Presenter Information

Kassandra L. Boshae, UofMFollow

Presentation Type

Presentation

Faculty Mentor’s Full Name

Philippe Diaz

Faculty Mentor’s Department

Biomedical and Pharmaceutical Sciences

Abstract

Retinoic acid (atRA) is used throughout the body as a signaling molecule, and is important in cellular differentiation for many different cell types. atRA is a product of the retinol metabolism pathway. atRA is generally produced within cells by conversion of Retinol (ROL) into Retinaldehyde (RAL). RAL is then either irreversibly converted to atRA or is converted back to ROL via aldo-keto-reductase 1B10 (AKR1B10), thereby lowering the concentration of atRA. This makes AKR1B10 an interesting target for therapies and treatments for many different diseases and conditions. We hypothesized that natural and synthetic compounds would inhibit AKR1B10 activity and increase concentrations of endogenous atRA. To test this, we performed enzyme kinetic assays and RT-qPCR. Specifically, E. coli were transformed with an AKR1B10 plasmid, ABM-pPB-N-His, under kanamycin selection. To induce AKR1B10 expression, cultures were then treated with 100uM IPTG and FPLC was used to purify AKR1B10 protein. Gel electrophoresis confirmed the purified protein was ~37 kDa. We then evaluated our panel of natural and synthetic compounds for inhibition of AKR1B10 in kinetic studies measuring the conversion of NADPH to NADP+. Additionally, healthy adult keratinocyte monolayers were cultured with our panel of AKR1B10 inhibitors for RT-qPCR analysis of differentiation and proliferation markers including KRT10, Involucrin, Ki67, AKR1B10 and atRA responsive genes including KLK13, STRA6, and NRIP1. Interestingly, AKR1B10 expression was modulated by culture calcium concentration and retinoids, suggesting differentiation-state dependent effects. Together, we have found a small number of compounds that show some promising AKR1B10 inhibition, which would increase endogenous atRA concentrations. Further study is warranted to better understand AKR1B10 expression in the skin and to determine overall efficacy.

Category

Life Sciences

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The Study of AKR1B10 Enzymatic Activity and Expression for Increasing Endogenous all trans-Retinoic Acid in the Skin

Retinoic acid (atRA) is used throughout the body as a signaling molecule, and is important in cellular differentiation for many different cell types. atRA is a product of the retinol metabolism pathway. atRA is generally produced within cells by conversion of Retinol (ROL) into Retinaldehyde (RAL). RAL is then either irreversibly converted to atRA or is converted back to ROL via aldo-keto-reductase 1B10 (AKR1B10), thereby lowering the concentration of atRA. This makes AKR1B10 an interesting target for therapies and treatments for many different diseases and conditions. We hypothesized that natural and synthetic compounds would inhibit AKR1B10 activity and increase concentrations of endogenous atRA. To test this, we performed enzyme kinetic assays and RT-qPCR. Specifically, E. coli were transformed with an AKR1B10 plasmid, ABM-pPB-N-His, under kanamycin selection. To induce AKR1B10 expression, cultures were then treated with 100uM IPTG and FPLC was used to purify AKR1B10 protein. Gel electrophoresis confirmed the purified protein was ~37 kDa. We then evaluated our panel of natural and synthetic compounds for inhibition of AKR1B10 in kinetic studies measuring the conversion of NADPH to NADP+. Additionally, healthy adult keratinocyte monolayers were cultured with our panel of AKR1B10 inhibitors for RT-qPCR analysis of differentiation and proliferation markers including KRT10, Involucrin, Ki67, AKR1B10 and atRA responsive genes including KLK13, STRA6, and NRIP1. Interestingly, AKR1B10 expression was modulated by culture calcium concentration and retinoids, suggesting differentiation-state dependent effects. Together, we have found a small number of compounds that show some promising AKR1B10 inhibition, which would increase endogenous atRA concentrations. Further study is warranted to better understand AKR1B10 expression in the skin and to determine overall efficacy.