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 Statement: The 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 BiP's 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 FicD's 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 FicD's role in maintaining cellular resilience and open avenues for therapeutic strategies targeting UPR-associated diseases.

AMPylation of small GTPases by Fic enzymes.

Small GTPases orchestrate numerous cellular pathways, acting as molecular switches and regulatory hubs to transmit molecular signals and because of this, they are often the target of pathogens. During infection, pathogens manipulate host cellular networks using post-translational modifications (PTMs). AMPylation, the modification of proteins with AMP, has been identified as a common PTM utilized by pathogens to hijack GTPase signalling during infection. AMPylation is primarily carried out by enzymes with a filamentation induced by cyclic-AMP (Fic) domain. Modification of small GTPases by AMP renders GTPases impervious to upstream regulatory inputs, resulting in unregulated downstream effector outputs for host cellular processes. Here, we overview Fic-mediated AMPylation of small GTPases by pathogens and other related PTMs catalysed by Fic enzymes on GTPases.

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 become overwhelmed. However, prolonged or strong UPR responses can result in elevated inflammation and cellular damage. Previously, we discovered that the enzyme filamentation induced by cyclic-AMP (Fic) can modulate the UPR response via posttranslational modification of binding immunoglobulin protein (BiP) by AMPylation during homeostasis and deAMPylation during stress. 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 physiological 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. Based on these findings, we propose that repeated physiological stress in differentiated tissues requires this rheostat for tissue resilience and continued function over the lifetime of an animal.

Revisiting AMPylation through the lens of Fic enzymes.

AMPylation, a post-translational modification (PTM) first discovered in the late 1960s, is catalyzed by adenosine monophosphate (AMP)-transferring enzymes. The observation that filamentation-induced-by-cyclic-AMP (fic) enzymes are associated with this unique PTM revealed that AMPylation plays a major role in hijacking of host signaling by pathogenic bacteria during infection. Studies over the past decade showed that AMPylation is conserved across all kingdoms of life and, outside their role in infection, also modulates cellular functions. Many aspects of AMPylation are yet to be uncovered. In this review we present the advancement in research on AMPylation and Fic enzymes as well as other distinct classes of enzymes that catalyze AMPylation.

ProteoVision: web server for advanced visualization of ribosomal proteins.

ProteoVision is a web server designed to explore protein structure and evolution through simultaneous visualization of multiple sequence alignments, topology diagrams and 3D structures. Starting with a multiple sequence alignment, ProteoVision computes conservation scores and a variety of physicochemical properties and simultaneously maps and visualizes alignments and other data on multiple levels of representation. The web server calculates and displays frequencies of amino acids. ProteoVision is optimized for ribosomal proteins but is applicable to analysis of any protein. ProteoVision handles internally generated and user uploaded alignments and connects them with a selected structure, found in the PDB or uploaded by the user. It can generate de novo topology diagrams from three-dimensional structures. All displayed data is interactive and can be saved in various formats as publication quality images or external datasets or PyMol Scripts. ProteoVision enables detailed study of protein fragments defined by Evolutionary Classification of protein Domains (ECOD) classification. ProteoVision is available at http://proteovision.chemistry.gatech.edu/.

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

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 determine its crystal structure. The complex reveals that the TPR-motifs of FICD bind specifically to the conserved hydrophobic linker of BiP and thus mediate specificity for the domain-docked conformation of BiP. Furthermore, we show that both AMPylation and deAMPylation of BiP are not directly regulated by the presence of unfolded proteins. Together, combining chemical biology, crystallography and biochemistry, our study provides structural insights into a key regulatory mechanism that safeguards ER homeostasis.

Current Advances in Covalent Stabilization of Macromolecular Complexes for Structural Biology.

Structural characterization of macromolecular assemblies is often limited by the transient nature of the interactions. The development of specific chemical tools to covalently tether interacting proteins to each other has played a major role in various fundamental discoveries in recent years. To this end, protein engineering techniques such as mutagenesis, incorporation of unnatural amino acids, and methods using synthetic substrate/cosubstrate derivatives were employed. In this review, we give an overview of both commonly used and recently developed biochemical methodologies for covalent stabilization of macromolecular complexes enabling structural investigation via crystallography, nuclear magnetic resonance, and cryo-electron microscopy. We divided the strategies into nonenzymatic- and enzymatic-driven cross-linking and further categorized them in either naturally occurring or engineered covalent linkage. This review offers a compilation of recent advances in diverse scientific fields where the structural characterization of macromolecular complexes was achieved by the aid of intermolecular covalent linkage.

Rab1-AMPylation by Legionella DrrA is allosterically activated by Rab1.

Legionella pneumophila infects 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 post-translational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP), and this process is referred to as AMPylation. Here, we use a chemical approach to stabilise low-affinity Rab:DrrA complexes in a site-specific manner to gain insight into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The crystal structure of the Rab:DrrA complex reveals a previously unknown non-conventional Rab-binding site (NC-RBS). Biochemical characterisation demonstrates allosteric stimulation of the AMPylation activity of DrrA via Rab binding to the NC-RBS. We speculate that allosteric control of DrrA could in principle prevent random and potentially cytotoxic AMPylation in the host, thereby perhaps ensuring efficient infection by Legionella.

Identification of targets of AMPylating Fic enzymes by co-substrate-mediated covalent capture.

Various pathogenic bacteria use post-translational modifications to manipulate the central components of host cell functions. Many of the enzymes released by these bacteria belong to the large Fic family, which modify targets with nucleotide monophosphates. The lack of a generic method for identifying the cellular targets of Fic family enzymes hinders investigation of their role and the effect of the post-translational modification. Here, we establish an approach that uses reactive co-substrate-linked enzymes for proteome profiling. We combine synthetic thiol-reactive nucleotide derivatives with recombinantly produced Fic enzymes containing strategically placed cysteines in their active sites to yield reactive binary probes for covalent substrate capture. The binary complexes capture their targets from cell lysates and permit subsequent identification. Furthermore, we determined the structures of low-affinity ternary enzyme-nucleotide-substrate complexes by applying a covalent-linking strategy. This approach thus allows target identification of the Fic enzymes from both bacteria and eukarya.

Sortase-Mediated Quantifiable Enzyme Immobilization on Magnetic Nanoparticles.

Protein immobilization has gained high interest in recent years for its valuable applications in life sciences involving drug delivery and protein arrays. Herein, we combine sortase-mediated protein immobilization with the versatility of magnetic nanoparticles and a sensitive GFP-based quantification system. Using this method, we successfully immobilized and quantified the amount of coupled enzymes by fluorescence spectroscopy and assessed their activity by kinetic measurements. We show that sortase-mediated coupling of enzymes enables preparation of biological samples with a high demand of purity as demonstrated by single-molecule FRET. Here, we report that sortase-mediated protein ligation allows both N- and C-terminal site-specific protein immobilization. Additionally, we demonstrate that sortase-mediated protein immobilization is suitable for direct protein immobilization from complex lysates. Direct immobilization from lysate allows study of enzyme functionality without the need of time-consuming enzyme purification, while magnetic nanoparticles permit easy addition and removal of coupled enzymes to and from a reaction mixture.

Ribosomal small subunit domains radiate from a central core.

The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2'OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit.

History of the ribosome and the origin of translation.

We present a molecular-level model for the origin and evolution of the translation system, using a 3D comparative method. In this model, the ribosome evolved by accretion, recursively adding expansion segments, iteratively growing, subsuming, and freezing the rRNA. Functions of expansion segments in the ancestral ribosome are assigned by correspondence with their functions in the extant ribosome. The model explains the evolution of the large ribosomal subunit, the small ribosomal subunit, tRNA, and mRNA. Prokaryotic ribosomes evolved in six phases, sequentially acquiring capabilities for RNA folding, catalysis, subunit association, correlated evolution, decoding, energy-driven translocation, and surface proteinization. Two additional phases exclusive to eukaryotes led to tentacle-like rRNA expansions. In this model, ribosomal proteinization was a driving force for the broad adoption of proteins in other biological processes. The exit tunnel was clearly a central theme of all phases of ribosomal evolution and was continuously extended and rigidified. In the primitive noncoding ribosome, proto-mRNA and the small ribosomal subunit acted as cofactors, positioning the activated ends of tRNAs within the peptidyl transferase center. This association linked the evolution of the large and small ribosomal subunits, proto-mRNA, and tRNA.

Secondary structures of rRNAs from all three domains of life.

Accurate secondary structures are important for understanding ribosomes, which are extremely large and highly complex. Using 3D structures of ribosomes as input, we have revised and corrected traditional secondary (2°) structures of rRNAs. We identify helices by specific geometric and molecular interaction criteria, not by co-variation. The structural approach allows us to incorporate non-canonical base pairs on parity with Watson-Crick base pairs. The resulting rRNA 2° structures are up-to-date and consistent with three-dimensional structures, and are information-rich. These 2° structures are relatively simple to understand and are amenable to reproduction and modification by end-users. The 2° structures made available here broadly sample the phylogenetic tree and are mapped with a variety of data related to molecular interactions and geometry, phylogeny and evolution. We have generated 2° structures for both large subunit (LSU) 23S/28S and small subunit (SSU) 16S/18S rRNAs of Escherichia coli, Thermus thermophilus, Haloarcula marismortui (LSU rRNA only), Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens. We provide high-resolution editable versions of the 2° structures in several file formats. For the SSU rRNA, the 2° structures use an intuitive representation of the central pseudoknot where base triples are presented as pairs of base pairs. Both LSU and SSU secondary maps are available (http://apollo.chemistry.gatech.edu/RibosomeGallery). Mapping of data onto 2° structures was performed on the RiboVision server (http://apollo.chemistry.gatech.edu/RiboVision).