Eukaryotic RNA-guided endonucleases evolved from a unique clade of bacterial enzymes.

RNA-guided endonucleases form the crux of diverse biological processes and technologies, including adaptive immunity, transposition, and genome editing. Some of these enzymes are components of insertion sequences (IS) in the IS200/IS605 and IS607 transposon families. Both IS families encode a TnpA transposase and a TnpB nuclease, an RNA-guided enzyme ancestral to CRISPR-Cas12s. In eukaryotes, TnpB homologs occur as two distinct types, Fanzor1s and Fanzor2s. We analyzed the evolutionary relationships between prokaryotic TnpBs and eukaryotic Fanzors, which revealed that both Fanzor1s and Fanzor2s stem from a single lineage of IS607 TnpBs with unusual active site arrangement. The widespread nature of Fanzors implies that the properties of this particular lineage of IS607 TnpBs were particularly suited to adaptation in eukaryotes. Biochemical analysis of an IS607 TnpB and Fanzor1s revealed common strategies employed by TnpBs and Fanzors to co-evolve with their cognate transposases. Collectively, our results provide a new model of sequential evolution from IS607 TnpBs to Fanzor2s, and Fanzor2s to Fanzor1s that details how genes of prokaryotic origin evolve to give rise to new protein families in eukaryotes.

Benzenesulfonamides Incorporating Hydantoin Moieties Effectively Inhibit Eukaryoticand Human Carbonic Anhydrases.

A series of novel 1-(4-benzenesulfonamide)-3-alkyl/benzyl-hydantoin derivatives were synthesized and evaluated for the inhibition of eukaryotic and human carbonic anhydrases (CAs, EC 4.2.1.1). The prepared compounds were screened for their hCA inhibitory activities against three cytosolic isoforms as well as two ß-CAs from fungal pathogens. The best inhibition was observed against hCA II and VII as well as Candida glabrata enzyme CgNce103. hCA I and Malassezia globosa MgCA enzymes were, on the other hand, less effectively inhibited by these compounds. The inhibitory potency of these compounds against CAs was found to be dependent on the electronic and steric effects of substituent groups on the N3-position of the hydantoin ring, which included alkyl, alkenyl and substituted benzyl moieties. The interesting results against CgNce103 make the compounds of interest for investigations in vivo as potential antifungals.

Core control principles of the eukaryotic cell cycle.

Cyclin-dependent kinases (CDKs) lie at the heart of eukaryotic cell cycle control, with different cyclin-CDK complexes initiating DNA replication (S-CDKs) and mitosis (M-CDKs)1,2. However, the principles on which cyclin-CDK complexes organize the temporal order of cell cycle events are contentious3. One model proposes that S-CDKs and M-CDKs are functionally specialized, with substantially different substrate specificities to execute different cell cycle events4-6. A second model proposes that S-CDKs and M-CDKs are redundant with each other, with both acting as sources of overall CDK activity7,8. In this model, increasing CDK activity, rather than CDK substrate specificity, orders cell cycle events9,10. Here we reconcile these two views of core cell cycle control. Using phosphoproteomic assays of in vivo CDK activity in fission yeast, we find that S-CDK and M-CDK substrate specificities are remarkably similar, showing that S-CDKs and M-CDKs are not completely specialized for S phase and mitosis alone. Normally, S-CDK cannot drive mitosis but can do so when protein phosphatase 1 is removed from the centrosome. Thus, increasing S-CDK activity in vivo is sufficient to overcome substrate specificity differences between S-CDK and M-CDK, and allows S-CDK to carry out M-CDK function. Therefore, we unite the two opposing views of cell cycle control, showing that the core cell cycle engine is largely based on a quantitative increase in CDK activity through the cell cycle, combined with minor and surmountable qualitative differences in catalytic specialization of S-CDKs and M-CDKs.

Aurora A kinase activation: Different means to different ends.

Aurora A is a serine/threonine kinase essential for mitotic entry and spindle assembly. Recent molecular studies have revealed the existence of multiple, distinct mechanisms of Aurora A activation, each occurring at specific subcellular locations, optimized for cellular context, and primed by signaling events including phosphorylation and oxidation.

Spots, stripes, and spiral waves in models for static and motile cells : GTPase patterns in cells.

The polarization and motility of eukaryotic cells depends on assembly and contraction of the actin cytoskeleton and its regulation by proteins called GTPases. The activity of GTPases causes assembly of filamentous actin (by GTPases Cdc42, Rac), resulting in protrusion of the cell edge. Mathematical models for GTPase dynamics address the spontaneous formation of patterns and nonuniform spatial distributions of such proteins in the cell. Here we revisit the wave-pinning model for GTPase-induced cell polarization, together with a number of extensions proposed in the literature. These include introduction of sources and sinks of active and inactive GTPase (by the group of A. Champneys), and negative feedback from F-actin to GTPase activity. We discuss these extensions singly and in combination, in 1D, and 2D static domains. We then show how the patterns that form (spots, waves, and spirals) interact with cell boundaries to create a variety of interesting and dynamic cell shapes and motion.

Inositol phosphate kinases in the eukaryote landscape.

Inositol phosphate encompasses a large multifaceted family of signalling molecules that originate from the combinatorial attachment of phosphate groups to the inositol ring. To date, four distinct inositol kinases have been identified, namely, IPK, ITPK, IPPK (IP5-2K), and PPIP5K. Although, ITPKs have recently been identified in archaea, eukaryotes have taken advantage of these enzymes to create a sophisticated signalling network based on inositol phosphates. However, it remains largely elusive what fundamental biochemical principles control the signalling cascade. Here, we present an evolutionary approach to understand the development of the 'inositol phosphate code' in eukaryotes. Distribution analyses of these four inositol kinase groups throughout the eukaryotic landscape reveal the loss of either ITPK, or of PPIP5K proteins in several species. Surprisingly, the loss of IPPK, an enzyme thought to catalyse the rate limiting step of IP6 (phytic acid) synthesis, was also recorded. Furthermore, this study highlights a noteworthy difference between animal (metazoan) and plant (archaeplastida) lineages. While metazoan appears to have a substantial amplification of IPK enzymes, archaeplastida genomes show a considerable increase in ITPK members. Differential evolution of IPK and ITPK between plant and animal lineage is likely reflective of converging functional adaptation of these two types of inositol kinases. Since, the IPK family comprises three sub-types IPMK, IP6K, and IP3-3K each with dedicated enzymatic specificity in metazoan, we propose that the amplified ITPK group in plant could be classified in sub-types with distinct enzymology.

Lipid flippases in polarized growth.

Polarized growth is required in eukaryotic cells for processes such as cell division, morphogenesis and motility, which involve conserved and interconnected signalling pathways controlling cell cycle progression, cytoskeleton reorganization and secretory pathway functioning. While many of the factors involved in polarized growth are known, it is not yet clear how they are coordinated both spatially and temporally. Several lines of evidence point to the important role of lipid flippases in polarized growth events. Lipid flippases, which mainly belong to the P4 subfamily of P-type ATPases, are active transporters that move different lipids to the cytosolic side of biological membranes at the expense of ATP. The involvement of the Saccharomyces cerevisiae plasma membrane P4 ATPases Dnf1p and Dnf2p in polarized growth and their activation by kinase phosphorylation were established some years ago. However, these two proteins do not seem to be responsible for the phosphatidylserine internalization required for early recruitment of proteins to the plasma membrane during yeast mating and budding. In a recent publication, we demonstrated that the Golgi-localized P4 ATPase Dnf3p has a preference for PS as a substrate, can reach the plasma membrane in a cell cycle-dependent manner, and is regulated by the same kinases that activate Dnf1p and Dnf2p. This finding solves a long-lasting enigma in the field of lipid flippases and suggests that tight and heavily coordinated spatiotemporal control of lipid translocation at the plasma membrane is important for proper polarized growth.

State-dependent sequential allostery exhibited by chaperonin TRiC/CCT revealed by network analysis of Cryo-EM maps.

The eukaryotic chaperonin TRiC/CCT plays a major role in assisting the folding of many proteins through an ATP-driven allosteric cycle. Recent structures elucidated by cryo-electron microscopy provide a broad view of the conformations visited at various stages of the chaperonin cycle, including a sequential activation of its subunits in response to nucleotide binding. But we lack a thorough mechanistic understanding of the structure-based dynamics and communication properties that underlie the TRiC/CCT machinery. In this study, we present a computational methodology based on elastic network models adapted to cryo-EM density maps to gain a deeper understanding of the structure-encoded allosteric dynamics of this hexadecameric machine. We have analysed several structures of the chaperonin resolved in different states toward mapping its conformational landscape. Our study indicates that the overall architecture intrinsically favours cooperative movements that comply with the structural variabilities observed in experiments. Furthermore, the individual subunits CCT1-CCT8 exhibit state-dependent sequential events at different states of the allosteric cycle. For example, in the ATP-bound state, subunits CCT5 and CCT4 selectively initiate the lid closure motions favoured by the overall architecture; whereas in the apo form of the heteromer, the subunit CCT7 exhibits the highest predisposition to structural change. The changes then propagate through parallel fluxes of allosteric signals to neighbours on both rings. The predicted state-dependent mechanisms of sequential activation provide new insights into TRiC/CCT intra- and inter-ring signal transduction events.

Oxidoreductases in Glycoprotein Glycosylation, Folding, and ERAD.

N-linked glycosylation and sugar chain processing, as well as disulfide bond formation, are among the most common post-translational protein modifications taking place in the endoplasmic reticulum (ER). They are essential modifications that are required for membrane and secretory proteins to achieve their correct folding and native structure. Several oxidoreductases responsible for disulfide bond formation, isomerization, and reduction have been shown to form stable, functional complexes with enzymes and chaperones that are involved in the initial addition of an N-glycan and in folding and quality control of the glycoproteins. Some of these oxidoreductases are selenoproteins. Recent studies also implicate glycan machinery-oxidoreductase complexes in the recognition and processing of misfolded glycoproteins and their reduction and targeting to ER-associated degradation. This review focuses on the intriguing cooperation between the glycoprotein-specific cell machineries and ER oxidoreductases, and highlights open questions regarding the functions of many members of this large family.

Escherichia coli YegI is a novel Ser/Thr kinase lacking conserved motifs that localizes to the inner membrane.

In bacteria, signaling phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys in phylogenetically diverse bacteria over the past decade have identified numerous proteins that are phosphorylated on Ser and/or Thr residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes such as in the Escherichia coli genome, which encodes at least three Ser/Thr kinases. Here, we identify a previously uncharacterized ORF, yegI, and demonstrate that it encodes a novel Ser/Thr kinase. YegI lacks several conserved motifs including residues important for Mg2+ binding seen in other bacterial Ser/Thr kinases, suggesting that the consensus may be too stringent. We further find that YegI is a two-pass membrane protein with both N- and C termini located intracellularly.

Genome-wide identification and characterization of eukaryotic protein kinases.

The tropical liver fluke, Fasciola gigantica is a food-borne parasite responsible for the hepatobiliary disease fascioliasis. The recent completion of F. gigantica genome sequencing by our group has provided a platform for the systematic analysis of the parasite genome. Eukaryotic protein kinases (ePKs) are regulators of cellular phosphorylation. In the present study, we used various computational and bioinformatics tools to extensively analyse the ePKs in F. gigantica (FgePKs) genome. A total of 455 ePKs were identified that represent ~2% of the parasite genome. Out of these, 214 ePKs are typical kinases (Ser/Thr- and Tyr-specific ePKs), and 241 were other kinases. Several FgePKs were found to possess unusual domain architectures, which suggests the diverse nature of the proteins that can be exploited for designing novel inhibitors. 115 kinases showed <35% query coverage when compared to human ePKs highlighting significant divergences in their respective kinomes, further providing a platform for novel structure-based drug designing. This study provides a platform that may open new avenues into our understanding of helminth biochemistry and drug discovery.

Structural variations of photosystem I-antenna supercomplex in response to adaptations to different light environments.

Photosystem I (PSI) is one of the two photosystems in photosynthesis, and generates reducing power required for carbon dioxide fixation. PSI exists as a reaction center core in cyanobacteria but is surrounded by light-harvesting antenna complexes (LHCI) to form PSI-LHCI supercomplexes in eukaryotic organisms. The structures of PSI core and PSI-LHCI have been reported from various organisms. We compare these structures and highlight the differences among different organisms. While the PSI core is more conserved, there are differences in its subunit composition and organization. Larger differences are found in the subunit composition, organization, and pigment binding in LHCI. All these changes can be explained in the framework of better adaptation to different light environment that each photosynthetic organism inhabits.

Divergent Evolution of Eukaryotic CC- and A-Adding Enzymes.

Synthesis of the CCA end of essential tRNAs is performed either by CCA-adding enzymes or as a collaboration between enzymes restricted to CC- and A-incorporation. While the occurrence of such tRNA nucleotidyltransferases with partial activities seemed to be restricted to Bacteria, the first example of such split CCA-adding activities was reported in Schizosaccharomyces pombe. Here, we demonstrate that the choanoflagellate Salpingoeca rosetta also carries CC- and A-adding enzymes. However, these enzymes have distinct evolutionary origins. Furthermore, the restricted activity of the eukaryotic CC-adding enzymes has evolved in a different way compared to their bacterial counterparts. Yet, the molecular basis is very similar, as highly conserved positions within a catalytically important flexible loop region are missing in the CC-adding enzymes. For both the CC-adding enzymes from S. rosetta as well as S. pombe, introduction of the loop elements from closely related enzymes with full activity was able to restore CCA-addition, corroborating the significance of this loop in the evolution of bacterial as well as eukaryotic tRNA nucleotidyltransferases. Our data demonstrate that partial CC- and A-adding activities in Bacteria and Eukaryotes are based on the same mechanistic principles but, surprisingly, originate from different evolutionary events.

The origin and evolution of cell-intrinsic antibacterial defenses in eukaryotes.

To survive in a world dominated by bacteria, eukaryotes have evolved numerous self-defense strategies. While some defenses are recent evolutionary innovations, others are ancient, with roots early in eukaryotic history. With a focus on antibacterial immunity, we highlight the evolution of pattern recognition receptors that detect bacteria, where diverse functional classes have been formed from the repeated use and reuse of a small set of protein domains. Next, we discuss core microbicidal strategies shared across eukaryotes, and how these systems may have been co-opted from ancient cellular mechanisms. We propose that studying antibacterial responses across diverse eukaryotes can reveal novel modes of defense, while highlighting the critical innovations that occurred early in the evolution of our own immune systems.

DEAD-box ATPases are global regulators of phase-separated organelles.

The ability of proteins and nucleic acids to undergo liquid-liquid phase separation has recently emerged as an important molecular principle of how cells rapidly and reversibly compartmentalize their components into membrane-less organelles such as the nucleolus, processing bodies or stress granules1,2. How the assembly and turnover of these organelles are controlled, and how these biological condensates selectively recruit or release components are poorly understood. Here we show that members of the large and highly abundant family of RNA-dependent DEAD-box ATPases (DDXs)3 are regulators of RNA-containing phase-separated organelles in prokaryotes and eukaryotes. Using in vitro reconstitution and in vivo experiments, we demonstrate that DDXs promote phase separation in their ATP-bound form, whereas ATP hydrolysis induces compartment turnover and release of RNA. This mechanism of membrane-less organelle regulation reveals a principle of cellular organization that is conserved from bacteria to humans. Furthermore, we show that DDXs control RNA flux into and out of phase-separated organelles, and thus propose that a cellular network of dynamic, DDX-controlled compartments establishes biochemical reaction centres that provide cells with spatial and temporal control of various RNA-processing steps, which could regulate the composition and fate of ribonucleoprotein particles.

Mechanics and pharmacology of substrate selection and transport by eukaryotic ABC exporters.

Much structural information has been amassed on ATP-binding cassette (ABC) transporters, including hundreds of structures of isolated domains and an increasing array of full-length transporters. The structures capture different steps in the transport cycle and have aided in the design and interpretation of computational simulations and biophysics experiments. These data provide a maturing, although still incomplete, elucidation of the protein dynamics and mechanisms of substrate selection and transit through the transporters. We present an updated view of the classical alternating-access mechanism as it applies to eukaryotic ABC transporters, focusing on type I exporters. Our model helps frame the progress in, and remaining questions about, transporter energetics, how substrates are selected and how ATP is consumed to perform work at the molecular scale. Many human ABC transporters are associated with disease; we highlight progress in understanding their pharmacology through the lens of structural biology and describe how this knowledge suggests approaches to pharmacologically targeting these transporters.

The multi-faceted regulation of nuclear tRNA gene transcription.

Transfer RNAs are among the most ancient molecules of life on earth. Beyond their crucial role in protein synthesis as carriers of amino acids, they are also important players in a plethora of other biological processes. Many debates in term of biogenesis, regulation and function persist around these fascinating non-coding RNAs. Our review focuses on the first step of their biogenesis in eukaryotes, i.e. their transcription from nuclear genes. Numerous and complementary ways have emerged during evolution to regulate transfer RNA gene transcription. Here, we will summarize the different actors implicated in this process: cis-elements, trans-factors, genomic contexts, epigenetic environments and finally three-dimensional organization of nuclear genomes. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1099-1108, 2019.

Eukaryotic Base Excision Repair: New Approaches Shine Light on Mechanism.

Genomic DNA is susceptible to endogenous and environmental stresses that modify DNA structure and its coding potential. Correspondingly, cells have evolved intricate DNA repair systems to deter changes to their genetic material. Base excision DNA repair involves a number of enzymes and protein cofactors that hasten repair of damaged DNA bases. Recent advances have identified macromolecular complexes that assemble at the DNA lesion and mediate repair. The repair of base lesions generally requires five enzymatic activities: glycosylase, endonuclease, lyase, polymerase, and ligase. The protein cofactors and mechanisms for coordinating the sequential enzymatic steps of repair are being revealed through a range of experimental approaches. We discuss the enzymes and protein cofactors involved in eukaryotic base excision repair, emphasizing the challenge of integrating findings from multiple methodologies. The results provide an opportunity to assimilate biochemical findings with cell-based assays to uncover new insights into this deceptively complex repair pathway.