We present the data and the technology, a combination of which allows us to determine the identity of proprotein convertases (PCs) related to the processing of specific protein targets including viral and bacterial pathogens. Our results, which support and extend the data of other laboratories, are required for the design of effective inhibitors of PCs because, in general, an inhibitor design starts with a specific substrate. Seven proteinases of the human PC family cleave the multibasic motifs R-X-(R/K/X)-R2 and, as a result, transform proproteins, including those from pathogens, into biologically active proteins and peptides. The precise cleavage preferences of PCs have not been known in sufficient detail; hence we were unable to determine the relative importance of the individual PCs in infectious diseases, thus making the design of specific inhibitors exceedingly difficult. To determine the cleavage preferences of PCs in more detail, we evaluated the relative efficiency of furin, PC2, PC4, PC5/6, PC7, and PACE4 in cleaving over 100 decapeptide sequences representing the R-X-(R/K/X)-R2 motifs of human, bacterial, and viral proteins. Our computer analysis of the data and the follow-on cleavage analysis of the selected full-length proteins corroborated our initial results thus allowing us to determine the cleavage preferences of the PCs and to suggest which PCs are promising drug targets in infectious diseases. Our results also suggest that pathogens, including anthrax PA83 and the avian influenza A H5N1 (bird flu) hemagglutinin precursor, evolved to be as sensitive to PC proteolysis as the most sensitive normal human proteins.
Pathogens or their toxins, including influenza virus, Pseudomonas, and anthrax toxins, require processing by host proprotein convertases (PCs) to enter host cells and to cause disease. Conversely, inhibiting PCs is likely to protect host cells from multiple furin-dependent, but otherwise unrelated, pathogens. To determine if this concept is correct, we designed specific nanomolar inhibitors of PCs modeled from the extended cleavage motif TPQRERRRKKR2GL of the avian influenza H5N1 hemagglutinin. We then confirmed the efficacy of the inhibitory peptides in vitro against the fluorescent peptide, anthrax protective antigen (PA83), and influenza hemagglutinin substrates and also in mice in vivo against two unrelated toxins, anthrax and Pseudomonas exotoxin. Peptides with Phe/Tyr at P1 were more selective for furin. Peptides with P1 Thr were potent against multiple PCs. Our strategy of basing the peptide sequence on a furin cleavage motif known for an avian flu virus shows the power of starting inhibitor design with a known substrate. Our results confirm that inhibiting furin-like PCs protects the host from the distinct furin-dependent infections and lay a foundation for novel, host cell-focused therapies against acute diseases.
RNA-capping enzymes are involved in the synthesis of the cap structure found at the 5'-end of eukaryotic mRNAs. The present study reports a detailed study on the thermodynamic parameters involved in the interaction of an RNA-capping enzyme with its ligands. Analysis of the interaction of the Saccharomyces cerevisiae RNA-capping enzyme (Ceg1) with GTP, RNA and manganese ions revealed significant differences between the binding forces that drive the interaction of the enzyme with its RNA and GTP substrates. Our thermodynamic analyses indicate that the initial association of GTP with the Ceg1 protein is driven by a favourable enthalpy change (DeltaH=-80.9 kJ/mol), but is also clearly associated with an unfavourable entropy change (TDeltaS=-62.9 kJ/mol). However, the interaction between Ceg1 and RNA revealed a completely different mode of binding, where binding to RNA is clearly dominated by a favourable entropic effect (TDeltaS=20.5 kJ/mol), with a minor contribution from a favourable enthalpy change (DeltaH=-5.3 kJ/mol). Fluorescence spectroscopy also allowed us to evaluate the initial binding of GTP to such an enzyme, thereby separating the GTP binding step from the concomitant metal-dependent hydrolysis of GTP that results in the formation of a covalent GMP-protein intermediate. In addition to the determination of the energetics of ligand binding, our study leads to a better understanding of the molecular basis of substrate recognition by RNA-capping enzymes.
Chez les eucaryotes, les ARN pré-messagers (ARN pré-m) transcrits par l'ARN polymérase II (ARN pol II) subissent plusieurs modifications co-transcriptionnelles avant d'être transportés sous forme mature (ARN messager [ARNm]) dans le cytoplasme pour y être traduits efficacement en différentes protéines. Chacune de ces modifications met en jeu de nombreux facteurs protéiques qui agissent au sein d'un vaste réseau d'interactions. On trouve, tout d'abord, l'ajout d'une structure coiffe à l'extrémité 5' des ARNm, l'épissage des séquences non codantes (introns) et la synthèse d'une queue de polyadénosines (queue de poly[A]) à l'extrémité 3'. Bien que chacune de ces étapes puisse être étudiée de façon indépendante, les données actuelles démontrent clairement que les modifications auxquelles elles conduisent sont fortement interconnectées et qu'elles influencent les fonctions particulières et l'efficacité de chacune d'elles. Puisque ces processus de modification ont lieu de façon coordonnée avec la transcription, l'ARN polymérase II (ARN pol II) joue également un rôle clé dans la régulation de ces événements. Maturation de l'ARNmSynthèse de la structure coiffe La maturation des ARN pré-m est un événement qui joue un rôle critique dans l'expression des gènes eucaryotes. Elle débute tout d'abord par l'ajout d'une structure coiffe, m7 GpppN-, à l'extrémité 5' des acides ribonucléiques néo-synthétisés. La coiffe est constituée d'un résidu guanosine méthylé en position N7 qui est relié au premier nucléotide retrouvé en 5' de l'ARNm via une liaison 5'-5' triphosphate ( Figure 1A) [1]. Mentionnons que chez les eucaryotes, les nucléotides adjacents à la coiffe ( m7 GpppNpN-) peuvent également être méthylés à hauteur de la position 2 de leur groupement ribose. Ces structures sont alors désignées coiffe 0 ( m7 GpppNpN-), coiffe 1 ( m7 Gppp m2 NpN-) ou coiffe 2 ( m7 Gppp m2 Np m2 N-) selon le nombre de nucléotides méthylés [2]. Bien que le rôle spécifique de ces méthylations sur les nucléotides adjacents à la coiffe demeure inconnu, certaines informations suggèrent que leur présence pourrait accroître la liaison des ribopolymères aux ribosomes [2]. > Chez les eucaryotes, les ARN messagers (ARNm) subissent de nombreuses modifications co-transcriptionnelles qui conduisent à leur maturation avant d'être transportés dans le cytoplasme pour être traduits efficacement en différentes protéi-nes. Parmi ces modifications, on trouve l'ajout d'une structure coiffe à l'extrémité 5' des ARNm, l'épissage des séquences non codantes (introns) et la synthèse d'une queue de polyadénosines à l'extrémité 3'. Malgré les découvertes distinctes de ces processus, il a été démontré qu'il existe une coopérativité entre ces différentes étapes de maturation et de transcription. MEDECINE/SCIENCES 2006 ; 22 : 626-32 Article disponible sur le site
No abstract
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.