Covalent bicyclization of protein complexes yields durable quaternary structures.

Proteins are essential biomolecules and central to biotechnological applications. In many cases, assembly into higher-order structures is a prerequisite for protein function. Under conditions relevant for applications, protein integrity is often challenged, resulting in disassembly, aggregation, and loss of function. The stabilization of quaternary structure has proven challenging, particularly for trimeric and higher-order complexes, given the complexity of involved inter- and intramolecular interaction networks. Here, we describe the chemical bicyclization of homotrimeric protein complexes, thereby increasing protein resistance toward thermal and chemical stress. This approach involves the structure-based selection of cross-linking sites, their variation to cysteine, and a subsequent reaction with a triselectrophilic agent to form a protein assembly with bicyclic topology. Besides overall increased stability, we observe resistance toward aggregation and greatly prolonged shelf life. This bicyclization strategy gives rise to unprecedented protein chain topologies and can enable new biotechnological and biomedical applications.

Re-evaluation of the hCMEC/D3 based in vitro BBB model for ABC transporter studies.

The blood-brain barrier (BBB) represents one of the biggest hurdles for CNS related drug delivery, preventing permeation of most molecules, and therefore poses a major challenge for researchers in finding effective treatments for CNS diseases. The low permeability of molecules through the BBB is linked on one hand to the extreme tightness by tight junction (TJ) formation limiting the paracellular transport, and on the other hand to the presence of ATP-driven efflux pumps which actively transport unwanted compounds out of the brain. In this study we evaluated the applicability of the immortalized human cell line hCMEC/D3 for ABC transporter studies, focusing on the most expressed ABC transporters at the human BBB: P-glycoprotein (PGP, ABCB1), multidrug resistance protein 4 (MRP4, ABCC4) and breast cancer resistance protein (BCRP, ABCG2). Therefore, a two-step screening method was applied, consisting of a regular uptake assay (96-well format) and bidirectional transport studies, using a transwell system as in vitro simulation of the human BBB. In conclusion, the hCMEC/D3 based in vitro BBB model is well suited to screen drug candidates for ABC transporter interactions on the basis of a regular uptake assay, but in terms of transcellular permeability studies the cell line is limited by a lack of sufficient junctional tightness.