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Structural basis of chaperone regulation by a post-translational modification

ABSTRACT: The Hsp70 chaperone BiP is covalently modified with adenosine monophosphate (referred to as AMPylation) in order to adapt its activity to the fluctuating folding load within the endoplasmic reticulum. This modification is catalyzed by the only human representative of the family of filamentation induced by cyclic adenosine monophosphate (Fic) enzymes HYPE/FICD. The structural basis for BiP binding and AMPylation has remained elusive due to the low affinity of enzyme substrate complexes.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Christoph Krisp

LAB HEAD: Prof. Dr. Aymelt Itzen

PROVIDER: PXD022869 | Pride | 2021-04-27

REPOSITORIES: Pride

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Specificity of AMPylation of the human chaperone BiP is mediated by TPR motifs of FICD.

Fauser Joel J Gulen Burak B Pogenberg Vivian V Pett Christian C Pourjafar-Dehkordi Danial D Krisp Christoph C Höpfner Dorothea D König Gesa G Schlüter Hartmut H Feige Matthias J MJ Zacharias Martin M Hedberg Christian C Itzen Aymelt A

Nature communications 20210423 1

To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determi ...[more]

PMID: 33893288

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FICD activity and AMPylation remodelling modulate human neurogenesis

Project description:The AMPylation is novel protein post-translation modification. In consist of attachment of the adenosine monophosphate onto the serine, threonine or tyrosine amino acid residues of the protein. This process is catalysed by bacterial effectors or in human by protein FICD (also called HYPE) which contain conserved fic domain. In our study we have focused on identification of the unknown AMPylated proteins and their function in human cells (HeLa, A549, SH-SY5Y, iPSCs, NPCs and neurons) and cerebral organoids (COs). For this purpose we designed and synthesized the small molecule N6-propargyl phosphoramidate adenosine (pro-N6pA) which was used for in situ labelling of the AMPylation in cells and COs. Subsequnetly the the propargyl tag was employed for preparation of chemical-proteomic samples which were measured by LC-MS/MS. This approach confirmed the known AMPylation of heat shock protein HSPA5 and found diverse group of novel AMPylation targets. Several cathepsins found as AMPylation targets were further characterized by identification of AMP binding site. Based on our chemical proteomic data we found out that neurons and neuronal like cells contain distinct AMPylatino pattern from other studied cell types. This also suggested the importance of the AMPylation in these cell types which were then studied in cerebral organoids by overexpressing the FICD wt and E234G highly active mutant. This led to the accelerated differentiation of progenitors into the neurons. Overall our approach is suitable for identification of the AMPylated proteins in living cells and led to characterization of FICD function.

2019-12-04 | PXD015062 | Pride

The DNA-binding induced (de)AMPylation activity of a Coxiella burnetii Fic enzyme targets Histone H3

Project description:Intracellular bacterial pathogens such as Coxiella burnetii evade their host's immune response by secreting effector proteins that bind and modify host proteins, thereby reshaping cell signaling pathways that help establish a replication-friendly niche. Previous research has identified Filamentation induced by cyclic AMP (Fic) enzymes as effector proteins in bacterial infections that modify their target proteins via the post-translational modification AMPylation. Fic enzymes use adenosine triphosphate (ATP) as co-substrate to transfer the adenosine monophosphate (AMP) group to a hydroxyl-containing side chain of their target protein. Many representatives of this enzyme class contain an autoinhibitory helix that suppresses AMPylation activity in vitro by contacting the active site. The mechanisms reversing this inhibition are incompletely understood. Extensive studies of the only human Fic protein FICD have revealed that FICD can switch between AMPylation and the reversal of this modification, deAMPylation, by a monomer-dimer transition. However, so far FICD remained the only example where deAMPylation by dimerization could be shown. Here we identify the protein product of C. burnetii CBU_0822, termed C. burnetii Fic 2 (CbFic2), to AMPylate host cell histone H3 at S10 and S28. We show that CbFic2 is a bifunctional enzyme, both capable of AMPylation as well as deAMPylation, and is regulated by the binding of DNA via its C-terminal helix-turn-helix domain. We propose that CbFic2 is capable of AMPylation in its monomeric form, and that binding to DNA induces a deAMPylating dimer.

2024-01-26 | PXD040330 | Pride

Diadenosine tetraphosphate as a cosubstrate for AMPylation mediated by FICD

Project description:Diadenosine polyphosphates (ApnAs) are noncanonical nucleotides that are ubiquitous across all forms of life. Due to their strong accumulation upon cellular stress, they are considered alarmones to signal homeostatic imbalance. Nonetheless, their cellular role is poorly understood. In this work, we assessed ApnAs for their usage as cosubstrate for protein AMPylation, a post-translational modification. In humans, AMPylation mediated by the AMPylator FICD with ATP as cosubstrate is a response to ER stress. Here, we demonstrate that Ap4A is proficiently consumed for AMPylation by FICD. By chemical proteomics using a new chemical probe, we identified new potential AMPylation targets. Interestingly, we found that AMPylation targets of FICD may differ depending on the nucleotide cosubstrate. These results may suggest that signaling at elevated Ap4A levels during cellular stress differs from when Ap4A is present at low concentrations allowing response to extracellular cues.

2023-01-27 | PXD036450 | Pride

HYPE mediated AMPylation site profiling with YnATP in human lysates

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2015-12-02 | PXD003053 | Pride

HYPE mediated AMPylation profiling in human lysates

Project description:We describe here the first example of global chemoproteomic screening and substrate validation for HYPE mediated AMPylation in mammalian cell lysate. Through quantitative mass spectrometry-based proteomics coupled with novel chemoproteomic tools providing MS/MS evidence of AMP modification we identified a total of 27 AMPylated proteins, including the previously validated substrate, ER chaperone BiP (HSPA5), along with novel substrates involved in pathways of gene expression, ATP biosynthesis and maintenance of cytoskeleton. This dataset represents the largest library of AMPylated human proteins reported to date and a foundation for substrate-specific investigations that can ultimately decipher the complex biological networks involved in eukaryotic AMPylation.

2015-12-02 | PXD002601 | Pride

Rab1b-AMPylation by Legionella DrrA is allosterically activated by Rab1

Project description:Legionella pneumophila infect eukaryotic cells by forming a replicative organelle – the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and posttranslational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP) – a process referred to as AMPylation. Here, we used a chemical approach for stabilizing low affinity Rab:DrrA complexes in a site-specific manner to gain insights into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The X-ray crystal structure of the Rab:DrrA complex revealed a previously unrecognized non-conventional Rab binding site (NC-RBS). Biochemical characterizations demonstrated allosteric stimulation of DrrA’s AMPylation activity via Rab-binding to the NC-RBS. We propose that allosteric control of DrrA not only prevents random and potentially cytotoxic AMPylation in the host, thereby ensuring an efficient infection process by Legionella, but also represents an unprecedented AMPylation activation mechanism.

2021-01-04 | PXD019043 | Pride

HYPE mediated AMPylation profiling in human lysates

2017-03-29 | MSV000080782 | MassIVE

P. aeruginosa PA14 WT in LB medium vs in LB with 10 mM adenosine medium

Project description:To investigate the impact of adenosine on gene expression of wild-type PA14. To investigate the impact of adenosine on gene expression of wild-type PA14, cells were harvested after incubating for 7 h in LB medium and LB with 10 mM adenosine medium, and RNA was extracted with the RNeasy Mini Kit (Qiagen, Valencia, CA) using a bead beater (Biospec, Bartlesville, OK ) with RNAlater buffer (Applied Biosystems, Foster City, CA) to stabilize the RNA.

2012-05-22 | E-GEOD-29665 | biostudies-arrayexpress

Adenosine-treated endothelial progenitor cells

Project description:The goal of this study was to investigate the effects of the cardioprotective nucleoside adenosine on gene expression in early and late endothelial progenitor cells. Adenosine mod Early outgrowth endothelial progenitor cells were obtained by adhesion of peripheral blood mononuclear cells of healthy volunteers. Late endothelial progenitor cells were obtained by purification of CD34+ peripheral blood cells and were cultured and amplified in endothelial-specific medium containing growth factors. Both cell types were treated by adenosine (10micromol/L) for 6 hours. Total RNA was extracted using conventional Trizol extraction, quantified by Nanodrop and analyzed by Bioanalyzer. 1microg of RNA was amplified using AminoAllyl MessageAmp kit. 5µg amino allyl-coupled RNA was labeled with Cy3 or Cy5 dyes. Dye coupling yield >5% was a prerequisite for further analysis. 750ng of labeled RNA was hybridized on 25,000 gene microarrays for 17 hours at 60°C. 4 arrays per sample were hybridized and scanned with the Genepix 4000B Scanner (Molecular Devices). Six independent experiments were performed. Microarray data quantification and pre-processing was performed with the MAIA software and intensity values were log-transformed. Gene expression values were standardized across experiments with mean = 0 and standard deviation = 1. The SAM tool was applied to identify differentially expressed genes.

2011-01-29 | E-GEOD-26744 | biostudies-arrayexpress

Transcription profiling by array of rat cerebellar granular neuronal precursor cells plated with Sonic Hedgehog (Shh) and then treated with the PKA activator DBA

Project description:Cerebellar granular neuronal precursors were plated in presence of Sonic Hedgehog (Shh) for 24h and then treated with the PKA activator Dibutyryl Cyclic Adenosine Monophosphate (DBA) for addittional 24h

2008-07-11 | E-MEXP-1668 | biostudies-arrayexpress

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