Morales Gallardo, J. | Search bioRxiv

bioRxiv2024

Expression, purification, and characterization of diacylated Lipo-YcjN from Escherichia coli

YcjN is a putative substrate-binding protein expressed from a cluster of genes involved in carbohydrate import and metabolism in Escherichia coli. Here, we determine the crystal structure of YcjN to a resolution of 1.95 [A], revealing that its three-dimensional structure is similar to substrate binding proteins in subcluster D-I, which includes the well-characterized maltose binding protein (MBP). Furthermore, we found that recombinant overexpression of YcjN results in the formation of a lipidated form of YcjN that is posttranslationally diacylated at cysteine 21. Comparisons of size-exclusion chromatography profiles and dynamic light scattering measurements of lipidated and non-lipidated YcjN proteins suggest that lipidated YcjN aggregates in solution via its lipid moiety. Additionally, bioinformatic analysis indicates that YcjN-like proteins may exist in both Bacteria and Archaea, potentially in both lipidated and non-lipidated forms. Together, our results provide a better understanding of the aggregation properties of recombinantly expressed bacterial lipoproteins in solution and establish a foundation for future studies that aim to elucidate the role of these proteins in bacterial physiology.

biophysics↗

bioRxiv2022

Prior parental experience attenuates hormonal stress responses and alters hippocampal glucocorticoid receptors in both sexes of the biparental rock dove

In the face of challenges, animals must balance investments in reproductive effort versus their own survival. Physiologically, this tradeoff may be mediated by glucocorticoid release by the hypothalamic-pituitary-adrenal (HPA) axis and prolactin release from the pituitary to maintain parental care. The degree to which animals react to, and recover from, stressors likely affects their ability to maintain parental behavior and ultimately, fitness. However, less is known about how the stress response changes when animals gain parental experience, and what mechanisms may underlie any effect of experience on hormonal stress responses. To address these questions, we measured the corticosterone (CORT) and prolactin (PRL) stress response in both sexes of the biparental rock dove (Columba livia) that had never raised chicks versus birds that had fledged at least one chick. We measured both CORT and PRL at baseline and after an acute stressor (30 minutes restraint). We also measured negative feedback ability by administering dexamethasone, a synthetic glucocorticoid that suppresses CORT release, and measuring CORT and PRL after 60 minutes. All hormones we measured when birds were not actively nesting, allowing us to assess any lasting effects of parental experience beyond the parental care period. Birds with parental experience had lower stress-induced and negative-feedback CORT, and higher stress-induced PRL than inexperienced birds. In a separate experiment, we measured glucocorticoid receptor subtype expression in the hippocampus, a key site of negative feedback regulation. We found that experienced birds expressed higher glucocorticoid receptors than inexperienced controls, which may mediate their ability to attenuate the hormonal stress response. Together, these results shed light on potential mechanisms by which gaining experience may improve parental performance and fitness. Summary statementExperienced rock dove parents show lower corticosterone and higher prolactin levels after an acute stressor than birds without parental experience and increased glucocorticoid receptor expression in the hippocampus may mediate this effect.

animal behavior and cognition↗