Ribosomal oxygenases are structurally conserved from prokaryotes to humans

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components1,2 and in the hydroxylation of transcription factors3 and splicing factor proteins4. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA5,6,7 and ribosomal proteins8 have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy9,10,11,12. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans8 raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone Nε-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases

Synapse Pathology in Psychiatric and Neurologic Disease

Inhibitory and excitatory synapses play a fundamental role in information processing in the brain. Excitatory synapses usually are situated on dendritic spines, small membrane protrusions that harbor glutamate receptors and postsynaptic density components and help transmit electrical signals. In recent years, it has become evident that spine morphology is intimately linked to synapse function—smaller spines have smaller synapses and support reduced synaptic transmission. The relationship between synaptic signaling, spine shape, and brain function is never more apparent than when the brain becomes dysfunctional. Many psychiatric and neurologic disorders, ranging from mental retardation and autism to Alzheimer’s disease and addiction, are accompanied by alterations in spine morphology and synapse number. In this review, we highlight the structure and molecular organization of synapses and discuss functional effects of synapse pathology in brain disease

Antimycobacterial drug discovery using Mycobacteria-infected amoebae identifies anti-infectives and new molecular targets

Tuberculosis remains a serious threat to human health world-wide, and improved efficiency of medical treatment requires a better understanding of the pathogenesis and the discovery of new drugs. In the present study, we performed a whole-cell based screen in order to complete the characterization of 168 compounds from the GlaxoSmithKline TB-set. We have established and utilized novel previously unexplored host-model systems to characterize the GSK compounds, i.e. the amoeboid organisms D. discoideum and A. castellanii, as well as a microglial phagocytic cell line, BV2. We infected these host cells with Mycobacterium marinum to monitor and characterize the anti-infective activity of the compounds with quantitative fluorescence measurements and high-content microscopy. In summary, 88.1% of the compounds were confirmed as antibiotics against M. marinum, 11.3% and 4.8% displayed strong anti-infective activity in, respectively, the mammalian and protozoan infection models. Additionally, in the two systems, 13-14% of the compounds displayed pro-infective activity. Our studies underline the relevance of using evolutionarily distant pathogen and host models in order to reveal conserved mechanisms of virulence and defence, respectively, which are potential "universal" targets for intervention. Subsequent mechanism of action studies based on generation of over-expresser M. bovis BCG strains, generation of spontaneous resistant mutants and whole genome sequencing revealed four new molecular targets, including FbpA, MurC, MmpL3 and GlpK

mTOR Controls Ovarian Follicle Growth by Regulating Granulosa Cell Proliferation

We have shown that inhibition of mTOR in granulosa cells and ovarian follicles results in compromised granulosa proliferation and reduced follicle growth. Further analysis here using spontaneously immortalized rat granulosa cells has revealed that mTOR pathway activity is enhanced during M-phase of the cell cycle. mTOR specific phosphorylation of p70S6 kinase and 4E-BP, and expression of Raptor are all enhanced during M-phase. The predominant effect of mTOR inhibition by the specific inhibitor Rapamycin (RAP) was a dose-responsive arrest in the G1 cell cycle stage. The fraction of granulosa cells that continued to divide in the presence of RAP exhibited a dose-dependent increase in aberrant mitotic figures known as anaphase bridges. Strikingly, estradiol consistently decreased the incidence of aberrant mitotic figures. In mice treated with RAP, the mitotic index was reduced compared to controls, and a similar increase in aberrant mitotic events was noted. RAP injected during a superovulation regime resulted in a dose-dependent reduction in the numbers of eggs ovulated. Implications for the real-time regulation of follicle growth and dominance, including the consequences of increased numbers of aneuploid granulosa cells, are discussed

Structural Insights into the Evolution of a Non-Biological Protein: Importance of Surface Residues in Protein Fold Optimization

Phylogenetic profiling of amino acid substitution patterns in proteins has led many to conclude that most structural information is carried by interior core residues that are solvent inaccessible. This conclusion is based on the observation that buried residues generally tolerate only conserved sequence changes, while surface residues allow more diverse chemical substitutions. This notion is now changing as it has become apparent that both core and surface residues play important roles in protein folding and stability. Unfortunately, the ability to identify specific mutations that will lead to enhanced stability remains a challenging problem. Here we discuss two mutations that emerged from an in vitro selection experiment designed to improve the folding stability of a non-biological ATP binding protein. These mutations alter two solvent accessible residues, and dramatically enhance the expression, solubility, thermal stability, and ligand binding affinity of the protein. The significance of both mutations was investigated individually and together, and the X-ray crystal structures of the parent sequence and double mutant protein were solved to a resolution limit of 2.8 and 1.65 Å, respectively. Comparative structural analysis of the evolved protein to proteins found in nature reveals that our non-biological protein evolved certain structural features shared by many thermophilic proteins. This experimental result suggests that protein fold optimization by in vitro selection offers a viable approach to generating stable variants of many naturally occurring proteins whose structures and functions are otherwise difficult to study

Vaccinia Virus Proteins A52 and B14 Share a Bcl-2–Like Fold but Have Evolved to Inhibit NF-κB rather than Apoptosis

Vaccinia virus (VACV), the prototype poxvirus, encodes numerous proteins that modulate the host response to infection. Two such proteins, B14 and A52, act inside infected cells to inhibit activation of NF-κB, thereby blocking the production of pro-inflammatory cytokines. We have solved the crystal structures of A52 and B14 at 1.9 Å and 2.7 Å resolution, respectively. Strikingly, both these proteins adopt a Bcl-2–like fold despite sharing no significant sequence similarity with other viral or cellular Bcl-2–like proteins. Unlike cellular and viral Bcl-2–like proteins described previously, A52 and B14 lack a surface groove for binding BH3 peptides from pro-apoptotic Bcl-2–like proteins and they do not modulate apoptosis. Structure-based phylogenetic analysis of 32 cellular and viral Bcl-2–like protein structures reveals that A52 and B14 are more closely related to each other and to VACV N1 and myxoma virus M11 than they are to other viral or cellular Bcl-2–like proteins. This suggests that a progenitor poxvirus acquired a gene encoding a Bcl-2–like protein and, over the course of evolution, gene duplication events have allowed the virus to exploit this Bcl-2 scaffold for interfering with distinct host signalling pathways

Rhabdovirus Matrix Protein Structures Reveal a Novel Mode of Self-Association

The matrix (M) proteins of rhabdoviruses are multifunctional proteins essential for virus maturation and budding that also regulate the expression of viral and host proteins. We have solved the structures of M from the vesicular stomatitis virus serotype New Jersey (genus: Vesiculovirus) and from Lagos bat virus (genus: Lyssavirus), revealing that both share a common fold despite sharing no identifiable sequence homology. Strikingly, in both structures a stretch of residues from the otherwise-disordered N terminus of a crystallographically adjacent molecule is observed binding to a hydrophobic cavity on the surface of the protein, thereby forming non-covalent linear polymers of M in the crystals. While the overall topology of the interaction is conserved between the two structures, the molecular details of the interactions are completely different. The observed interactions provide a compelling model for the flexible self-assembly of the matrix protein during virion morphogenesis and may also modulate interactions with host proteins

Clamp loader ATPases and the evolution of DNA replication machinery

Clamp loaders are pentameric ATPases of the AAA+ family that operate to ensure processive DNA replication. They do so by loading onto DNA the ring-shaped sliding clamps that tether the polymerase to the DNA. Structural and biochemical analysis of clamp loaders has shown how, despite differences in composition across different branches of life, all clamp loaders undergo the same concerted conformational transformations, which generate a binding surface for the open clamp and an internal spiral chamber into which the DNA at the replication fork can slide, triggering ATP hydrolysis, release of the clamp loader, and closure of the clamp round the DNA. We review here the current understanding of the clamp loader mechanism and discuss the implications of the differences between clamp loaders from the different branches of life