NanoB2 to monitor interactions of ligands with membrane proteins by combining nanobodies and NanoBRET.

The therapeutic potential of ligands targeting disease-associated membrane proteins is predicted by ligand-receptor binding constants, which can be determined using NanoLuciferase (NanoLuc)-based bioluminescence resonance energy transfer (NanoBRET) methods. However, the broad applicability of these methods is hampered by the restricted availability of fluorescent probes. We describe the use of antibody fragments, like nanobodies, as universal building blocks for fluorescent probes for use in NanoBRET. Our nanobody-NanoBRET (NanoB2) workflow starts with the generation of NanoLuc-tagged receptors and fluorescent nanobodies, enabling homogeneous, real-time monitoring of nanobody-receptor binding. Moreover, NanoB2 facilitates the assessment of receptor binding of unlabeled ligands in competition binding experiments. The broad significance is illustrated by the successful application of NanoB2 to different drug targets (e.g., multiple G protein-coupled receptors [GPCRs] and a receptor tyrosine kinase [RTK]) at distinct therapeutically relevant binding sites (i.e., extracellular and intracellular).

The Mitochondrial Hsp90 TRAP1 and Alzheimer's Disease.

Alzheimer's Disease (AD) is the most common form of dementia, characterised by intra- and extracellular protein aggregation. In AD, the cellular protein quality control (PQC) system is derailed and fails to prevent the formation of these aggregates. Especially the mitochondrial paralogue of the conserved Hsp90 chaperone class, tumour necrosis factor receptor-associated protein 1 (TRAP1), is strongly downregulated in AD, more than other major PQC factors. Here, we review molecular mechanism and cellular function of TRAP1 and subsequently discuss possible links to AD. TRAP1 is an interesting paradigm for the Hsp90 family, as it chaperones proteins with vital cellular function, despite not being regulated by any of the co-chaperones that drive its cytosolic paralogues. TRAP1 encloses late folding intermediates in a non-active state. Thereby, it is involved in the assembly of the electron transport chain, and it favours the switch from oxidative phosphorylation to glycolysis. Another key function is that it ensures mitochondrial integrity by regulating the mitochondrial pore opening through Cyclophilin D. While it is still unclear whether TRAP1 itself is a driver or a passenger in AD, it might be a guide to identify key factors initiating neurodegeneration.