Alpha-Synuclein is a Target of Fic-mediated Adenylylation/AMPylation: Implications for Parkinson’s Disease

Sanyal, Anwesha; Dutta, Sayan Deb; Chandran, Aswathy; Koller, Antonius; Camara, Ali; Watson, Ben G.; Sengupta, Ranjan; Ysselstein, Daniel; Montenegro, Paola; Cannon, Jason R.; Rochet, Jean-Christophe; Mattoo, Seema

doi:10.1101/525659

Cited by 3 publications

References 53 publications

(41 reference statements)

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AMPylation is a specific lysosomal protein posttranslational modification in neuronal maturation

Becker

Cappel

et al. 2021

Preprint

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Protein AMPylation is a pervasive posttranslational modification with an emerging role in neurodevelopment. In metazoans the two highly conserved protein AMP-transferases together with a diverse group of AMPylated proteins have been identified using chemical proteomics and biochemical techniques. However, the function of this modification remains largely unknown. Particularly problematic is the localization of thus far identified AMPylated proteins and putative AMP-transferases. Here, we uncover protein AMPylation as a novel posttranslational modification of luminal lysosomal proteins characteristic in differentiating neurons. Through a combination of chemical proteomics, advanced gel-based separation of modified and unmodified proteins and activity assay, we show that an AMPylated, lysosomal soluble form of exonuclease PLD3 increases dramatically during neuronal maturation and that AMPylation inhibits its catalytic activity. Together, our findings unveil so far unknown lysosomal posttranslational modification, its connection to neuronal differentiation and putatively provide a novel molecular rationale to design of therapeutics for lysosomal storage diseases.

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AMPylation is a specific lysosomal protein posttranslational modification in neuronal maturation

Becker

Cappel

et al. 2021

Preprint

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Transforming chemical proteomics enrichment into high-throughput method using SP2E workflow

Becker

Wiest

Telek

et al. 2022

Preprint

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Protein post-translational modifications (PTMs) play a critical role in regulation of protein catalytic activity, localization and protein-protein interactions. An attachment of PTMs onto proteins significantly diversifies their structure and function resulting in so-called proteoforms. However, the sole identification of post-translationally modified proteins, which are often cell type and disease specific, is still a highly challenging task. Sub-stoichiometric amounts and modification of low abundant proteins necessitate purification or enrichment of the modified proteins. Although the introduction of the mass spectrometry-based chemical proteomic strategy has enabled to screen protein PTMs with increased throughput, sample preparation has remained highly time consuming and tedious. Here, we report an optimized workflow for enrichment of PTM proteins in 96-well plate format which can be possible extended to robotic automatization. This platform allows to significantly lower the input of total protein, which opens up the opportunity to screen specialized and difficult to culture cell lines in high-throughput manner. The presented SP2E protocol is robust, time- and cost-effective as well as suitable for large-scale screening of proteoforms. Application of the SP2E protocol will thus enable the characterization of proteoforms in various processes such as neurodevelopment, neurodegeneration and cancer and may contribute to an overall acceleration of the recently launched Human Proteoform Project.

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A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD

Camara

George

Hebner

et al. 2020

IJMS

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The covalent transfer of the AMP portion of ATP onto a target protein—termed adenylylation or AMPylation—by the human Fic protein HYPE/FICD has recently garnered attention as a key regulatory mechanism in endoplasmic reticulum homeostasis, neurodegeneration, and neurogenesis. As a central player in such critical cellular events, high-throughput screening (HTS) efforts targeting HYPE-mediated AMPylation warrant investigation. Herein, we present a dual HTS assay for the simultaneous identification of small-molecule activators and inhibitors of HYPE AMPylation. Employing the fluorescence polarization of an ATP analog fluorophore—Fl-ATP—we developed and optimized an efficient, robust assay that monitors HYPE autoAMPylation and is amenable to automated, high-throughput processing of diverse chemical libraries. Challenging our pilot screen with compounds from the LOPAC, Spectrum, MEGx, and NATx libraries yielded 0.3% and 1% hit rates for HYPE activators and inhibitors, respectively. Further, these hits were assessed for dose-dependency and validated via orthogonal biochemical AMPylation assays. We thus present a high-quality HTS assay suitable for tracking HYPE’s enzymatic activity, and the resultant first small-molecule manipulators of HYPE-promoted autoAMPylation.

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Alpha-Synuclein is a Target of Fic-mediated Adenylylation/AMPylation: Implications for Parkinson’s Disease

Cited by 3 publications

References 53 publications

AMPylation is a specific lysosomal protein posttranslational modification in neuronal maturation

AMPylation is a specific lysosomal protein posttranslational modification in neuronal maturation

Transforming chemical proteomics enrichment into high-throughput method using SP2E workflow

A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD

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