Foldamers Reveal and Validate Therapeutic Targets Associated with Toxic α-Synuclein Self-Assembly

Parkinson’s disease (PD) is a progressive neurodegenerative disorder for which there is no successful prevention or intervention. The pathological hallmark for PD involves the self-assembly of functional Alpha-Synuclein (αS) into non-functional amyloid structures. One of the potential therapeutic interventions against PD is the effective inhibition of αS aggregation. However, the bottleneck towards achieving this goal is the identification of αS domains/sequences that are essential for aggregation. Using a protein mimetic approach, we have identified αS sequences-based targets that are essential for aggregation and will have significant therapeutic implications. An extensive array of in vitro, ex vivo, and in vivo assays is utilized to validate αS sequences and their structural characteristics that are essential for aggregation and propagation of PD phenotypes. The study aids in developing significant mechanistic and therapeutic insights into various facets of αS aggregation, which will pave the way for effective treatments for PD

Macromolecule-Assisted<em> de novo</em> Protein Folding

In the processes of protein synthesis and folding, newly synthesized polypeptides are tightly connected to the macromolecules, such as ribosomes, lipid bilayers, or cotranslationally folded domains in multidomain proteins, representing a hallmark of <em>de novo</em> protein folding environments <em>in vivo</em>. Such linkage effects on the aggregation of endogenous polypeptides have been largely neglected, although all these macromolecules have been known to effectively and robustly solubilize their linked heterologous proteins in fusion or display technology. Thus, their roles in the aggregation of linked endogenous polypeptides need to be elucidated and incorporated into the mechanisms of <em>de novo</em> protein folding <em>in vivo</em>. In the classic hydrophobic interaction-based stabilizing mechanism underlying the molecular chaperone-assisted protein folding, it has been assumed that the macromolecules connected through a simple linkage without hydrophobic interactions and conformational changes would make no effect on the aggregation of their linked polypeptide chains. However, an increasing line of evidence indicates that the intrinsic properties of soluble macromolecules, especially their surface charges and excluded volume, could be important and universal factors for stabilizing their linked polypeptides against aggregation. Taken together, these macromolecules could act as folding helpers by keeping their linked nascent chains in a folding-competent state. The folding assistance provided by these macromolecules in the linkage context would give new insights into <em>de novo</em> protein<em> </em>folding inside the cell

RNA‐dependent Chaperone (chaperna) as an Engineered Pro‐region for the Folding of Recombinant Microbial Transglutaminase

Transglutaminase (TGase) induces the cross‐linking of proteins by catalyzing an acyl transfer reaction. TGase is a zymogen, activated by the removal of its pro‐region. Because the pro‐region is crucial for folding and inhibition of the TGase activity, the recombinant expression of the mature TGase (mTGase) without the pro‐region, usually results in inactive inclusion bodies or low protein yield. Here, Streptomyces netropsis TGase was fused with Escherichia coli lysyl‐tRNA synthetase (LysRS), as a module with chaperoning activity in an RNA dependent manner (chaperna). The TGase activity from purified fusion protein induced via the removal of LysRS by tev protease in vitro. Moreover, active mTGase was produced in E. coli via an intracellular cleavage system, wherein LysRS‐mTGase was cleaved by the coexpressed tev protease in vivo. The results suggest that LysRS essentially mimics pro‐region, which exerts a dual function—folding of TGase into active conformation and keeping it as dormant state—in an RNA‐dependent manner. Thus, trans‐acting RNAs, prompt the cis‐acting chaperone function of LysRS, while being mechanistically similar to the intramolecular chaperone function of the pro‐region. These results could be implemented and extended for the folding of “difficult‐to‐express” recombinant proteins, by harnessing the chaperna function

Pan-Influenza A Protection by Prime–Boost Vaccination with Cold-Adapted Live-Attenuated Influenza Vaccine in a Mouse Model

Influenza virus infections continually pose a major public health threat with seasonal epidemics and sporadic pandemics worldwide. While currently licensed influenza vaccines provide only strain-specific protection, antigenic drift and shift occasionally render the viruses resistant to the host immune responses, which highlight the need for a vaccine that provides broad protection against multiple subtypes. In this study, we suggest a vaccination strategy using cold-adapted, live attenuated influenza vaccines (CAIVs) to provide a broad, potent, and safe cross-protection covering antigenically distinct hemagglutinin (HA) groups 1 and 2 influenza viruses. Using a mouse model, we tested different prime–boost combinations of CAIVs for their ability to induce humoral and T-cell responses, and protective efficacy against H1 and H5 (HA group 1) as well as H3 and H7 (HA group 2) influenza viruses. Notably, even in the absence of antibody-mediated neutralizing activity or HA inhibitory activity in vitro, CAIVs provided a potent protection against heterologous and heterosubtypic lethal challenges in vivo. Heterologous combination of prime (H1)–boost (H5) vaccine strains showed the most potent cross-protection efficacy. In vivo depletion experiments demonstrated not only that T cells and natural killer cells contributed to the cross-protection, but also the involvement of antibody-dependent mechanisms for the cross-protection. Vaccination-induced antibodies did not enhance the infectivity of heterologous viruses, and prime vaccination did not interfere with neutralizing antibody generation by the boost vaccination, allaying vaccine safety concerns associated with heterogeneity between the vaccines and challenge strains. Our data show that CAIV-based strategy can serve as a simple but powerful option for developing a “truly” universal influenza vaccine providing pan-influenza A protection, which has not been achieved yet by other vaccine strategies. The promising results of potency, breadth, and safety demonstrated in the mouse model support further studies in higher animal models for clinical relevance

Stabilization of Intrinsically Disordered DKK2 Protein by Fusion to RNA-Binding Domain

Intrinsic disorders are a common feature of hub proteins in eukaryotic interactomes controlling the signaling pathways. The intrinsically disordered proteins (IDPs) are prone to misfolding, and maintaining their functional stability remains a major challenge in validating their therapeutic potentials. Considering that IDPs are highly enriched in RNA-binding proteins (RBPs), here we reasoned and confirmed that IDPs could be stabilized by fusion to RBPs. Dickkopf2 (DKK2), Wnt antagonist and a prototype IDP, was fused with lysyl-tRNA synthetase (LysRS), with or without the fragment crystallizable (Fc) domain of an immunoglobulin and expressed predominantly as a soluble form from a bacterial host. The functional competence was confirmed by in vitro Wnt signaling reporter and tube formation in human umbilical vein endothelial cells (HUVECs) and in vivo Matrigel plug assay. The removal of LysRS by site-specific protease cleavage prompted the insoluble aggregation, confirming that the linkage to RBP chaperones the functional competence of IDPs. While addressing to DKK2 as a key modulator for cancer and ischemic vascular diseases, our results suggest the use of RBPs as stabilizers of disordered proteinaceous materials for acquiring and maintaining the structural stability and functional competence, which would impact the druggability of a variety of IDPs from human proteome

Green Tea Catechin-Inactivated Viral Vaccine Platform

Traditionally, chemical agents such as formalin (FA) and β-propiolactone (BPL) have long been used for the preparation of inactivated vaccines or toxoids. It has been shown that FA extensively modifies vaccine antigens and thus affects immunogenicity profiles, sometimes compromising the protective efficacy of the vaccines or even exacerbating the disease upon infection. In this study, we show that natural catechins from green tea extracts (GT) can be used as an inactivating agent to prepare inactivated viral vaccines. GT treatment resulted in complete and irreversible inactivation of influenza virus as well as dengue virus. In contrast to FA that reacted extensively with multiple amino acids including lysine, a major anchor residue for epitope binding to MHC molecules, GT catechin epigallocatechin-3-gallate (EGCG) crosslinked primarily with cysteine residues and thus preserved the major epitopes of the influenza hemagglutinin. In a mouse model, vaccination with GT-inactivated influenza virus (GTi virus) elicited higher levels of viral neutralizing antibodies than FA-inactivated virus (FAi virus). The vaccination completely protected the mice from a lethal challenge and restricted the challenge viral replication in the lungs. Of note, the quality of antibody responses of GTi virus was superior to that with FAi virus, in terms of the magnitude of antibody titer, cross-reactivity to hetero-subtypes of influenza viruses, and the avidity to viral antigens. As the first report of using non-toxic natural compounds for the preparation of inactivated viral vaccines, the present results could be translated into a clinically relevant vaccine platform with improved efficacy, safety, productivity, and public acceptance

Chaperna-Mediated Assembly of Ferritin-Based Middle East Respiratory Syndrome-Coronavirus Nanoparticles

The folding of monomeric antigens and their subsequent assembly into higher ordered structures are crucial for robust and effective production of nanoparticle (NP) vaccines in a timely and reproducible manner. Despite significant advances in in silico design and structure-based assembly, most engineered NPs are refractory to soluble expression and fail to assemble as designed, presenting major challenges in the manufacturing process. The failure is due to a lack of understanding of the kinetic pathways and enabling technical platforms to ensure successful folding of the monomer antigens into regular assemblages. Capitalizing on a novel function of RNA as a molecular chaperone (chaperna: chaperone + RNA), we provide a robust protein-folding vehicle that may be implemented to NP assembly in bacterial hosts. The receptor-binding domain (RBD) of Middle East respiratory syndrome-coronavirus (MERS-CoV) was fused with the RNA-interaction domain (RID) and bacterioferritin, and expressed in Escherichia coli in a soluble form. Site-specific proteolytic removal of the RID prompted the assemblage of monomers into NPs, which was confirmed by electron microscopy and dynamic light scattering. The mutations that affected the RNA binding to RBD significantly increased the soluble aggregation into amorphous structures, reducing the overall yield of NPs of a defined size. This underscored the RNA-antigen interactions during NP assembly. The sera after mouse immunization effectively interfered with the binding of MERS-CoV RBD to the cellular receptor hDPP4. The results suggest that RNA-binding controls the overall kinetic network of the antigen folding pathway in favor of enhanced assemblage of NPs into highly regular and immunologically relevant conformations. The concentration of the ion Fe2+, salt, and fusion linker also contributed to the assembly in vitro, and the stability of the NPs. The kinetic “pace-keeping” role of chaperna in the super molecular assembly of antigen monomers holds promise for the development and delivery of NPs and virus-like particles as recombinant vaccines and for serological detection of viral infections