DNA Topoisomerases are essential to resolve topological problems during DNA metabolism in all species. However, the prevalence and function of RNA topoisomerases remain uncertain. Here, we show that RNA topoisomerase activity is prevalent in Type IA topoisomerases from bacteria, archaea, and eukarya. Moreover, this activity always requires the conserved Type IA core domains and the same catalytic residue used in DNA topoisomerase reaction; however, it does not absolutely require the non-conserved carboxyl-terminal domain (CTD), which is necessary for relaxation reactions of supercoiled DNA. The RNA topoisomerase activity of human Top3β differs from that of Escherichia coli topoisomerase I in that the former but not the latter requires the CTD, indicating that topoisomerases have developed distinct mechanisms during evolution to catalyze RNA topoisomerase reactions. Notably, Top3β proteins from several animals associate with polyribosomes, which are units of mRNA translation, whereas the Top3 homologs from E. coli and yeast lack the association. The Top3β-polyribosome association requires TDRD3, which directly interacts with Top3β and is present in animals but not bacteria or yeast. We propose that RNA topoisomerases arose in the early RNA world, and that they are retained through all domains of DNA-based life, where they mediate mRNA translation as part of polyribosomes in animals.

In humans, the olfactory epithelium is located in two narrow passages, the olfactory clefts, at the upper part of the nasal cavities. The olfactory epithelium is covered by a mucus layer which is essential for the function of the olfactory neurons that are directly connected with the brain through the cribriform plate. This anatomical weakness of the brain protection may be the source of infection. Little is known about the composition of this mucus in humans. Previous proteomic analyses have been performed on washes of the entire nasal cavities and therefore might better correspond to the mucus over the respiratory epithelium than to the mucus covering the olfactory epithelium. In the present study, we sampled the olfactory mucus directly from the clefts of 16 healthy adult volunteers, and 83 proteins were identified in the samples using two-dimensional gel electrophoresis, MALDI-TOF, RPLC, and Edman sequencing. Forty-three proteins were not previously observed either in nasal mucus sampled through washings, saliva, tear, or cerebrospinal fluid. In Accordance with the data in the protein databases, the most abundant proteins are secreted, whereas some others correspond to intracellular proteins covering a large range of functions: anti-inflammatory, antimicrobial, protease inhibition, antioxidant, transport, transcription, transduction, cytoskeletal, regulation, binding, and metabolism of odorant molecules. This study clearly demonstrates the complexity of the mucus covering the human olfactory epithelium, which might comprise potential markers for characterizing pathophysiological states.

Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. Highresolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-Å-deep two-chamber cavity at the interface of variable light (V L ) and variable heavy (V H ) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereoand chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes.catalytic antibodies | crystal structure | insecticide | organophosphate hydrolysis