Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors

Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological and pathophysiological conditions, but its use as a redox marker suffers from the lack of tools to detect and quantify MetO within cells. In this work, we created a pair of complementary stereospecific genetically-encoded mechanism-based ratiometric fluorescent sensors of MetO by inserting a circularly yellow fluorescent protein between yeast methionine sulfoxide reductases and thioredoxins. The two sensors, named MetSOx and MetROx for their ability to detect S and R-forms of MetO, respectively, were utilized for targeted analysis of protein oxidation, regulation and repair, as well as for monitoring MetO in bacterial and mammalian cells, analyzing compartment-specific changes in MetO, and examining responses to physiological stimuli

Super-complexes of adhesion GPCRs and neural guidance receptors.

Latrophilin adhesion-GPCRs (Lphn1-3 or ADGRL1-3) and Unc5 cell guidance receptors (Unc5A-D) interact with FLRT proteins (FLRT1-3), thereby promoting cell adhesion and repulsion, respectively. How the three proteins interact and function simultaneously is poorly understood. We show that Unc5D interacts with FLRT2 in cis, controlling cell adhesion in response to externally presented Lphn3. The ectodomains of the three proteins bind cooperatively. Crystal structures of the ternary complex formed by the extracellular domains reveal that Lphn3 dimerizes when bound to FLRT2:Unc5, resulting in a stoichiometry of 1:1:2 (FLRT2:Unc5D:Lphn3). This 1:1:2 complex further dimerizes to form a larger 'super-complex' (2:2:4), using a previously undescribed binding motif in the Unc5D TSP1 domain. Molecular dynamics simulations, point-directed mutagenesis and mass spectrometry demonstrate the stability and molecular properties of these complexes. Our data exemplify how receptors increase their functional repertoire by forming different context-dependent higher-order complexes

Secreted amyloid-β precursor protein functions as a GABABR1a ligand to modulate synaptic transmission

Amyloid-β precursor protein (APP) is central to the pathogenesis of Alzheimer's disease, yet its physiological function remains unresolved. Accumulating evidence suggests that APP has a synaptic function mediated by an unidentified receptor for secreted APP (sAPP). Here we show that the sAPP extension domain directly bound the sushi 1 domain specific to the γ-aminobutyric acid type B receptor subunit 1a (GABABR1a). sAPP-GABABR1a binding suppressed synaptic transmission and enhanced short-term facilitation in mouse hippocampal synapses via inhibition of synaptic vesicle release. A 17-amino acid peptide corresponding to the GABABR1a binding region within APP suppressed in vivo spontaneous neuronal activity in the hippocampus of anesthetized Thy1-GCaMP6s mice. Our findings identify GABABR1a as a synaptic receptor for sAPP and reveal a physiological role for sAPP in regulating GABABR1a function to modulate synaptic transmission

Secreted amyloid-b precursor protein functions as a GABA B R1a ligand to modulate synaptic transmission

Amyloid-b precursor protein (APP) is central to the pathogenesis of Alzheimer’s disease, yet its physiological function remains unresolved. Accumulating evidence suggests that APP has a synaptic function mediated by an unidentified receptor for secreted APP (sAPP). Here we show that the sAPP extension domain directly bound the sushi 1 domain specific to the g-aminobutyric acid type B receptor subunit 1a (GABA B R1a). sAPP-GABA B R1a binding suppressed synaptic transmission and enhanced short-term facilitation in mouse hippocampal synapses via inhibition of synaptic vesicle release. A 17–amino acid peptide corresponding to the GABA B R1a binding region within APP suppressed in vivo spontaneous neuronal activity in the hippocampus of anesthetized Thy1-GCaMP6s mice. Our findings identify GABA B R1a as a synaptic receptor for sAPP and reveal a physiological role for sAPP in regulating GABA B R1a function to modulate synaptic transmission