purification-refolding-to-amyloid-fibrils-his-6-tagged-recombinant

Purification and Refolding to Amyloid Fibrils of (His)<sub>6</sub>-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in<em> E. coli</em>

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...

INRA

The influenza viruses contain a segmented, negative stranded RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP). X-ray structures have shown that NP contains well-structured domains juxtaposed with regions of missing electron densities corresponding to loops. In this study, we tested if these flexible loops gated or promoted RNA binding and RNA-induced oligomerization of NP. We first performed molecular dynamics simulations of wt NP monomer and trimer in comparison with the R361A protein mutated in the RNA binding groove, using the H1N1 NP as the initial structure. Calculation of the root-mean-square fluctuations highlighted the presence of two flexible loops in NP trimer: loop 1 (73-90), loop 2 (200-214). In NP, loops 1 and 2 formed a 10-15 Å-wide pinch giving access to the RNA binding groove. Loop 1 was stabilized by interactions with K113 of the adjacent β-sheet 1 (91-112) that interacted with the RNA grove (linker 360-373) via multiple hydrophobic contacts. In R361A, a salt bridge formed between E80 of loop 1 and R208 of loop 2 driven by hydrophobic contacts between L79 and W207, due to a decreased flexibility of loop 2 and loop 1 unfolding. Thus, RNA could not access its binding groove in R361A; accordingly, R361A had a much lower affinity for RNA than NP. Disruption of the E80-R208 interaction in the triple mutant R361A-E80A-E81A increased its RNA binding affinity and restored its oligomerization back to wt levels in contrast with impaired levels of R361A. Our data suggest that the flexibility of loops 1 and 2 is required for RNA sampling and binding which likely involve conformational change(s) of the nucleoprotein.

Molecular dynamics studies of the nucleoprotein of influenza A virus: role of the protein flexibility in RNA binding.

Lipid membrane can enhance prion protein (PrP) pathological fibrillogenesis. A neuronal paralog of PrP, named Shadoo (Sho), is localized to similar membrane environment as PrP and can also convert to amyloid-like fibrilles. To gain insight into the role of Sho in prion diseases, we studied Sho interactions with cellular membrane models. Sho was found to bind anionic lipid vesicles. Spectroscopic and microscopic data showed that membrane-associated Sho slowly converted into amyloid fibers. Furthermore, binding of Sho to anionic liposomes has a disruptive effect on the integrity of the lipid bilayer leading to the formation of supramolecular lipid-protein complexes. In consequence, the role of Sho in prion diseases might depend on the oligomerization state of Sho but also the nature of these lipoprotein assembles.

Shadoo binds lipid membranes and undergoes aggregation and fibrillization.

Measurements of the presence of prions in biological tissues or fluids rely more and more on cell-free assays. Although protein misfolding cyclic amplification (PMCA) has emerged as a valuable, sensitive tool, it is currently hampered by its lack of robustness and rapidity for high-throughput purposes. Here, we made a number of improvements making it possible to amplify the maximum levels of scrapie prions in a single 48-h round and in a microplate format. The amplification rates and the infectious titer of the PMCA-formed prions appeared similar to those derived from the in vivo laboratory bioassays. This enhanced technique also amplified efficiently prions from different species, including those responsible for human variant Creutzfeldt-Jakob disease. This new format should help in developing ultrasensitive, high-throughput prion assays for cognitive, diagnostic, and therapeutic applications. IMPORTANCE The method developed here allows large-scale, fast, and reliable cell-free amplification of subinfectious levels of prions from different species. The sensitivity and rapidity achieved approach or equal those of other recently developed prion-seeded conversion assays. Our simplified assay may be amenable to high-throughput, automated purposes and serve in a complementary manner with other recently developed assays for urgently needed antemortem diagnostic tests, by using bodily fluids containing small amounts of prion infectivity. Such a combination of assays is of paramount importance to reduce the transfusion risk in the human population and to identify asymptomatic carriers of variant Creutzfeldt-Jakob disease.

Highly infectious prions generated by a single round of microplate-based protein misfolding cyclic amplification.

The M2-1 protein of the important pathogen human respiratory syncytial virus is a zinc-binding transcription antiterminator that is essential for viral gene expression. We present the crystal structure of full-length M2-1 protein in its native tetrameric form at a resolution of 2.5 Å. The structure reveals that M2-1 forms a disk-like assembly with tetramerization driven by a long helix forming a four-helix bundle at its center, further stabilized by contact between the zinc-binding domain and adjacent protomers. The tetramerization helix is linked to a core domain responsible for RNA binding activity by a flexible region on which lie two functionally critical serine residues that are phosphorylated during infection. The crystal structure of a phosphomimetic M2-1 variant revealed altered charge density surrounding this flexible region although its position was unaffected. Structure-guided mutagenesis identified residues that contributed to RNA binding and antitermination activity, revealing a strong correlation between these two activities, and further defining the role of phosphorylation in M2-1 antitermination activity. The data we present here identify surfaces critical for M2-1 function that may be targeted by antiviral compounds.

Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation.

PB1-F2 is a nonstructural accessory protein of Influenza A virus described to enhance the mortality and the morbidity of the virus in a host-dependent manner. In this work, an electrochemical biosensor based on an immunodetection system was developed to follow the oligomerization of PB1-F2 during the viral cycle. The immunosensor was based on conductive polypyrrole modified with ferrocenyl groups as a redox marker for enhancing signal detection. Antibodies specific for monomeric or oligomeric PB1-F2 forms were immobilized on polypyrrole matrix via biotin/streptavidin layer. We demonstrated that this electrochemical biosensor sensitively detects PB1-F2 in both conformational forms. The linear range extends from 5 nM to 1.5 μM and from 5 nM to 0.5 μM for monomeric and oligomeric PB1-F2, respectively. The calculated limit of detection was 0.42 nM for monomeric PB1-F2 and 16 nM for oligomers. The biosensor platform allows the detection and quantification of PB1-F2 in lysates of infected cells during viral cycle. We show that at early stages of viral cycle, PB1-F2 is mainly monomeric but switched to amyloid-like structures at a later stage of infection. The quantification of two protein structural forms points out that PB1-F2 expression profiles and kinetics of oligomerization are cell-type-dependent.

Electrochemical detection of the oligomerization of PB1-F2 influenza A virus protein in infected cells.

The RNA genome of respiratory syncytial virus (RSV) is constitutively encapsidated by the viral nucleoprotein N, thus forming a helical nucleocapsid. Polymerization of N along the genomic and antigenomic RNAs is concomitant to replication and requires the preservation of an unassembled monomeric nucleoprotein pool. To this end, and by analogy with Paramyxoviridae and Rhabdoviridae, it is expected that the viral phosphoprotein P acts as a chaperone protein, forming a soluble complex with the RNA-free form of N (N(0)-P complex). Here, we have engineered a mutant form of N that is monomeric, is unable to bind RNA, still interacts with P, and could thus mimic the N(0) monomer. We used this N mutant, designated N(mono), as a substitute for N(0) in order to characterize the P regions involved in the N(0)-P complex formation. Using a series of P fragments, we determined by glutathione S-transferase (GST) pulldown assays that the N and C termini of P are able to interact with N(mono). We analyzed the functional role of amino-terminal residues of P by site-directed mutagenesis, using an RSV polymerase activity assay based on a human RSV minireplicon, and found that several residues were critical for viral RNA synthesis. Using GST pulldown and surface plasmon resonance assays, we showed that these critical residues are involved in the interaction between P[1-40] peptide and N(mono) in vitro. Finally, we showed that overexpression of the peptide P[1-29] can inhibit the polymerase activity in the context of the RSV minireplicon, thus demonstrating that targeting the N(0)-P interaction could constitute a potential antiviral strategy.

Identification and characterization of the binding site of the respiratory syncytial virus phosphoprotein to RNA-free nucleoprotein.

Prion diseases are characterized by conformational changes of a cellular prion protein (PrP(C)) into a β-sheet-enriched and aggregated conformer (PrP(Sc)). Shadoo (Sho), a member of the prion protein family, is expressed in the central nervous system (CNS) and is highly conserved among vertebrates. On the basis of histoanatomical colocalization and sequence similarities, it is suspected that Sho and PrP may be functionally related. The downregulation of Sho expression during prion pathology and the direct interaction between Sho and PrP, as revealed by two-hybrid analysis, suggest a relationship between Sho and prion replication. Using biochemical and biophysical approaches, we demonstrate that Sho forms a 1:1 complex with full-length PrP with a dissociation constant in the micromolar range, and this interaction consequently modifies the PrP-folding pathway. Using a truncated PrP that mimics the C-terminal C1 fragment, an allosteric binding behavior with a Hill number of 4 was observed, suggesting that at least a tetramerization state occurs. A cell-based prion titration assay performed with different concentrations of Sho revealed an increase in the PrP(Sc) conversion rate in the presence of Sho. Collectively, our observations suggest that Sho can affect the prion replication process by (i) acting as a holdase and (ii) interfering with the dominant-negative inhibitor effect of the C1 fragment.

Interaction between Shadoo and PrP Affects the PrP-Folding Pathway.

PB1-F2 is a small accessory protein encoded by an alternative open reading frame in PB1 segments of most influenza A virus. PB1-F2 is involved in virulence by inducing mitochondria mediated immune cells apoptosis, increasing inflammation, and enhancing predisposition to secondary bacterial infections. Using biophysical approaches we characterized membrane disruptive activity of the full-length PB1-F2 (90aa), its N-terminal domain (52aa), expressed by currently circulating H1N1 viruses, and its C-terminal domain (38aa). Both full-length and N-terminal domain of PB1-F2 are soluble at pHs ≤ 6, while the C-terminal fragment was found soluble only at pH ≤ 3. All three peptides are intrinsically disordered. At pH ≥7, the C-terminal part of PB1-F2 spontaneously switches to amyloid oligomers while full-length and N-terminal domain of PB1-F2 aggregate to amorphous structures. When incubated with anionic liposomes at pH 5, full-length and C-terminal part of PB1-F2 assemble into amyloid structures and disrupt membrane at nM concentrations. PB1-F2 and its C-terminal exhibit no significant antimicrobial activity. When added in the culture medium of mammalian cells, PB1-F2 amorphous aggregates show no cytotoxicity, while PB1-F2 pre-assembled into amyloid oligomers or fragmented nanoscaled fibrils was highly cytotoxic. Furthermore, the formation of PB1-F2 amyloid oligormers in infected cells was directly reflected by membrane disruption and cell death as observed in U937 and A549 cells. Altogether our results demonstrate that membrane-lytic activity of PB1-F2 is closely linked to supramolecular organization of the protein of the targeted membranes.

Article	Year
Molecular dynamics studies of the nucleoprotein of influenza A virus: role of the protein flexibility in RNA binding. PloS one\| PubMed ID: 22272272	2012
Shadoo binds lipid membranes and undergoes aggregation and fibrillization. Biochemical and biophysical research communications\| PubMed ID: 23911790	2013
Highly infectious prions generated by a single round of microplate-based protein misfolding cyclic amplification. mBio\| PubMed ID: 24381300	2014
Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 24434552	2014
Electrochemical detection of the oligomerization of PB1-F2 influenza A virus protein in infected cells. Analytical chemistry\| PubMed ID: 25051456	2014
Identification and characterization of the binding site of the respiratory syncytial virus phosphoprotein to RNA-free nucleoprotein. Journal of virology\| PubMed ID: 25568210	2015
Interaction between Shadoo and PrP Affects the PrP-Folding Pathway. Journal of virology\| PubMed ID: 25855735	2015
Amyloid Assemblies of Influenza A Virus PB1-F2 Protein Damage Membrane and Induce Cytotoxicity. The Journal of biological chemistry\| PubMed ID: 26601953	2015

Charles-Adrien Richard

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