Yannick Bessin

Direct interaction between the Rice yellow mottle virus (RYMV) VPg and the central domain of the rice eIF(iso)4G1 factor correlates with rice susceptibility and RYMV virulence.

The adaptation of Rice yellow mottle virus (RYMV) to recessive resistance mediated by the rymv1-2 allele has been reported as a model to study the emergence and evolution of virulent variants. The resistance and virulence factors have been identified as eukaryotic translation initiation factor eIF(iso)4G1 and viral genome-linked protein (VPg), respectively, but the molecular mechanisms involved in their interaction are still unknown. In this study, we demonstrated a direct interaction between RYMV VPg and the central domain of rice eIF(iso)4G1 both in vitro, using recombinant proteins, and in vivo, using a yeast two-hybrid assay. Insertion of the E309K mutation in eIF(iso)4G1, conferring resistance in planta, strongly diminished the interaction with avirulent VPg. The efficiency of the major virulence mutations at restoring the interaction with the resistance protein was assessed. Our results explain the prevalence of virulence mutations fixed during experimental evolution studies and are consistent with the respective viral RNA accumulation levels of avirulent and virulent isolates. Our results also explain the origin of the residual multiplication of wild-type isolates in rymv1-2-resistant plants and the role of genetic context in the poor adaptability of the S2/S3 strain. Finally, the strategies of RYMV and members of family Potyviridae to overcome recessive resistance were compared.

Degradable Hybrid Materials Based on Cationic Acylhydrazone Dynamic Covalent Polymers Promote DNA Complexation through Multivalent Interactions

The design of smart nonviral vectors for gene delivery is of prime importance for the successful implementation of gene therapies. In particular, degradable analogues of macromolecules represent promising targets as they would combine the multivalent presentation of multiple binding units that is necessary for achieving effective complexation of therapeutic oligonucleotides with the controlled degradation of the vector that would in turn trigger drug release. Toward this end, we have designed and synthesized hybrid polyacylhydrazone-based dynamic materials that combine bis-functionalized cationic monomers with ethylene oxide containing monomers. Polymer formation was characterized by (1) H and DOSY NMR spectroscopy and was found to take place at high concentration, whereas macrocycles were predominantly formed at low concentration. HPLC monitoring of solutions of these materials in aqueous buffers at pH values ranging from 5.0 to 7.0 revealed their acid-catalyzed degradation. An ethidium bromide displacement assay and gel electrophoresis clearly demonstrated that, despite being dynamic, these materials are capable of effectively complexing dsDNA in aqueous buffer and biological serum at N/P ratios comparable to polyethyleneimine polymers. The self-assembly of dynamic covalent polymers through the incorporation of a reversible covalent bond within their main chain is therefore a promising strategy for generating degradable materials that are capable of establishing multivalent interactions and effectively complexing dsDNA in biological media.

Structural Insight into the Mycobacterium tuberculosis Rv0020c Protein and Its Interaction with the PknB Kinase

The protein Rv0020c from Mycobacterium tuberculosis, also called FhaA, is one of the major substrates of the essential Ser/Thr protein kinase (STPK) PknB. The protein is composed of three domains and is phosphorylated on a unique site in its N terminus. We solved the solution structure of both N- and C-terminal domains and demonstrated that the approximately 300 amino acids of the intermediate domain are not folded. We present evidence that the FHA, a phosphospecific binding domain, of Rv0020c does not interact with the phosphorylated catalytic domains of PknB, but with the phosphorylated juxtamembrane domain that links the catalytic domain to the mycobacterial membrane. We also demonstrated that the degree and the pattern of phosphorylation of this juxtamembrane domain modulates the affinity of the substrate (Rv0020c) toward its kinase (PknB).

Dynamic Expression of DNA Complexation with Self-assembled Biomolecular Clusters†

We report herein the implementation of a dynamic covalent chemistry approach to the generation of multivalent clusters for DNA recognition. We show that biomolecular clusters can be expressed in situ by a programmed self-assembly process using chemoselective ligations. The cationic clusters are shown, by fluorescence displacement assay, gel electrophoresis and isothermal titration calorimetry, to effectively complex DNA through multivalent interactions. The reversibility of the ligation was exploited to demonstrate that template effects occur, whereby DNA imposes component selection in order to favor the most active DNA-binding clusters. Furthermore, we show that a chemical effector can be used to trigger DNA release through component exchange reactions.

First evidence of the pore-forming properties of a keratin from skin mucus of rainbow trout (Oncorhynchus mykiss, formerly Salmo gairdneri)

The epidermis of fish is covered with a layer of mucus, which contributes to the defence of the species against parasites, bacteria and fungi. We have previously extracted glycoproteins from various mucus samples from fish and have shown that they present pore-forming activities well correlated with strong antibacterial properties [Ebran, Julien, Orange, Saglio, Lemaitre and Molle(2000) Biochim. Biophys. Acta 1467, 271-280]. The present study focuses on the 65 kDa glycoprotein, Tr65, from the rainbow trout (Oncorhynchus mykiss, formerly Salmo gairdneri).Enzymatic digestion of Tr65 yielded a fragment pattern with strong homology with that of trout type II cytokeratin. Sequence analysis of the cDNA clone obtained by PCR confirmed this homology. We thus constructed a plasmid to overproduce the recombinant Tr65. We extracted and purified this recombinant Tr65, using it for multichannel and single-channel experiments in azolectin bilayers. Our results with recombinant Tr65 confirmed the pore-forming properties already shown with native antibacterial Tr65. These findings offer new insights into the function of keratin proteins present in various mucosal surfaces of animals and human beings.

Combination of Noncovalent Mass Spectrometry and Traveling Wave Ion Mobility Spectrometry Reveals Sugar-Induced Conformational Changes of Central Glycolytic Genes Repressor/DNA Complex

The central glycolytic genes repressor (CggR) is a 37 kDa transcriptional repressor protein which plays a key role in Bacillus subtilis glycolysis by regulating the transcription of the gapA operon. Fructose-1,6-bisphosphate (FBP), identified as the effector sugar, has been shown to abolish the binding cooperativity of CggR to its DNA target and to modify the conformational dynamics of the CggR/DNA complex. In the present study, noncovalent mass spectrometry (MS) was used to obtain deeper insights into FBP-dependent CggR/DNA interactions. The effect of FBP binding on CggR alone and on CggR/DNA complexes was examined using automated chip-based nanoelectrospray MS and traveling wave ion mobility mass spectrometry (IM-MS). Our results revealed that tetrameric CggR dissociates into dimers upon FBP binding. Moreover, FBP binding to CggR/DNA complexes triggers disruption of intermolecular protein/protein interactions within the complex, significantly modifying its conformation as evidenced by a 5% increase of its collision cross section. For the first time, the use of IM-MS is reported to probe ligand-induced conformational modifications of a protein/DNA complex with an emphasis on the comparison with solution-based techniques.

The RYMV-Encoded Viral Suppressor of RNA Silencing P1 Is a Zinc-Binding Protein with Redox-Dependent Flexibility

Viral suppressors of RNA interference (VSRs) target host gene silencing pathways, thereby operating important roles in the viral cycle and in host cells, in which they counteract host innate immune responses. However, the molecular mechanisms of VSRs are poorly understood. We provide here biochemical and biophysical features of the dual suppressor/activator VSR P1 protein encoded by the rice yellow mottle virus. In silico analyses of P1 suggested common features with zinc finger proteins and native mass spectrometry unambiguously confirmed that recombinant P1 binds reversibly two zinc atoms, each with a different strength. Additionally, we demonstrate that the reaction of P1 with H2O2 leads to zinc release, disulfide bond formation, and protein oligomerization. A reversible protein modification by redox alterations has only been described for a limited number of zinc finger proteins and has never been reported for VSRs. Those reported here for P1 might be a general feature of Cys-rich VSRs and could be a key regulatory mechanism for the control of RNA silencing.

A metal-free synthetic approach to peptide-based iminosugar clusters as novel multivalent glycosidase inhibitors

Multivalent bioconjugates represent emerging tools for enzyme inhibition. In particular, iminosugar clusters have recently shown promising results for the inhibition of glycosidases. However, most of them are prepared by copper-mediated click reactions. We report herein a metal-free approach based on oxime ligation for preparing iminosugar clusters using cyclic and linear tetra-aldehyde peptide scaffolds and oxyamine iminosugars (40–70% yield). Glycosidase inhibition assays revealed the superiority of the clusters made of the linear scaffold, displaying up to a 77-fold increase of relative potency per iminosugar. Thus, this metal-free approach provides a rapid access to structurally-diverse iminosugar clusters for establishing structure–activity relationships in the context of multivalent glycosidase inhibition.

One-Pot Self-Assembly of Peptide-Based Cage-Type Nanostructures using Orthogonal Ligations

The designed arrangement of biomolecular entities within monodisperse nanostructures is an important challenge toward functional biomaterials. We report herein a method for the formation of water-soluble peptide-based cages using orthogonal ligation reactions-acylhydrazone condensation and thiol-maleimide addition. The results show that using preorganized cyclic peptides and heterobifunctional spacers as building blocks and a set of orthogonal and chemoselective ligation reactions enable cage formation in one pot from six components and through eight reactions. Molecular modelling simulations reveal the structural dynamics of these structures. Finally, we exploited the reactional dynamics of the acylhydrazone by demonstrating the controlled dissociation of the cage through directed component exchange.

Switching Multivalent DNA Complexation using Metal-Controlled Cationic Supramolecular Self-Assemblies

We provide a proof-of-principle that coordination chemistry drives the in situ self-assembly of an inactive ligand into a multivalent cluster capable of effectively complexing DNA. We show that metal coordination and scavenging can be used to switch the multivalency of the system. Thus, controlled DNA complexation and decomplexation could be achieved.