Almost all membrane proteins are inserted or translocated across membranes by the universally conserved Sec translocon. Despite its central role, experimental access to Sec function has remained limited. Here, we present ProSecCO (Protein Secretion in Cell-free via synthetic Operons), which is a cell-free protein synthesis platform that inserts SecYEG into synthetic vesicles, enabling direct testing of Sec in real-time and high-throughput, circumventing longstanding viability constraints. Screening 300 Sec variants in a single experiment, we consolidate three decades of Sec research, while vastly expanding mutant diversity for structure-function insights. Mapping over 30 functionally critical regions that modulate Sec activity across three orders of magnitude, we uncover dozens of super-active translocation variants and one variant of improved insertion activity. We further leverage ProSecCO to increase membrane protein quality and nanobody export, highlighting the potential of our system for advancing applications in synthetic biology and biotechnology.
M. Meier, Scott A. Scholz, Leo von Bank et al.· Science Advances· 2 citations
Cholesterol is a key determinant of membrane stability and fluidity in both natural and synthetic lipid systems. Although widely used, its content varies greatly among commercial liposomes, and its regulatory effects on biological interactions remain insufficiently understood. Here, we reveal that cholesterol content critically governs the protein corona composition and binding affinity of liposomes, thereby dictating their cellular interactions and in vivo fate. Complement proteins exhibit a biphasic adsorption pattern that modulates monocyte and Kupffer cell uptake and systemic clearance. High cholesterol levels enrich apolipoproteins, particularly ApoE, promoting hepatocyte targeting, liver accumulation, and enhanced uptake by dendritic cells in blood and spleen. In contrast, low-cholesterol liposomes form albumin-dominated coronas that favor uptake by liver sinusoidal and pulmonary endothelial cells, leading to preferential lung accumulation. Notably, cholesterol-rich liposomes significantly suppress hepatocellular carcinoma progression through improved liver targeting and prolonged circulation. These findings identify cholesterol as a pivotal regulator of serum protein interactions and biodistribution, providing valuable insights for the rational design of efficient, organ-specific liposomal drug delivery systems.
Fangqin Fu, Yuting Ge, Xuemei Hu et al.· ACS Nano· 0 citations
Amyloid polymorphism is important in neurodegenerative and metabolic disease. Understanding fibril molecular polymorphism is critical to understanding why certain protein aggregates made from the same protein are pathogenic while others are benign. Here, we generate two polymorphs of human insulin, as a model system, induced by a thermal change in the nucleation (lag) phase. Both amyloid strains predominantly exhibit β-sheet structures; however, the cold-induced fibrils (formed under a two-h cold (4 °C) shock) display a more heterogeneous structure compared to the more uniform content in the conventional warm fibrils (formed at 37 °C the whole time). These structural variations highlight the complexity of the nucleation and growth mechanisms, with cold fibrils potentially trapped in non-equilibrium states due to a higher nucleation barrier. We find that the functional activity of cold/warm fibrils is unique in in vitro seeding/amyloid propagation, stability against inhibition, and cellular cytotoxicity. Our results show how amyloid polymorphs could differentially modulate pathogenicity. Moreover, the importance of environmental conditions on the molecular features of amyloid nucleation may be critical for design and stability of future protein biologics beyond therapeutic insulin.
Elnaz Hosseini, Saeid Hadi Alijanvand, Y. Kan et al.· Communications Chemistry· 0 citations
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