Steric trapping reveals a cooperativity network in the intramembrane protease GlpG.

Membrane proteins are assembled through balanced interactions among proteins, lipids and water. Studying their folding while maintaining the native lipid environment is necessary but challenging. Here we present methods for analyzing key elements of membrane protein folding including thermodynamic stability, compactness of the unfolded state and folding cooperativity under native conditions. The methods are based on steric trapping, which couples the unfolding of a doubly biotinylated protein to the binding of monovalent streptavidin (mSA). We further advanced this technology for general application by developing versatile biotin probes possessing spectroscopic reporters that are sensitized by mSA binding or protein unfolding. By applying these methods to the Escherichia coli intramembrane protease GlpG, we elucidated a widely unraveled unfolded state, subglobal unfolding of the region encompassing the active site, and a network of cooperative and localized interactions to maintain stability. These findings provide crucial insights into the folding energy landscape of membrane proteins

Lipopeptide-Coated Iron Oxide Nanoparticles as Potential Glycoconjugate-Based Synthetic Anticancer Vaccines

Although iron oxide magnetic nanoparticles (NPs) have been widely utilized in molecular imaging and drug delivery studies, they have not been evaluated as carriers for glycoconjugate-based anticancer vaccines. Tumor-associated carbohydrate antigens (TACAs) are attractive targets for the development of anticancer vaccines. Due to the weak immunogenicity of these antigens, it is highly challenging to elicit strong anti-TACA immune responses. With their high biocompatibilities and large surface areas, magnetic NPs were synthesized for TACA delivery. The magnetic NPs were coated with phospholipid-functionalized TACA glycopeptides through hydrophobic–hydrophobic interactions without the need for any covalent linkages. Multiple copies of glycopeptides were presented on NPs, potentially leading to enhanced interactions with antibody-secreting B cells through multivalent binding. Mice immunized with the NPs generated strong antibody responses, and the glycopeptide structures important for high antibody titers were identified. The antibodies produced were capable of recognizing both mouse and human tumor cells expressing the glycopeptide, resulting in tumor cell death through complement-mediated cytotoxicities. These results demonstrate that magnetic NPs can be a new and simple platform for multivalently displaying TACA and boosting anti-TACA immune responses without the need for a typical protein carrier

Growth enhancement of porcine epidemic diarrhea virus (PEDV) in Vero E6 cells expressing PEDV nucleocapsid protein.

More recently emerging strains of porcine epidemic diarrhea virus (PEDV) cause severe diarrhea and especially high mortality rates in infected piglets, leading to substantial economic loss to worldwide swine industry. These outbreaks urgently call for updated and effective PEDV vaccines. Better understanding in PEDV biology and improvement in technological platforms for virus production can immensely assist and accelerate PEDV vaccine development. In this study, we explored the ability of PEDV nucleocapsid (N) protein in improving viral yields in cell culture systems. We demonstrated that PEDV N expression positively affected both recovery of PEDV from infectious clones and PEDV propagation in cell culture. Compared to Vero E6 cells, Vero E6 cells expressing PEDV N could accelerate growth of a slow-growing PEDV strain to higher peak titers by 12 hours or enhance the yield of a vaccine candidate strain by two orders of magnitude. Interestingly, PEDV N also slightly enhances replication of porcine reproductive and respiratory virus, a PEDV relative in the Nidovirales order. These results solidify the importance of N in PEDV recovery and propagation and suggest a potentially useful consideration in designing vaccine production platforms for PEDV or closely related pathogens

Lipopeptide-Coated Iron Oxide Nanoparticles as Potential Glycoconjugate-Based Synthetic Anticancer Vaccines

Although iron oxide magnetic nanoparticles (NPs) have been widely utilized in molecular imaging and drug delivery studies, they have not been evaluated as carriers for glycoconjugate-based anticancer vaccines. Tumor-associated carbohydrate antigens (TACAs) are attractive targets for the development of anticancer vaccines. Due to the weak immunogenicity of these antigens, it is highly challenging to elicit strong anti-TACA immune responses. With their high biocompatibilities and large surface areas, magnetic NPs were synthesized for TACA delivery. The magnetic NPs were coated with phospholipid-functionalized TACA glycopeptides through hydrophobic–hydrophobic interactions without the need for any covalent linkages. Multiple copies of glycopeptides were presented on NPs, potentially leading to enhanced interactions with antibody-secreting B cells through multivalent binding. Mice immunized with the NPs generated strong antibody responses, and the glycopeptide structures important for high antibody titers were identified. The antibodies produced were capable of recognizing both mouse and human tumor cells expressing the glycopeptide, resulting in tumor cell death through complement-mediated cytotoxicities. These results demonstrate that magnetic NPs can be a new and simple platform for multivalently displaying TACA and boosting anti-TACA immune responses without the need for a typical protein carrier

SARS-CoV-2 Delta (B.1.617.2) variant replicates and induces syncytia formation in human induced pluripotent stem cell-derived macrophages

Alveolar macrophages are tissue-resident immune cells that protect epithelial cells in the alveoli from invasion by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, the interaction between macrophages and SARS-CoV-2 is inevitable. However, little is known about the role of macrophages in SARS-CoV-2 infection. Here, we generated macrophages from human induced pluripotent stem cells (hiPSCs) to investigate the susceptibility of hiPSC-derived macrophages (iMΦ) to the authentic SARS-CoV-2 Delta (B.1.617.2) and Omicron (B.1.1.529) variants as well as their gene expression profiles of proinflammatory cytokines during infection. With undetectable angiotensin-converting enzyme 2 (ACE2) mRNA and protein expression, iMΦ were susceptible to productive infection with the Delta variant, whereas infection of iMΦ with the Omicron variant was abortive. Interestingly, Delta induced cell-cell fusion or syncytia formation in iMΦ, which was not observed in Omicron-infected cells. However, iMΦ expressed moderate levels of proinflammatory cytokine genes in response to SARS-CoV-2 infection, in contrast to strong upregulation of these cytokine genes in response to polarization by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Overall, our findings indicate that the SARS-CoV-2 Delta variant can replicate and cause syncytia formation in macrophages, suggesting that the Delta variant can enter cells with undetectable ACE2 levels and exhibit greater fusogenicity

Antitumor Humoral and T Cell Responses by Mucin‑1 Conjugates of Bacteriophage Qβ in Wild-type Mice

Mucin-1 (MUC1) is one of the top ranked tumor associated antigens. In order to generate effective anti-MUC1 immune responses as potential anticancer vaccines, MUC1 peptides and glycopeptides have been covalently conjugated to bacteriophage Qβ. Immunization of mice with these constructs led to highly potent antibody responses with IgG titers over one million, which are among the highest anti-MUC1 IgG titers reported to date. Furthermore, the high IgG antibody levels persisted for more than six months. The constructs also elicited MUC1 specific cytotoxic T cells, which can selectively kill MUC1 positive tumor cells. The unique abilities of Qβ-MUC1 conjugates to powerfully induce both antibody and cytotoxic T cell immunity targeting tumor cells bode well for future translation of the constructs as anticancer vaccines

Steric trapping reveals a cooperativity network in the intramembrane protease GlpG

Membrane proteins are assembled through balanced interactions among protein, lipids and water. Studying their folding while maintaining the native lipid environment is necessary but challenging. Here we present methods for analyzing key elements in membrane protein folding including thermodynamic stability, compactness of the unfolded state and folding cooperativity under native conditions. The methods are based on steric trapping which couples unfolding of a doubly-biotinylated protein to binding of monovalent streptavidin (mSA). We further advanced this technology for general application by developing versatile biotin probes possessing spectroscopic reporters that are sensitized by mSA binding or protein unfolding. By applying these methods to an intramembrane protease GlpG of Escherichia coli, we elucidated a widely unraveled unfolded state, subglobal unfolding of the region encompassing the active site, and a network of cooperative and localized interactions to maintain the stability. These findings provide crucial insights into the folding energy landscape of membrane proteins