Factors affecting protein recovery during Hsp40 affinity profiling.

The identification and quantification of misfolded proteins from complex mixtures is important for biological characterization and disease diagnosis, but remains a major bioanalytical challenge. We have developed Hsp40 Affinity Profiling as a bioanalytical approach to profile protein stability in response to cellular stress. In this assay, we ectopically introduce the Hsp40 FlagDNAJB8H31Q into cells and use quantitative proteomics to determine how protein affinity for DNAJB8 changes in the presence of cellular stress, without regard for native clients. Herein, we evaluate potential approaches to improve the performance of this bioanalytical assay. We find that although intracellular crosslinking increases recovery of protein interactors, this is not enough to overcome the relative drop in DNAJB8 recovery. While the J-domain promotes Hsp70 association, it does not affect the yield of protein association with DNAJB8 under basal conditions. By contrast, crosslinking and J-domain ablation both substantially increase relative protein interactor recovery with the structurally distinct Class B Hsp40 DNAJB1 but are completely compensated by poorer yield of DNAJB1 itself. Cellular thermal stress promotes increased affinity between DNAJB8H31Q and interacting proteins, as expected for interactions driven by recognition of misfolded proteins. DNAJB8WT does not demonstrate such a property, suggesting that under stress misfolded proteins are handed off to Hsp70. Hence, we find that DNAJB8H31Q is still our most effective recognition element for the recovery of destabilized client proteins following cellular stress.

Filamentous anti-influenza agents wrapping around viruses.

Owing to the emerging resistance to current anti-influenza therapies, strategies for blocking virus-cell interaction with agents that mimic interactions with host cell receptors are garnering interest. In this context, a multivalent presentation of sialyl groups on various types of scaffold materials such as dendrimers, liposomes, nanoparticles, and natural/synthetic polymers has been investigated for the inhibition of influenza A virus infection. However, the development of versatile antiviral agents based on monodisperse scaffolds capable of precise molecular design remains challenging. Whether an anisotropically extended filamentous nanostructure can serve as an effective scaffold for maximum inhibition of viral cell attachment has not been investigated. In this study, the preparation of a series of sialyllactose-conjugated filamentous bacteriophages (SLPhages), with controlled loading levels, ligand valencies, and two types of sialyllactose (α2,3' and α2,6'), is demonstrated. With optimal ligand loading and valency, SLPhages showed inhibitory activity (in vitro) against influenza A viruses at concentrations of tens of picomolar. This remarkable inhibition is due to the strong interaction between the SLPhage and the virus; this interaction is adequately potent to compensate for the cost of the bending and wrapping of the SLPhage around the influenza virus. Our study may open new avenues for the development of filamentous anti-viral agents, in which virus-wrapping or aggregation is the primary feature responsible for the blocking of cell entry.