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Protein AMPylation, the covalent addition of adenosine monophosphate (AMP) to protein substrates, has been known as a post translational modification for over 50 years. Research in this field is largely underdeveloped due to the lack of tools that enable the systematic identification of AMPylated substrates. Here, we address this gap by developing an enrichment technique to isolate and study AMPylated proteins using a nucleotide-binding protein, hinT. Cryo-EM reconstruction of an AMPylated protein bound to hinT provides a structural basis for AMP selectivity. Using structure guided mutagenesis, we optimize enrichment to identify novel substrates of the evolutionarily conserved AMPylase, Selenoprotein O. We show that mammalian Selenoprotein O regulates metabolic flux through AMPylation of key mitochondrial proteins including glutamate dehydrogenase and pyruvate dehydrogenase. Our findings highlight the broader significance of AMPylation, an emerging post translational modification with critical roles in signal transduction and disease pathology. Furthermore, we establish a powerful enrichment platform for the discovery of novel AMPylated proteins to study the mechanisms and significance of protein AMPylation in cellular function. Protein AMPylation regulates cellular processes. through the covalent addition of AMP to target proteins. Here, the authors develop a versatile enrichment strategy to profile AMPylated proteins and identify substrates of the mammalian AMPylase, Selenoprotein O.

Key Points On infection, bacterial pathogens interact with host membranes to trigger various cellular processes through different mechanisms. These processes include alterations to the dynamics between the plasma membrane and the actin cytoskeleton, and subversion of the membrane-associated pathways that are involved in vesicle trafficking. Many bacterial effectors manipulate phosphoinositide (PI) homeostasis at the plasma membrane to destabilize actin dynamics and alter the morphology of the membrane. This facilitates the entry of pathogens or, in other cases, damages the cells by disrupting membrane integrity and eventually leading to rapid cell lysis in the later stage of infection. Some pathogens use bacterial phosphatases or PI adaptor proteins to form intracellular vacuoles that are derived from host membranes in order to establish a replicative niche. Altered PI levels at the surfaces of these vacuoles as a result of the activity of bacterial phosphatases can block phagosomal maturation to avoid lysosomal fusion. The GTPase signalling pathway is often targeted by bacterial pathogens to manipulate the actin cytoskeleton and endosomal trafficking. RAB GTPases , which have an important role in vesicular trafficking pathways, are recruited to bacterium-containing vacuoles, where their active state can be differentially regulated by effectors. Bacterial effectors mimic GTPase-activating protein (GAP) or guanine nucleotide exchange factor (GEF) activity to target RHO-family GTPases that are key regulators of actin dynamics. This results in loss of cell shape, motility and ability to phagocytose pathogens. Autophagy is one of the cellular defence mechanisms against the invasion of pathogenic bacteria. However, some pathogens have evolved strategies to subvert autophagy to their own advantage by establishing autophagic vesicles as their replicative niche. This allows them to survive inside host cells and avoid lysosomal degradation. Some bacterial effectors are speculated to induce autophagy during infection. This may not only protect the bacteria from degradative enzymes and immune responses, but also provide nutrients from cellular debris. For extracellular pathogens, inducing autophagy helps prevent phagocytosis. Bacterial pathogens secrete a range of effector proteins to target the signalling pathways that regulate host cell membranes. Here, Orth and colleagues describe the bacterial effectors that target phosphoinositide signalling, GTPase signalling and autophagy, and discuss how targeting these pathways can alter host membrane dynamics. Bacterial pathogens interact with host membranes to trigger a wide range of cellular processes during the course of infection. These processes include alterations to the dynamics between the plasma membrane and the actin cytoskeleton, and subversion of the membrane-associated pathways involved in vesicle trafficking. Such changes facilitate the entry and replication of the pathogen, and prevent its phagocytosis and degradation. In this Review, we describe the manipulation of host membranes by numerous bacterial effectors that target phosphoinositide metabolism, GTPase signalling and autophagy.

Defects in normal autophagic pathways are implicated in numerous human diseases—such as neurodegenerative diseases, cancer, and cardiomyopathy—highlighting the importance of autophagy and its proper regulation. Herein we show that Vibrio parahaemolyticus uses the type III effector VopQ (Vibrio outer protein Q) to alter autophagic flux by manipulating the partitioning of small molecules and ions in the lysosome. This effector binds to the conserved Vo domain of the vacuolar-type H+ -ATPase and causes deacidification of the lysosomes within minutes of entering the host cell. VopQ forms a gated channel ~18 Å in diameter that facilitates outward flux of ions across lipid bilayers. The electrostatic interactions of this type 3 secretion system effector with target membranes dictate its preference for host vacuolar-type H+ -ATPase-containing membranes, indicating that its pore-forming activity is specific and not promiscuous. As seen with other effectors, VopQ is exploiting a eukaryotic mechanism, in this case manipulating lysosomal homeostasis and autophagic flux through transmembrane permeation.

The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. Here, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.

Vesicle fusion governs many important biological processes, and imbalances in the regulation of membrane fusion can lead to a variety of diseases such as diabetes and neurological disorders. Here we show that the Vibrio parahaemolyticus effector protein VopQ is a potent inhibitor of membrane fusion based on an in vitro yeast vacuole fusion model. Previously, we demonstrated that VopQ binds to the V ₒ domain of the conserved V-type H ⁺-ATPase (V-ATPase) found on acidic compartments such as the yeast vacuole. VopQ forms a nonspecific, voltage-gated membrane channel of 18 Å resulting in neutralization of these compartments. We now present data showing that VopQ inhibits yeast vacuole fusion. Furthermore, we identified a unique mutation in VopQ that delineates its two functions, deacidification and inhibition of membrane fusion. The use of VopQ as a membrane fusion inhibitor in this manner now provides convincing evidence that vacuole fusion occurs independently of luminal acidification in vitro. Significance Fusion of intracellular membranes is involved in many critical cellular processes, such as neurotransmission, protein trafficking, and in the lysosomal degradation of invading bacterial pathogens. Accordingly, some intracellular bacterial pathogens use protein effectors to alter host membrane fusion directly as a survival mechanism. In this study, we show that the Vibrio secreted effector, VopQ, is a potent inhibitor of yeast homotypic vacuole fusion in vitro. Although VopQ was shown to deacidify yeast vacuoles via its known V-type H ⁺-ATPase (V-ATPase)-binding and channel-forming activities, its ability to inhibit vacuole fusion does not depend on channel-forming activity. Our studies suggest that yeast vacuole fusion is not regulated by lumenal acidification and identify a reagent to study the V-ATPase role in some membrane fusion events.

Vesicle fusion governs many important biological processes, and imbalances in the regulation of membrane fusion can lead to a variety of diseases such as diabetes and neurological disorders. Here we show that theVibrio parahaemolyticuseffector protein VopQ is a potent inhibitor of membrane fusion based on an in vitro yeast vacuole fusion model. Previously, we demonstrated that VopQ binds to the Vₒ domain of the conserved V-type H⁺-ATPase (V-ATPase) found on acidic compartments such as the yeast vacuole. VopQ forms a nonspecific, voltage-gated membrane channel of 18 Å resulting in neutralization of these compartments. We now present data showing that VopQ inhibits yeast vacuole fusion. Furthermore, we identified a unique mutation in VopQ that delineates its two functions, deacidification and inhibition of membrane fusion. The use of VopQ as a membrane fusion inhibitor in this manner now provides convincing evidence that vacuole fusion occurs independently of luminal acidification in vitro.

The transfer of a phosphate from ATP to a protein substrate, a modification known as phosphorylation, is catalyzed by protein kinases. Protein kinases play a crucial role in virtually every cellular activity. Recent studies of atypical protein kinases have highlighted the structural similarity of the kinase superfamily despite notable differences in primary amino acid sequence. We searched for putative protein kinases in the intracellular bacterial pathogen, Legionella pneumophila and identified the Type-4 secretion system (T4SS) effector, Lpg2603 as a remote member of the protein kinase superfamily. We show that Lpg2603 is an active protein kinase with several atypical structural features. Importantly, we find that the eukaryotic-specific host signaling molecule, inositol hexakisphosphate (IP6) is required for Lpg2603 kinase activity. Crystal structures of Lpg2603 in the apo-form and bound to IP6 reveal active site rearrangement that allows for ATP binding and catalysis. Our results on the structure and activity of Lpg2603 reveal a unique mode of regulation of protein kinases and will aid future work into the function of this effector during Legionella pathogenesis.

The configuration of Dye Sensitized solar cells (DSSC), consists of sintered nanoparticle titanium dioxide film, dyes, electrolyte and counter electrodes. Upon the absorption of photons by the dye molecules, excitons are generated, subsequently electrons are injected into the TiO2 photoanode. Afterward the electrons injected into the TiO2 photoanode, to produce photocurrent, scavenged by redox couple, and the hole transport to the photo cathode. The power conversion efficiency of the device depends on the amount of dye adsorbed by the photoanode. This paper explores in enhancing the efficiency of the device by controlled microwave exposure. With same exposure time, the photoanode is exposed at three different frequencies. SEM analysis is carried out to find the porosity of the photoanode on exposure. Current density is found to have an effect on microwave exposure.