Protein AMPylation Research Articles

Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O.

Defining the biological importance of proteins with unknown functions poses a significant obstacle in understanding cellular processes. Although bioinformatic and structural predictions have contributed to the study of unknown proteins, in vitro experimental validations are often hampered by the optimal conditions and cofactors required for biochemical activity. Cofactor binding is not only essential for the activity of some enzymes but may also enhance the thermal stability of the protein. One practical application of this phenomenon lies in utilizing the change in thermal stability, as measured by alterations in the protein's melting temperature, to probe ligand binding. Thermal shift assay (TSA) can be used to analyze the binding of different ligands to the protein of interest or find a stabilizing condition to perform experiments such as X-ray crystallography. Here we will describe a protocol for TSA utilizing the pseudokinase, Selenoprotein O (SelO), for a simple and high-throughput method for testing metal and nucleotide binding. In contrast to canonical kinases, SelO binds ATP in an inverted orientation to catalyze the transfer of AMP to the hydroxyl side chains of proteins in a posttranslational modification known as protein AMPylation. By leveraging the shift in the melting temperatures, we provide crucial insights into the molecular interactions underlying SelO function.

Small molecule FICD inhibitors suppress endogenous and pathologic FICD-mediated protein AMPylation.

The AMP transferase, FICD, is an emerging drug target finetuning stress signaling in the endoplasmic reticulum (ER). FICD is a bi-functional enzyme, catalyzing both AMP addition (AMPylation) and removal (deAMPylation) from the ER resident chaperone BiP/GRP78. Despite increasing evidence linking excessive BiP/GRP78 AMPylation to human diseases, small molecules to inhibit pathogenic FICD variants are lacking. Using an in-vitro high-throughput screen, we identify two small-molecule FICD inhibitors, C22 and C73. Both molecules significantly inhibit FICD-mediated BiP/GRP78 AMPylation in intact cells while only weakly inhibiting BiP/GRP78 deAMPylation. C22 and C73 also efficiently inhibit pathogenic FICD variants and improve proinsulin processing in β cells. Our study identifies and validates FICD inhibitors, highlighting a novel therapeutic avenue against pathologic protein AMPylation.

Legionella maintains host cell ubiquitin homeostasis by effectors with unique catalytic mechanisms

The intracellular bacterial pathogen Legionella pneumophila modulates host cell functions by secreting multiple effectors with diverse biochemical activities. In particular, effectors of the SidE family interfere with host protein ubiquitination in a process that involves production of phosphoribosyl ubiquitin (PR-Ub). Here, we show that effector LnaB converts PR-Ub into ADP-ribosylated ubiquitin, which is further processed to ADP-ribose and functional ubiquitin by the (ADP-ribosyl)hydrolase MavL, thus maintaining ubiquitin homeostasis in infected cells. Upon being activated by actin, LnaB also undergoes self-AMPylation on tyrosine residues. The activity of LnaB requires a motif consisting of Ser, His and Glu (SHxxxE) present in a large family of toxins from diverse bacterial pathogens. Thus, our study sheds light on the mechanisms by which a pathogen maintains ubiquitin homeostasis and identifies a family of enzymes capable of protein AMPylation.

Legionella maintains host cell ubiquitin homeostasis by effectors with unique catalytic mechanisms.

The reversal of ubiquitination induced by members of the SidE effector family of Legionella pneumophila produces phosphoribosyl ubiquitin (PR-Ub) that is potentially detrimental to host cells. Here we show that the effector LnaB functions to transfer the AMP moiety from ATP to the phosphoryl moiety of PR-Ub to convert it into ADP-ribosylated ubiquitin (ADPR-Ub), which is further processed to ADP-ribose and functional ubiquitin by the (ADP-ribosyl)hydrolase MavL, thus maintaining ubiquitin homeostasis in infected cells. Upon being activated by Actin, LnaB also undergoes self-AMPylation on tyrosine residues. The activity of LnaB requires a motif consisting of Ser, His and Glu (S-HxxxE) present in a large family of toxins from diverse bacterial pathogens. Our study not only reveals intricate mechanisms for a pathogen to maintain ubiquitin homeostasis but also identifies a new family of enzymes capable of protein AMPylation, suggesting that this posttranslational modification is widely used in signaling during host-pathogen interactions.

The Alarmone Diadenosine Tetraphosphate as a Cosubstrate for Protein AMPylation.

Diadenosine polyphosphates (Apn As) are non-canonical nucleotides whose cellular concentrations increase during stress and are therefore termed alarmones, signaling homeostatic imbalance. Their cellular role is poorly understood. In this work, we assessed Apn As for their usage as cosubstrates for protein AMPylation, a post-translational modification in which adenosine monophosphate (AMP) is transferred to proteins. In humans, AMPylation mediated by the AMPylator FICD with ATP as a cosubstrate is a response to ER stress. Herein, we demonstrate that Ap4 A 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 Ap4 A levels during cellular stress differs from when Ap4 A is present at low concentrations, allowing response to extracellular cues.

Das Alarmon Diadenosintetraphosphat als Cosubstrat für die AMPylierung von Proteinen

AbstractDiadenosinpolyphosphate (ApnAs) sind nicht‐kanonische Nukleotide, deren zelluläre Konzentrationen bei Stress als Signal eines homöostatischen Ungleichgewichts ansteigen und die daher als Alarmone bezeichnet werden. Ihre zelluläre Rolle ist nur unzureichend verstanden. In dieser Arbeit haben wir untersucht, ob ApnAs als Cosubstrat für die AMPylierung von Proteinen verwendet werden. AMPylierung ist eine posttranslationale Modifikation, bei der Adenosinmonophosphat (AMP) auf Proteine übertragen wird. Beim Menschen ist die AMPylierung, die durch den AMPylator FICD mit ATP als Cosubstrat vermittelt wird, eine Reaktion auf ER‐Stress. Hier zeigen wir, dass Ap4A für die AMPylierung durch FICD effizient verwendet wird. Durch chemische Proteomik unter Verwendung einer neuen chemischen Sonde haben wir neue potenzielle AMPylierungsziele identifiziert. Interessanterweise haben wir festgestellt, dass sich die AMPylierungsziele von FICD je nach Nukleotid‐Cosubstrat unterscheiden können. Diese Ergebnisse könnten darauf hindeuten, dass sich die Signalübertragung bei erhöhten Ap4A‐Spiegeln während zellulärem Stress von derjenigen bei niedrigen Ap4A Konzentrationen unterscheidet, und damit eine Reaktion auf extrazelluläre Signale ermöglicht.

A convolutional neural network based tool for predicting protein AMPylation sites from binary profile representation

AMPylation is an emerging post-translational modification that occurs on the hydroxyl group of threonine, serine, or tyrosine via a phosphodiester bond. AMPylators catalyze this process as covalent attachment of adenosine monophosphate to the amino acid side chain of a peptide. Recent studies have shown that this post-translational modification is directly responsible for the regulation of neurodevelopment and neurodegeneration and is also involved in many physiological processes. Despite the importance of this post-translational modification, there is no peptide sequence dataset available for conducting computation analysis. Therefore, so far, no computational approach has been proposed for predicting AMPylation. In this study, we introduce a new dataset of this distinct post-translational modification and develop a new machine learning tool using a deep convolutional neural network called DeepAmp to predict AMPylation sites in proteins. DeepAmp achieves 77.7%, 79.1%, 76.8%, 0.55, and 0.85 in terms of Accuracy, Sensitivity, Specificity, Matthews Correlation Coefficient, and Area Under Curve for AMPylation site prediction task, respectively. As the first machine learning model, DeepAmp demonstrate promising results which highlight its potential to solve this problem. Our presented dataset and DeepAmp as a standalone predictor are publicly available at https://github.com/MehediAzim/DeepAmp.

AMPylation profiling during neuronal differentiation reveals extensive variation on lysosomal proteins

SummaryProtein AMPylation is a posttranslational modification with an emerging role in neurodevelopment. In metazoans 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 AMPylation remains largely unknown. Particularly problematic is the localization of thus far identified AMPylated proteins and putative AMP-transferases. We show that protein AMPylation is likely a posttranslational modification of luminal lysosomal proteins characteristic in differentiating neurons. Through a combination of chemical proteomics, gel-based separation of modified and unmodified proteins, and an activity assay, we determine that the modified, lysosomal soluble form of exonuclease PLD3 increases dramatically during neuronal maturation and that AMPylation correlates with its catalytic activity. Together, our findings indicate that AMPylation is a so far unknown lysosomal posttranslational modification connected to neuronal differentiation and it may provide a molecular rationale behind lysosomal storage diseases and neurodegeneration.

Over-expression of the constitutive AMPylase FIC-1(E274G) does not deplete cellular ATP pools in C. elegans.

Protein AMPylation has emerged as a posttranslational protein modification regulating cellular proteostasis. AMPylation is conferred by Fic AMPylases, which catalyze the covalent attachment of AMP to target proteins at the expense of ATP. Over-expression of constitutive-active Fic AMPylases is toxic. Here, we test the hypothesis that excessive Fic AMPylase activity could deplete cellular ATP pools, leading to cell death. We find that increased/decreased Fic AMPylase activity only alters cellular ATP concentrations by approximately 15%. This suggests that hyper-AMPylation-mediated cell death is likely not the consequence of cellular ATP depletion.

Fic and non-Fic AMPylases: protein AMPylation in metazoans.

Protein AMPylation refers to the covalent attachment of an AMP moiety to the amino acid side chains of target proteins using ATP as nucleotide donor. This process is catalysed by dedicated AMP transferases, called AMPylases. Since this initial discovery, several research groups have identified AMPylation as a critical post-translational modification relevant to normal and pathological cell signalling in both bacteria and metazoans. Bacterial AMPylases are abundant enzymes that either regulate the function of endogenous bacterial proteins or are translocated into host cells to hijack host cell signalling processes. By contrast, only two classes of metazoan AMPylases have been identified so far: enzymes containing a conserved filamentation induced by cAMP (Fic) domain (Fic AMPylases), which primarily modify the ER-resident chaperone BiP, and SelO, a mitochondrial AMPylase involved in redox signalling. In this review, we compare and contrast bacterial and metazoan Fic and non-Fic AMPylases, and summarize recent technological and conceptual developments in the emerging field of AMPylation.

Role of Selenoproteins in Redox Regulation of Signaling and the Antioxidant System: A Review.

Selenium is a vital trace element present as selenocysteine (Sec) in proteins that are, thus, known as selenoproteins. Humans have 25 selenoproteins, most of which are functionally characterized as oxidoreductases, where the Sec residue plays a catalytic role in redox regulation and antioxidant activity. Glutathione peroxidase plays a pivotal role in scavenging and inactivating hydrogen and lipid peroxides, whereas thioredoxin reductase reduces oxidized thioredoxins as well as non-disulfide substrates, such as lipid hydroperoxides and hydrogen peroxide. Selenoprotein R protects the cell against oxidative damage by reducing methionine-R-sulfoxide back to methionine. Selenoprotein O regulates redox homeostasis with catalytic activity of protein AMPylation. Moreover, endoplasmic reticulum (ER) membrane selenoproteins (SelI, K, N, S, and Sel15) are involved in ER membrane stress regulation. Selenoproteins containing the CXXU motif (SelH, M, T, V, and W) are putative oxidoreductases that participate in various cellular processes depending on redox regulation. Herein, we review the recent studies on the role of selenoproteins in redox regulation and their physiological functions in humans, as well as their role in various diseases.

A Pronucleotide Probe for Live-Cell Imaging of Protein AMPylation.

Conjugation of proteins to AMP (AMPylation) is a prevalent post‐translational modification (PTM) in human cells, involved in the regulation of unfolded protein response and neural development. Here we present a tailored pronucleotide probe suitable for in situ imaging and chemical proteomics profiling of AMPylated proteins. Using straightforward strain‐promoted azide–alkyne click chemistry, the probe provides stable fluorescence labelling in living cells.

Protein AMPylation by an evolutionarily conserved pseudokinase

Protein AMPylation by an evolutionarily conserved pseudokinase

Protein AMPylation by an Evolutionarily Conserved Pseudokinase

Approximately 10% of human protein kinases are believed to be inactive and named pseudokinases because they lack residues required for catalysis. Here, we show that the highly conserved pseudokinase selenoprotein-O (SelO) transfers AMP from ATP to Ser, Thr, and Tyr residues on protein substrates (AMPylation), uncovering a previously unrecognized activity for a member of the protein kinase superfamily. The crystal structure of a SelO homolog reveals a protein kinase-like fold with ATP flipped in the active site, thus providing a structural basis for catalysis. SelO pseudokinases localize to the mitochondria and AMPylate proteins involved in redox homeostasis. Consequently, SelO activity is necessary for the proper cellular response to oxidative stress. Our results suggest that AMPylation may be a more widespread post-translational modification than previously appreciated and that pseudokinases should be analyzed for alternative transferase activities.

RAMPing Up Stress Signaling: Protein AMPylation in Metazoans.

Protein AMPylation - the covalent attachment of an AMP residue to amino acid side chains using ATP as the donor - is a post-translational modification (PTM) increasingly appreciated as relevant for both normal and pathological cell signaling. In metazoans single copies of filamentation induced by cAMP (fic)-domain-containing AMPylases - the enzymes responsible for AMPylation - preferentially modify a set of dedicated targets and contribute to the perception of cellular stress and its regulation. Pathogenic bacteria can exploit AMPylation of eukaryotic target proteins to rewire host cell signaling machinery in support of their propagation and survival. We review endogenous as well as parasitic protein AMPylation in metazoans and summarize current views of how fic-domain-containing AMPylases contribute to cellular proteostasis.

In vitro AMPylation Assays Using Purified, Recombinant Proteins.

Post-translational protein modifications (PTMs) orchestrate the activity of individual proteins and ensure their proper function. While modifications such as phosphorylation or glycosylation are well understood, more unusual modifications, including nitrosylation or AMPylation remain comparatively poorly characterized. Research on protein AMPylation-which refers to the covalent addition of an AMP moiety to the side chains of serine, threonine or tyrosine-has undergone a renaissance (Yarbrough et al., 2009; Engel et al., 2012; Ham et al., 2014; Woolery et al., 2014; Preissler et al., 2015; Sanyal et al., 2015; Truttmann et al., 2016; Truttmann et al., 2017). The identification and characterization of filamentation (fic) domain-containing AMPylases sparked new interest in this PTM (Kinch et al., 2009; Yarbrough et al., 2009). Based on recent in vivo and in vitro studies, we now know that secreted bacterial AMPylases covalently attach AMP to members of the Rho family of GTPases, while metazoan AMPylases modify HSP70 family proteins in the cytoplasm and the endoplasmic reticulum (ER) (Itzen et al., 2011; Hedberg and Itzen, 2015; Truttmann and Ploegh, 2017). AMPylation is thought to trap HSP70 in a primed yet transiently disabled state that cannot participate in protein refolding reactions (Preissler et al., 2015). In vitro AMPylation experiments are key to assess the activity, kinetics and specificity of protein AMPylation catalyzed by pro- and eukaryotic enzymes. These simple assays require recombinant AMPylases, target proteins (Rho GTPases, HSP70s), as well as ATP as a nucleotide source. Here, we describe strategies to qualitatively and quantitatively study protein AMPylation in vitro.

The Caenorhabditis elegans Protein FIC-1 Is an AMPylase That Covalently Modifies Heat-Shock 70 Family Proteins, Translation Elongation Factors and Histones.

Protein AMPylation by Fic domain-containing proteins (Fic proteins) is an ancient and conserved post-translational modification of mostly unexplored significance. Here we characterize the Caenorhabditis elegans Fic protein FIC-1 in vitro and in vivo. FIC-1 is an AMPylase that localizes to the nuclear surface and modifies core histones H2 and H3 as well as heat shock protein 70 family members and translation elongation factors. The three-dimensional structure of FIC-1 is similar to that of its human ortholog, HYPE, with 38% sequence identity. We identify a link between FIC-1-mediated AMPylation and susceptibility to the pathogen Pseudomonas aeruginosa, establishing a connection between AMPylation and innate immunity in C. elegans.

Synthesis and evaluation of 2-ethynyl-adenosine-5'-triphosphate as a chemical reporter for protein AMPylation.

Protein AMPylation is a posttranslational modification (PTM) defined as the transfer of an adenosine monophosphate (AMP) from adenosine triphosphate (ATP) to a hydroxyl side-chain of a protein substrate. One recently reported AMPylator enzyme, Vibrio outer protein S (VopS), plays a role in pathogenesis by AMPylation of Rho GTPases, which disrupts crucial signaling pathways, leading to eventual cell death. Given the resurgent interest in this modification, there is a critical need for chemical tools that better facilitate the study of AMPylation and the enzymes responsible for this modification. Herein we report the synthesis of 2-ethynyl-adenosine-5'-triphosphate () and its utilization as a non-radioactive chemical reporter for protein AMPylation.

Inhibiting AMPylation: A Novel Screen To Identify the First Small Molecule Inhibitors of Protein AMPylation

Enzymatic transfer of the AMP portion of ATP to substrate proteins has recently been described as an essential mechanism of bacterial infection for several pathogens. The first AMPylator to be discovered, VopS from Vibrio parahemolyticus, catalyzes the transfer of AMP onto the host GTPases Cdc42 and Rac1. Modification of these proteins disrupts downstream signaling events, contributing to cell rounding and apoptosis, and recent studies have suggested that blocking AMPylation may be an effective route to stop infection. To date, however, no small molecule inhibitors have been discovered for any of the AMPylators. Therefore, we developed a fluorescence-polarization-based high-throughput screening assay and used it to discover the first inhibitors of protein AMPylation. Herein we report the discovery of the first small molecule VopS inhibitors (e.g., calmidazolium, GW7647, and MK886) with Ki's ranging from 6 to 50 μM and upward of 30-fold selectivity versus HYPE, the only known human AMPylator.

A New Chemical Handle for Protein AMPylation at the Host–Pathogen Interface

Tagging protein AMPylation: A new chemical reporter for AMPylation, recently identified as a key post-translational modification during bacterial infection, is a robust tool for detecting and identifying AMPylated proteins in vitro.