Biology subjects

Servage, K. A.

Publications and source records attributed to Servage, K. A..

7 recordsLinked to original sources

bioRxiv2026

Pseudomonas effector AvrC is a rhamnosyltransferase with broad substrate specificity

Plant cells can directly or indirectly detect bacterial effectors, triggering a hypersensitive response to defend against pathogen infection. One extensively studied effector, AvrB, is a Fido (Fic, Doc, AvrB) domain-containing protein that acts as a glycosyltransferase. AvrC is an elusive Pseudomonas syringae avirulence effector protein with significant sequence and structural similarity to AvrB. Combining biochemistry, mass spectrometry, and AlphaFold prediction tools, we show that AvrC is a glycosyltransferase with auto-rhamnosylation activity. Like AvrB, AvrC can rhamnosylate a threonine residue (T166) on the A. thaliana guardee protein RIN4. In vitro assays revealed rhamnosylation substrates for AvrC also include plant coatomer subunits COPE1 and COPZ1. Collectively, our findings indicate that AvrC is a rhamnosyltransferase with broad substrate specificity. Our experimental strategies and findings provide valuable insights into future studies on the characterization of other Fido proteins.

biochemistry↗

bioRxiv2025

Bacterial ubiquitin ligase engineered for small molecule and protein target identification

The Legionella SidE effectors ubiquitinate host proteins independently of the canonical E1-E2 cascade. Here we engineer the SidE ligases to develop a modular proximity ligation approach for the identification of targets of small molecules and proteins, which we call SidBait. We validate the method with known small molecule-protein interactions and use it to identify CaMKII as an off-target interactor of the breast cancer drug ribociclib. Structural analysis and activity assays confirm that ribociclib binds the CaMKII active site and inhibits its activity. We further customize SidBait to identify protein-protein interactions, including substrates for enzymes, and discover the F-actin capping protein (CapZ) as a target of the Legionella effector RavB during infection. Structural and biochemical studies indicate that RavB allosterically binds CapZ and decaps actin, thus functionally mimicking eukaryotic CapZ interacting proteins. Collectively, our results establish SidBait as a reliable tool for identifying targets of small molecules and proteins.

molecular biology↗

bioRxiv2024

Covalent inhibition of the SARS-CoV-2 NiRAN domain via an active-site cysteine

The kinase-like NiRAN domain of nsp12 in SARS-CoV-2 catalyzes the formation of the 5 RNA cap structure. This activity is required for viral replication, offering a new target for the development of antivirals. Here, we develop a high-throughput assay to screen for small molecule inhibitors targeting the SARS-CoV-2 NiRAN domain. We identified NCI-2, a compound with a reactive chloromethyl group that covalently binds to an active site cysteine (Cys53) in the NiRAN domain, inhibiting its activity. NCI-2 can enter cells, bind to, and inactivate ectopically expressed nsp12. A cryo-EM reconstruction of the SARS-CoV-2 replication-transcription complex (RTC) bound to NCI-2 offers a detailed structural blueprint for rational drug design. Although NCI-2 showed limited potency against SARS-CoV-2 replication in cells, our work lays the groundwork for developing more potent and selective inhibitors targeting the NiRAN domain. This approach presents a promising therapeutic strategy for effectively combating COVID-19 and potentially mitigating future coronavirus outbreaks.

biochemistry↗

bioRxiv2024

Biochemical and structural insights into a 5' to 3' RNA ligase reveal a potential role in tRNA ligation

ATP-grasp superfamily enzymes contain a hand-like ATP-binding fold and catalyze a variety of reactions using a similar catalytic mechanism. More than 30 protein families are categorized in this superfamily, and they are involved in a plethora of cellular processes and human diseases. Here we identify C12orf29 as an atypical ATP-grasp enzyme that ligates RNA. Human C12orf29 and its homologs auto-adenylate on an active site Lys residue as part of a reaction intermediate that specifically ligates RNA halves containing a 5-phosphate and a 3-hydroxyl. C12orf29 binds tRNA in cells and can ligate tRNA within the anticodon loop in vitro. Genetic depletion of c12orf29 in female mice alters global tRNA levels in brain. Furthermore, crystal structures of a C12orf29 homolog from Yasminevirus bound to nucleotides reveal a minimal and atypical RNA ligase fold with a unique active site architecture that participates in catalysis. Collectively, our results identify C12orf29 as an RNA ligase and suggest its involvement in tRNA biology. Significance StatementATP-grasp enzymes share an atypical ATP-binding fold and catalyze a diverse set of reactions involved in many essential cellular processes. We identified C12orf29 as an atypical ATP-grasp enzyme. Our biochemical and structural characterizations reveal this enzyme to be a 5 to 3 RNA ligase, structurally and functionally similar to the phage T4 RNA ligase. C12orf29 can ligate tRNAs in vitro and C12orf29 knockout female mice have altered tRNA levels in brain. We also report structures of C12orf29, which have revealed critical insights into the mode of ATP binding and catalysis. Our work suggests that C12orf29 may be a new player in the regulation of tRNAs.

biochemistry↗

bioRxiv2024

FicD Sensitizes Cellular Response to Glucose Fluctuations in Mouse Embryonic Fibroblasts

During homeostasis, the endoplasmic reticulum (ER) maintains productive transmembrane and secretory protein folding that is vital for proper cellular function. The ER-resident HSP70 chaperone, BiP, plays a pivotal role in sensing ER stress to activate the unfolded protein response (UPR). BiP function is regulated by the bifunctional enzyme FicD that mediates AMPylation and deAMPylation of BiP in response to changes in ER stress. AMPylated BiP acts as a molecular rheostat to regulate UPR signaling, yet little is known about the molecular consequences of FicD loss. In this study, we investigate the role of FicD in mouse embryonic fibroblast (MEF) response to pharmacologically and metabolically induced ER stress. We find differential BiP AMPylation signatures when comparing robust chemical ER stress inducers to physiological glucose starvation stress and recovery. Wildtype MEFs respond to pharmacological ER stress by downregulating BiP AMPylation. Conversely, BiP AMPylation in wildtype MEFs increases upon metabolic stress induced by glucose starvation. Deletion of FicD results in widespread gene expression changes under baseline growth conditions. In addition, FicD null MEFs exhibit dampened UPR signaling, altered cell stress recovery response, and unconstrained protein secretion. Taken together, our findings indicate that FicD is important for tampering UPR signaling, stress recovery, and the maintenance of secretory protein homeostasis. Significance StatementThe chaperone BiP plays a key quality control role in the endoplasmic reticulum, the cellular location for the production, folding, and transport of secreted proteins. The enzyme FicD regulates BiPs activity through AMPylation and deAMPylation. Our study unveils the importance of FicD in regulating BiP and the unfolded protein response (UPR) during stress. We identify distinct BiP AMPylation signatures for different stressors, highlighting FicDs nuanced control. Deletion of FicD causes widespread gene expression changes, disrupts UPR signaling, alters stress recovery, and perturbs protein secretion in cells. These observations underscore the pivotal contribution of FicD for preserving secretory protein homeostasis. Our findings deepen the understanding of FicDs role in maintaining cellular resilience and open avenues for therapeutic strategies targeting UPR-associated diseases.

molecular biology↗

bioRxiv2022

Fic-mediated AMPylation tempers the Unfolded Protein Response during physiological stress

The proper balance of synthesis, folding, modification and degradation of proteins, also known as protein homeostasis, is vital to cellular health and function. The unfolded protein response (UPR) is activated when the mechanisms maintaining protein homeostasis in the endoplasmic reticulum (ER) become overwhelmed. However, prolonged or strong UPR responses can result in elevated inflammation and cellular damage. Previously, we discovered that the bifunctional enzyme Fic can modulate the UPR response via post-translational modification of BiP by AMPylation and deAMPylation. Loss of fic in Drosophila leads to vision defects and altered UPR activation in the fly eye. To investigate the importance of Fic-mediated AMPylation in a mammalian system, we generated a conditional null allele of Fic in mice and characterized the effect of Fic loss on the exocrine pancreas. Compared to controls, Fic-/- mice exhibit elevated serum markers for pancreatic dysfunction and display enhanced UPR signaling in the exocrine pancreas in response to physiologic and pharmacological stress. In addition, both fic-/- flies and Fic-/- mice show reduced capacity to recover from damage by stress that triggers the UPR. These findings show that Fic- mediated AMPylation acts as a molecular rheostat that is required to temper the UPR response in the mammalian pancreas during physiological stress.

physiology↗

bioRxiv2020

A Legionella effector ADP-ribosyltransferase inactivates glutamate dehydrogenase

ADP-ribosyltransferases (ARTs) are a widespread superfamily of enzymes frequently employed in pathogenic strategies of bacteria. Legionella pneumophila, the causative agent of Legionnaires disease, has acquired over 330 translocated effectors that showcase remarkable biochemical and structural diversity. Here we took a bioinformatic approach to search the Legionella effector repertoire for additional divergent members of the ART superfamily and identified an ART domain in Lpg0181. We show that L. pneumophila Lpg0181 targets a specific class of 120-kDa NAD+-dependent glutamate dehydrogenase (GDH) enzymes found in fungi and protists, including many natural hosts of Legionella. Lpg0181 targets a conserved arginine residue in the NAD+ -binding pocket of GDH, thereby blocking oxidative deamination of glutamate. While intracellular pathogens employ diverse virulence mechanisms to overcome host-limited nutrient availability, Lpg0181 is--to the best of our knowledge--the first example of a Legionella effector which directly targets a host metabolic enzyme.

biochemistry↗