Photolysis of Trenbolone Acetate Metabolites in the Presence of Nucleophiles: Evidence for Metastable Photoaddition Products and Reversible Associations with Dissolved Organic Matter.

Photolysis of trenbolone acetate (TBA) metabolites in the presence of various nitrogen-, sulfur-, or oxygen-containing nucleophiles (e.g., azide, ammonia, or thiosulfate, respectively) results in rapid (half-lives ∼20-60 min), photochemically induced nucleophile incorporation across the parent steroid's trienone moiety. The formation of such nucleophile adducts limits formation of photohydrates, suggesting competition between the nucleophile and water for photochemical addition into the activated steroid structure. Analogous to previously reported photohydration outcomes, LC/MS analyses suggest that such photonucleophilic addition reactions are reversible, with more rapid elimination rates than thermal dehydration of photohydrates, and regenerate parent steroid structures. Beyond photonucleophilic addition pathways, we also found that hydroxylamine and presumed nucleophilic moieties in model dissolved organic matter (DOM; Fluka humic acid) can react via thermal substitution with TBA metabolite photohydrates, although this reaction with model DOM was only observed for photohydrates of trendione. Most nucleophile addition products [i.e., formed via (photo)reaction with thiosulfate, hydroxylamine, and ammonia] are notably more polar relative to the parent metabolite and photohydration products. Thus, if present, both nucleophilic adducts and bound residues in organic matter will facilitate transport and help mask detection of TBA metabolites in surface waters and treatment systems.

Proteome-Scale Screening to Identify High-Expression Signal Peptides with Minimal N-Terminus Biases via Yeast Display.

Signal peptides are critical for the efficient expression and routing of extracellular and secreted proteins. Most protein production and screening technologies rely upon a relatively small set of signal peptides. Despite their central role in biotechnology, there are limited studies comprehensively examining the interplay between signal peptides and expressed protein sequences. Here, we describe a high-throughput method to screen novel signal peptides that maintain a high degree of surface expression across a range of protein scaffolds with highly variable N-termini. We find that the canonical signal peptide used in yeast surface display, derived from Aga2p, fails to achieve high surface expression for 42.5% of constructs containing diverse N-termini. To circumvent this, we have identified two novel signal peptides derived from endogenous yeast proteins, SRL1 and KISH, which are highly tolerant to diverse N-terminal sequences. This pipeline can be used to expand our understanding of signal peptide function, identify improved signal peptides for protein expression, and refine the computational tools used for signal peptide prediction.