8750 Characterization of 11βHSD1-Like Protein: A Novel Species-Restricted Member of the 11βHSD Enzyme Family

Abstract Disclosure: E. Valbe: None. J. Ng: None. P.J. Fuller: None. T.J. Cole: None. The 11β-hydroxysteroid dehydrogenases (11bHSD) are a subfamily of short-chain dehydrogenase/reductase enzymes, which regulate cortisol metabolism and their physiological actions in vivo. Although much is known about the biology and function of 11bHSD1 and 11bHSD2, little is known of a recently discovered third member of the 11bHSD family, 11bHSD1L, which is expressed in primates, fish, plants, and other mammals, but is absent in most rodent species. Current expression profiling in human, sheep and marmoset tissues suggest that 11bHSD1L may be linked to the regulation of reproduction through the hypothalamic-pituitary-gonadal (HPG) axis and/or to an unknown regulatory role in the central nervous system. The definitive physiological substrate for 11bHSD1L is unknown. We have assessed the expression of 11bHSD1L in the non-human primate marmoset and show that 11bHSD1L mRNA and protein levels are highest in regions of the brain and reproductive organs, which parallels expression patterns in human tissues. Localization of 11bHSD1L protein in marmoset tissue sections by immunohistochemistry identified several cell populations previously unreported for 11bHSD1L, that included the Sertoli cells in the testis, prostate epithelial cells and theca cells in the ovary, further implicating 11bHSD1L as a potential novel regulator of the HPG Axis. Furthermore, we have measured expression of 11bHSD1L in sub-regions of the foetal sheep brain and other tissues, where mRNA levels were elevated in neural-derived tissues. There is some evidence that 11bHSD1L has a functional role in cortisol turnover, as a cortisol oxidase. Using a dual-luciferase based reporter system and a quantitative LC-MS/MS technique with HSD1L overexpressing cells, we show that cortisol is unlikely to be a substrate of 11bHSD1L. We have also demonstrated via CRISPR-CAS9 mediated gene knockout that 11bHSD1L may play a role in cell proliferation or survival in the human neuroblastoma cell line SH-SY5Y, which expresses very high levels of HSD1L. Upon knockout of both 11bHSD1L alleles, SH-SY5Y cells become unviable. This may further implicate 11bHSD1L as a relevant gene in cancer cell growth, particularly considering its increased expression in many tumour cell lines. Finally, we discuss our progress in purifying 11bHSD1L as a recombinant protein for in vitro substrate and structural studies. Presentation: 6/3/2024