Ion Mobility-Mass Spectrometry Reveals a Dipeptide That Acts as a Molecular Chaperone for Amyloid β

Previously, we discovered and structurally characterized a complex between amyloid β 1–40 and the neuropeptide leucine enkephalin. This work identified leucine enkephalin as a potentially useful starting point for the discovery of peptide-related biotherapeutics for Alzheimer’s disease. In order to better understand such complexes that are formed <i>in vitro</i>, we describe here the analysis of a series of site-directed amino acid substitution variants of both peptides, covering the leucine enkephalin sequence in its entirety and a large number of selected residues of amyloid β 1–40 (residues: D1, E3, F4, R5, H6, Y10, E11, H13, H14, Q15, K16, E22, K28, and V40). Ion mobility–mass spectrometry measurements and molecular dynamics simulations reveal that the hydrophobic C-terminus of leucine enkephalin (Phe-Leu, FL) is crucial for the formation of peptide complexes. As such, we explore here the interaction of the dipeptide FL with both wildtype and variant forms of amyloid β in order to structurally characterize the complexes formed. We find that FL binds preferentially to amyloid β oligomers and attaches to amyloid β within the region between its N-terminus and its hydrophobic core, most specifically at residues Y10 and Q15. We further show that FL is able to prevent fibril formation

Evidence for a 1,3-Dipolar Cyclo-addition Mechanism in the Decarboxylation of Phenylacrylic Acids Catalyzed by Ferulic Acid Decarboxylase

Ferulic acid decarboxylase catalyzes the decarboxylation of phenylacrylic acid using a newly identified cofactor, prenylated flavin mononucleotide (prFMN). The proposed mechanism involves the formation of a putative pentacyclic intermediate formed by a 1,3 dipolar cyclo-addition of prFMN with the α–β double bond of the substrate, which serves to activate the substrate toward decarboxylation. However, enzyme-catalyzed 1,3 dipolar cyclo-additions are unprecedented and other mechanisms are plausible. Here we describe the use of a mechanism-based inhibitor, 2-fluoro-2-nitrovinylbenzene, to trap the putative cyclo-addition intermediate, thereby demonstrating that prFMN can function as a dipole in a 1,3 dipolar cyclo-addition reaction as the initial step in a novel type of enzymatic reaction

Affinity-Based Selectivity Profiling of an In-Class Selective Competitive Inhibitor of Acyl Protein Thioesterase 2

Activity-based protein profiling (ABPP) has revolutionized the discovery and optimization of active-site ligands across distinct enzyme families, providing a robust platform for in-class selectivity profiling. Nonetheless, this approach is less straightforward for profiling reversible inhibitors and does not access proteins outside the ABPP probe’s target profile. While the active-site competitive acyl protein thioesterase 2 inhibitor ML349 (<i>K</i>_i = 120 nM) is highly selective within the serine hydrolase enzyme family, it could still interact with other cellular targets. Here we present a chemoproteomic workflow to enrich and profile candidate ML349-binding proteins. In human cell lysates, biotinylated-ML349 enriches a recurring set of proteins, including metabolite kinases and flavin-dependent oxidoreductases that are potentially enhanced by avidity-driven multimeric interactions. Confirmatory assays by native mass spectrometry and fluorescence polarization quickly rank-ordered these weak off-targets, providing justification to explore ligand interactions and stoichiometry beyond ABPP