BackgroundFlexuous rod-shaped nanoparticles made of the coat protein (CP) of papaya mosaic virus (PapMV) have been shown to trigger innate immunity through engagement of toll-like receptor 7 (TLR7). PapMV nanoparticles can also serve as a vaccine platform as they can increase the immune response to fused peptide antigens. Although this approach shows great potential, fusion of antigens directly to the CP open reading frame (ORF) is challenging because the fused peptides can alter the structure of the CP and its capacity to self assemble into nanoparticles—a property essential for triggering an efficient immune response to the peptide. This represents a serious limitation to the utility of this approach as fusion of small peptides only is tolerated.ResultsWe have developed a novel approach in which peptides are fused directly to pre-formed PapMV nanoparticles. This approach is based on the use of a bacterial transpeptidase (sortase A; SrtA) that can attach the peptide directly to the nanoparticle. An engineered PapMV CP harbouring the SrtA recognition motif allows efficient coupling. To refine our engineering, and to predict the efficacy of coupling with SrtA, we modeled the PapMV structure based on the known structure of PapMV CP and on recent reports revealing the structure of two closely related potexviruses: pepino mosaic virus (PepMV) and bamboo mosaic virus (BaMV). We show that SrtA can allow the attachment of long peptides [Influenza M2e peptide (26 amino acids) and the HIV-1 T20 peptide (39 amino acids)] to PapMV nanoparticles. Consistent with our PapMV structural model, we show that around 30% of PapMV CP subunits in each nanoparticle can be fused to the peptide antigen. As predicted, engineered nanoparticles were capable of inducing a strong antibody response to the fused antigen. Finally, in a challenge study with influenza virus, we show that mice vaccinated with PapMV-M2e are protected from infection.ConclusionsThis technology will allow the development of vaccines harbouring long peptides containing several B and/or T cell epitopes that can contribute to a broad and robust protection from infection. The design can be fast, versatile and can be adapted to the development of vaccines for many infectious diseases as well as cancer vaccines.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-017-0289-y) contains supplementary material, which is available to authorized users.
The RNA-binding protein p54(nrb) is involved in many nuclear processes including transcription, RNA processing, and retention of hyperedited RNAs. In interphase cells, p54(nrb) localizes to the nucleoplasm and concentrates with protein partners in the paraspeckles via an interaction with the non-coding RNA Neat1. During mitosis, p54(nrb) becomes multiphosphorylated and the effects of this modification are not known. In the present study, we show that p54(nrb) phosphorylation does not affect the interactions with its protein partners but rather diminishes its general RNA-binding ability. Biochemical assays indicate that in vitro phosphorylation of a GST-p54(nrb) construct by CDK1 abolishes the interaction with 5' splice site RNA sequence. Site-directed mutagenesis shows that the threonine 15 residue, located N-terminal to the RRM tandem domains of p54(nrb), is involved in this inhibition. In vivo analysis reveals that Neat1 ncRNA co-immunoprecipitates with p54(nrb) in either interphase or mitotic cells, suggesting that p54(nrb)-Neat1 interaction is not modulated by phosphorylation. Accordingly, in vitro phosphorylated GST-p54(nrb) still interacts with PIR-1 RNA, a G-rich Neat1 sequence known to interact with p54(nrb). In vitro RNA binding assays show that CDK1-phosphorylation of a GST-p54(nrb) construct abolishes its interaction with homoribopolymers poly(A), poly(C), and poly(U) but not with poly(G). These data suggest that p54(nrb) interaction with RNA could be selectively modulated by phosphorylation during mitosis.
START domain proteins are conserved α/β helix-grip fold that play a role in the non-vesicular and intracellular transport of lipids and sterols. The mechanism and conformational changes permitting the entry of the ligand into their buried binding sites is not well understood. Moreover, their functions and the identification of cognate ligands is still an active area of research. Here, we report the solution structure of STARD6 and the characterization of its backbone dynamics on multiple time-scales through 15N spin-relaxation and amide exchange studies. We reveal for the first time the presence of concerted fluctuations in the Ω1 loop and the C-terminal helix on the microsecond-millisecond time-scale that allows for the opening of the binding site and ligand entry. We also report that STARD6 binds specifically testosterone. Our work represents a milestone for the study of ligand binding mechanism by other START domains and the elucidation of the biological function of STARD6.
Caspases are cysteinyl peptidases involved in inflammation and apoptosis during which hundreds of proteins are cleaved by executioner caspase-3 and -7. Despite the fact that caspase-3 has a higher catalytic activity, caspase-7 is more proficient at cleaving poly(ADP ribose) polymerase 1 (PARP1) because it uses an exosite within its N-terminal domain (NTD). Here, we demonstrate that molecular determinants also located in the NTD enhance the recognition and proteolysis of the Hsp90 co-chaperone p23. Structure-activity relationship analyses using mutagenesis of the caspase-7 NTD and kinetics show that residues 36-45 of caspase-7, which overlap with residues necessary for efficacious PARP1 cleavage, participate in p23 recognition. We also demonstrate using chimeric and truncated proteins that the caspase-7 NTD binds close to the cleavage site in the C-terminal tail of p23. Moreover, because p23 is cleaved at a site bearing a P4 Pro residue (PEVD↓G), which is far from the optimal sequence, we tested all residues at that position and found notable differences in the preference of caspase-7 and magnitude of differences between residues compared to the results of studies that have used small peptidic substrate libraries. Finally, bioinformatics shows that the regions we identified in caspase-7 and p23 are intrinsically disordered regions that contain molecular recognition features that permit a transient interaction between these two proteins. In summary, we characterized the binding mode for a caspase that is tailored to the specific recognition and cleavage of a substrate, highlighting the importance of studying the peptidase-substrate pair to understand the modalities of substrate recognition by caspases.
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