A Modern Mode of Activation for Nucleic Acid Enzymes

Through evolution, enzymes have developed subtle modes of activation in order to ensure the sufficiently high substrate specificity required by modern cellular metabolism. One of these modes is the use of a target-dependent module (i.e. a docking domain) such as those found in signalling kinases. Upon the binding of the target to a docking domain, the substrate is positioned within the catalytic site. The prodomain acts as a target-dependent module switching the kinase from an off state to an on state. As compared to the allosteric mode of activation, there is no need for the presence of a third partner. None of the ribozymes discovered to date have such a mode of activation, nor does any other known RNA. Starting from a specific on/off adaptor for the hepatitis delta virus ribozyme, that differs but has a mechanism reminiscent of this signalling kinase, we have adapted this mode of activation, using the techniques of molecular engineering, to both catalytic RNAs and DNAs exhibiting various activities. Specifically, we adapted three cleaving ribozymes (hepatitis delta virus, hammerhead and hairpin ribozymes), a cleaving 10-23 deoxyribozyme, a ligating hairpin ribozyme and an artificially selected capping ribozyme. In each case, there was a significant gain in terms of substrate specificity. Even if this mode of control is unreported for natural catalytic nucleic acids, its use needs not be limited to proteinous enzymes. We suggest that the complexity of the modern cellular metabolism might have been an important selective pressure in this evolutionary process

Typical investigational medicinal products follow relatively uniform regulations in 10 European Clinical Research Infrastructures Network (ECRIN) countries

<p>Abstract</p> <p>Background</p> <p>In order to facilitate multinational clinical research, regulatory requirements need to become international and harmonised. The EU introduced the Directive 2001/20/EC in 2004, regulating investigational medicinal products in Europe.</p> <p>Methods</p> <p>We conducted a survey in order to identify the national regulatory requirements for major categories of clinical research in ten European Clinical Research Infrastructures Network (ECRIN) countries-Austria, Denmark, France, Germany, Hungary, Ireland, Italy, Spain, Sweden, and United Kingdom-covering approximately 70% of the EU population. Here we describe the results for regulatory requirements for typical investigational medicinal products, in the ten countries.</p> <p>Results</p> <p>Our results show that the ten countries have fairly harmonised definitions of typical investigational medicinal products. Clinical trials assessing typical investigational medicinal products require authorisation from a national competent authority in each of the countries surveyed. The opinion of the competent authorities is communicated to the trial sponsor within the same timelines, i.e., no more than 60 days, in all ten countries. The authority to which the application has to be sent to in the different countries is not fully harmonised.</p> <p>Conclusion</p> <p>The Directive 2001/20/EC defined the term 'investigational medicinal product' and all regulatory requirements described therein are applicable to investigational medicinal products. Our survey showed, however, that those requirements had been adopted in ten European countries, not for investigational medicinal products overall, but rather a narrower category which we term 'typical' investigational medicinal products. The result is partial EU harmonisation of requirements and a relatively navigable landscape for the sponsor regarding typical investigational medicinal products.</p

BILBO1 Is a Scaffold Protein of the Flagellar Pocket Collar in the Pathogen Trypanosoma brucei

The flagellar pocket (FP) of the pathogen Trypanosoma brucei is an important single copy structure that is formed by the invagination of the pellicular membrane. It is the unique site of endo- and exocytosis and is required for parasite pathogenicity. The FP consists of distinct structural sub-domains with the least explored being the annulus/horseshoe shaped flagellar pocket collar (FPC). To date the only known component of the FPC is the protein BILBO1, a cytoskeleton protein that has a N-terminus that contains an ubiquitin-like fold, two EF-hand domains, plus a large C-terminal coiled-coil domain. BILBO1 has been shown to bind calcium, but in this work we demonstrate that mutating either or both calcium-binding domains prevents calcium binding. The expression of deletion or mutated forms of BILBO1 in trypanosomes and mammalian cells demonstrate that the coiled-coil domain is necessary and sufficient for the formation of BILBO1 polymers. This is supported by Yeast two-hybrid analysis. Expression of full-length BILBO1 in mammalian cells induces the formation of linear polymers with comma and globular shaped termini, whereas mutation of the canonical calcium-binding domain resulted in the formation of helical polymers and mutation in both EF-hand domains prevented the formation of linear polymers. We also demonstrate that in T. brucei the coiled-coil domain is able to target BILBO1 to the FPC and to form polymers whilst the EF-hand domains influence polymers shape. This data indicates that BILBO1 has intrinsic polymer forming properties and that binding calcium can modulate the form of these polymers. We discuss whether these properties can influence the formation of the FPC