pharmacologically inhibiting DEPDC1/EEF1A1–FOXO3a axis may serve a promising strategy for targeting human osteosarcoma.

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RasGRP1 is a causal factor in the development of l-DOPA–induced dyskinesia in Parkinson’s disease

Science Advances ◽

10.1126/sciadv.aaz7001 ◽

2020 ◽

Vol 6 (18) ◽

pp. eaaz7001 ◽

Cited By ~ 7

Author(s):

Mehdi Eshraghi ◽

Uri Nimrod Ramírez-Jarquín ◽

Neelam Shahani ◽

Tommaso Nuzzo ◽

Arianna De Rosa ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Molecular Mechanisms ◽

Involuntary Movement ◽

Mammalian Target Of Rapamycin ◽

Mass Spectrometry Analysis ◽

Therapeutic Effects ◽

Nucleotide Exchange ◽

Spectrometry Analysis ◽

Macaque Model

The therapeutic effects of l-3,4-dihydroxyphenylalanine (l-DOPA) in patients with Parkinson’s disease (PD) severely diminishes with the onset of abnormal involuntary movement, l-DOPA–induced dyskinesia (LID). However, the molecular mechanisms that promote LID remain unclear. Here, we demonstrated that RasGRP1 [(guanine nucleotide exchange factor (GEF)] controls the development of LID. l-DOPA treatment rapidly up-regulated RasGRP1 in the striatum of mouse and macaque model of PD. The lack of RasGRP1 in mice (RasGRP1−/−) dramatically diminished LID without interfering with the therapeutic effects of l-DOPA. Besides acting as a GEF for Ras homolog enriched in the brain (Rheb), the activator of the mammalian target of rapamycin kinase (mTOR), RasGRP1 promotes l-DOPA–induced extracellular signal-regulated kinase (ERK) and the mTOR signaling in the striatum. High-resolution tandem mass spectrometry analysis revealed multiple RasGRP1 downstream targets linked to LID vulnerability. Collectively, the study demonstrated that RasGRP1 is a critical striatal regulator of LID.

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Identification of DNAJA1 as a novel interacting partner and a substrate of human transglutaminase 2

Biochemical Journal ◽

10.1042/bcj20160440 ◽

2016 ◽

Vol 473 (21) ◽

pp. 3889-3901 ◽

Cited By ~ 1

Author(s):

Elvan Ergülen ◽

Bálint Bécsi ◽

István Csomós ◽

László Fésüs ◽

Kajal Kanchan

Keyword(s):

Molecular Mechanisms ◽

Transglutaminase 2 ◽

Mass Spectrometry Analysis ◽

Catalytic Core ◽

Spectrometry Analysis ◽

Inactive Form ◽

In Situ Conditions ◽

And Migration ◽

Regulated Functions

Transglutaminase 2 (TG2) is a ubiquitously expressed multifunctional member of the transglutaminase enzyme family. It has been implicated to have roles in many physiological and pathological processes such as differentiation, apoptosis, signal transduction, adhesion and migration, wound healing and inflammation. Previous studies revealed that TG2 has various intra- and extra-cellular interacting partners, which contribute to these processes. In the present study, we identified a molecular co-chaperone, DNAJA1, as a novel interacting partner of human TG2 using a GST pull-down assay and subsequent mass spectrometry analysis, and further confirmed this interaction via ELISA and surface plasmon resonance measurements. Interaction studies were also performed with domain variants of TG2 and results suggest that the catalytic core domain of TG2 is essential for the TG2–DNAJA1 interaction. Cross-linking activity was not essential for the interaction since DNAJA1 was also found to interact with the catalytically inactive form of TG2. Furthermore, we have showed that DNAJA1 interacts with the open form of TG2 and regulates its transamidation activity under both in vitro and in situ conditions. We also found that DNAJA1 is a glutamine donor substrate of TG2. Since DNAJA1 and TG2 are reported to regulate common pathological conditions such as neurodegenerative disorders and cancer, the findings in the present paper open up possibilities to explore molecular mechanisms behind TG2-regulated functions.

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CircLIFR synergizes with MSH2 to attenuate chemoresistance via MutSα/ATM-p73 axis in bladder cancer

Molecular Cancer ◽

10.1186/s12943-021-01360-4 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Hui Zhang ◽

Xingyuan Xiao ◽

Wenjie Wei ◽

Chao Huang ◽

Miao Wang ◽

...

Keyword(s):

Bladder Cancer ◽

Molecular Mechanisms ◽

Mass Spectrometry Analysis ◽

Tumor Xenograft ◽

Circular Rnas ◽

Spectrometry Analysis ◽

Xenograft Models ◽

Pdx Models

Abstract Background Cisplatin (CDDP) has become a standard-of-care treatment for muscle-invasive bladder cancer (MIBC), while chemoresistance remains a major challenge. Accumulating evidence indicates that circular RNAs (circRNAs) are discrete functional entities. However, the regulatory functions as well as complexities of circRNAs in modulating CDDP-based chemotherapy in bladder cancer are yet to be well revealed. Methods Through analyzing the expression profile of circRNAs in bladder cancer tissues, RNA FISH, circRNA pull-down assay, mass spectrometry analysis and RIP, circLIFR was identified and its interaction with MSH2 was confirmed. The effects of circLIFR and MSH2 on CDDP-based chemotherapy were explored by flow cytometry and rescue experiments. Co-IP and Western blot were used to investigate the molecular mechanisms underlying the functions of circLIFR and MSH2. Biological implications of circLIFR and MSH2 in bladder cancer were implemented in tumor xenograft models and PDX models. Results CircLIFR was downregulated in bladder cancer and expression was positively correlated with favorable prognosis. Moreover, circLIFR synergizing with MSH2, which was a mediator of CDDP sensitivity in bladder cancer cells, positively modulated sensitivity to CDDP in vitro and in vivo. Mechanistically, circLIFR augmented the interaction between MutSα and ATM, ultimately contributing to stabilize p73, which triggered to apoptosis. Importantly, MIBC with high expression of circLIFR and MSH2 was more sensitive to CDDP-based chemotherapy in tumor xenograft models and PDX models. Conclusions CircLIFR could interact with MSH2 to positively modulate CDDP-sensitivity through MutSα/ATM-p73 axis in bladder cancer. CircLIFR and MSH2 might be act as promising therapeutic targets for CDDP-resistant bladder cancer.

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Phytochemicals of Minthostachys diffusa Epling and Their Health-Promoting Bioactivities

Foods ◽

10.3390/foods9020144 ◽

2020 ◽

Vol 9 (2) ◽

pp. 144

Author(s):

Immacolata Faraone ◽

Daniela Russo ◽

Lucia Chiummiento ◽

Eloy Fernandez ◽

Alka Choudhary ◽

...

Keyword(s):

Ethyl Acetate ◽

Radical Scavenging ◽

Ethyl Acetate Fraction ◽

Mass Spectrometry Analysis ◽

Antioxidant Power ◽

Spectrometry Analysis ◽

In Silico Docking ◽

Aerial Parts ◽

Health Promoting

The genus Minthostachys belonging to the Lamiaceae family, and is an important South American mint genus used commonly in folk medicine as an aroma in cooking. The phytochemical-rich samples of the aerial parts of Minthostachys diffusa Epling. were tested for pharmacological and health-promoting bioactivities using in vitro chemical and enzymatic assays. A range of radical scavenging activities of the samples against biological radicals such as nitric oxide and superoxide anion and against synthetic 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radicals, the ferric reducing antioxidant power and the lipid peroxidation inhibition were determined and ranked using the ‘relative antioxidant capacity index’ (RACI). The ethyl acetate fraction showed the highest RACI of +1.12. Analysis of the various fractions’ inhibitory ability against enzymes involved in diabetes (α-amylase and α-glucosidase), and against enzymes associated with Parkinson’s or Alzheimer’s diseases (acetylcholinesterase and butyrylcholinesterase) also suggested that the ethyl acetate fraction was the most active. Liquid chromatography–tandem mass spectrometry analysis of the ethyl acetate fraction showed more than 30 polyphenolic compounds, including triterpenes. The inhibitory cholinesterase effects of the triterpenes identified from M. diffusa were further analysed by in silico docking of these compounds into 3D-structures of acetylcholinesterase and butyrylcholinesterase. This is the first study on pharmacological activities and phytochemical profiling of the aerial parts of M. diffusa, showing that this plant, normally used as food in South America, is also rich in health-promoting phytochemicals.

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A Comparative Study of the Antioxidative Effects of Helichrysum italicum and Helichrysum arenarium Infusions

Antioxidants ◽

10.3390/antiox10030380 ◽

2021 ◽

Vol 10 (3) ◽

pp. 380

Author(s):

Katja Kramberger ◽

Zala Jenko Pražnikar ◽

Alenka Baruca Arbeiter ◽

Ana Petelin ◽

Dunja Bandelj ◽

...

Keyword(s):

Oxidative Stress ◽

High Performance ◽

Radical Scavenging Activity ◽

Radical Scavenging ◽

Morphological Type ◽

Mass Spectrometry Analysis ◽

In Vitro Assays ◽

Spectrometry Analysis ◽

Stress Related Genes

Helichrysum arenarium (L.) Moench (abbrev. as HA) has a long tradition in European ethnomedicine and its inflorescences are approved as a herbal medicinal product. In the Mediterranean part of Europe, Helichrysum italicum (Roth) G. Don (abbrev. as HI) is more common. Since infusions from both plants are traditionally used, we aimed to compare their antioxidative potential using in vitro assays. Two morphologically distinct HI plants, HIa and HIb, were compared to a commercially available HA product. Genetic analysis using microsatellites confirmed a clear differentiation between HI and HA and suggested that HIb was a hybrid resulting from spontaneous hybridization from unknown HI subspecies. High-performance liquid chromatography–mass spectrometry analysis showed the highest amounts of hydroxycinnamic acids and total arzanol derivatives in HIa, whereas HIb was richest in monohydroxybenzoic acids, caffeic acids, and coumarins, and HA contained the highest amounts of flavonoids, especially flavanones. HIa exhibited the highest radical scavenging activity; it was more efficient in protecting different cell lines from induced oxidative stress and in inducing oxidative stress-related genes superoxide dismutase 1, catalase, and glutathione reductase 1. The antioxidative potential of HI was not only dependent on the morphological type of the plant but also on the harvest date, revealing important information for obtaining the best possible product. Considering the superior properties of HI compared to HA, the evaluation of HI as a medicinal plant could be recommended.

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Phytotoxicity of Essential Oils on Selected Weeds: Potential Hazard on Food Crops

Plants ◽

10.3390/plants7040079 ◽

2018 ◽

Vol 7 (4) ◽

pp. 79 ◽

Cited By ~ 12

Author(s):

María Ibáñez ◽

María Blázquez

Keyword(s):

Seed Germination ◽

Essential Oil ◽

Essential Oils ◽

Mass Spectrometry Analysis ◽

Gas Chromatography Mass Spectrometry ◽

Food Crops ◽

Potential Hazard ◽

Spectrometry Analysis

The chemical composition of winter savory, peppermint, and anise essential oils, and in vitro and in vivo phytotoxic activity against weeds (Portulaca oleracea, Lolium multiflorum, and Echinochloa crus-galli) and food crops (maize, rice, and tomato), have been studied. Sixty-four compounds accounting for between 97.67–99.66% of the total essential oils were identified by Gas Chromatography-Mass Spectrometry analysis. Winter savory with carvacrol (43.34%) and thymol (23.20%) as the main compounds produced a total inhibitory effect against the seed germination of tested weed. Menthol (48.23%), menthone (23.33%), and iso-menthone (16.33%) from peppermint only showed total seed germination inhibition on L. multiflorum, whereas no significant effects were observed with trans-anethole (99.46%) from anise at all concentrations (0.125–1 µL/mL). Low doses of peppermint essential oil could be used as a sustainable alternative to synthetic agrochemicals to control L. multiflorum. The results corroborate that in vivo assays with a commercial emulsifiable concentrate need higher doses of the essential oils to reproduce previous in vitro trials. The higher in vivo phytotoxicity of winter savory essential oil constitutes an eco-friendly and less pernicious alternative to weed control. It is possible to achieve a greater in vivo phytotoxicity if less active essential oil like peppermint is included with other active excipients.

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