RESUMEN
The vitamin A derivative, retinoid acid (RA) is key player in guiding adaptive mucosal immune responses. However, data on the uptake and metabolism of vitamin A within human immune cells has remained largely elusive because retinoids are small, lipophilic molecules which are difficult to detect. To overcome this problem and to be able to study the effect of vitamin A metabolism in human immune cell subsets, we have synthesized novel bio-orthogonal retinoid-based probes (clickable probes), which are structurally and functionally indistinguishable from vitamin A. The probes contain a functional group (an alkyne) to conjugate to a fluorogenic dye to monitor retinoid molecules in real-time in immune cells. We demonstrate, by using flow cytometry and microscopy, that multiple immune cells have the capacity to internalize retinoids to varying degrees, including human monocyte-derived dendritic cells (DCs) and naïve B lymphocytes. We observed that naïve B cells lack the enzymatic machinery to produce RA, but use exogenous retinoic acid to enhance CD38 expression. Furthermore, we showed that human DCs metabolize retinal into retinoic acid, which in co-culture with naïve B cells led to of the induction of CD38 expression. These data demonstrate that in humans, DCs can serve as an exogenous source of RA for naïve B cells. Taken together, through the use of clickable vitamins our data provide valuable insight in the mechanism of vitamin A metabolism and its importance for human adaptive immunity.
Asunto(s)
Linfocitos B/inmunología , Química Clic/métodos , Células Dendríticas/inmunología , Vitamina A/metabolismo , ADP-Ribosil Ciclasa 1/metabolismo , Inmunidad Adaptativa , Células Cultivadas , Técnicas de Cocultivo , Cobre/metabolismo , Citometría de Flujo , Colorantes Fluorescentes , Humanos , Tretinoina/química , Tretinoina/metabolismo , Regulación hacia Arriba , Vitamina A/químicaRESUMEN
Aldehyde dehydrogenases (ALDHs) convert aldehydes into carboxylic acids and are often upregulated in cancer. They have been linked to therapy resistance and are therefore potential therapeutic targets. However, only a few selective and potent inhibitors are currently available for this group of enzymes. Competitive activity-based protein profiling (ABPP) would aid the development and validation of new selective inhibitors. Herein, a broad-spectrum activity-based probe that reports on several ALDHs is presented. This probe was used in a competitive ABPP protocol against three ALDH inhibitors in lung cancer cells to determine their selectivity profiles and establish their target engagement.
Asunto(s)
Aldehído Deshidrogenasa/metabolismo , Inhibidores Enzimáticos/química , Aldehído Deshidrogenasa/antagonistas & inhibidores , Aldehído Deshidrogenasa/genética , Línea Celular Tumoral , Química Clic , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Colorantes Fluorescentes/química , Humanos , ProteómicaRESUMEN
Retinaldehyde dehydrogenases belong to a superfamily of enzymes that regulate cell differentiation and are responsible for detoxification of anticancer drugs. Chemical tools and methods are of great utility to visualize and quantify aldehyde dehydrogenase (ALDH) activity in health and disease. Here, we present the discovery of a first-in-class chemical probe based on retinal, the endogenous substrate of retinal ALDHs. We unveil the utility of this probe in quantitating ALDH isozyme activity in a panel of cancer cells via both fluorescence and chemical proteomic approaches. We demonstrate that our probe is superior to the widely used ALDEFLUOR assay to explain the ability of breast cancer (stem) cells to produce all-trans retinoic acid. Furthermore, our probe revealed the cellular selectivity profile of an advanced ALDH1A1 inhibitor, thereby prompting us to investigate the nature of its cytotoxicity. Our results showcase the application of substrate-based probes in interrogating pathologically relevant enzyme activities. They also highlight the general power of chemical proteomics in driving the discovery of new biological insights and its utility to guide drug discovery efforts.
RESUMEN
Lipids perform a wide range of functions inside the cell, ranging from structural building block of membranes and energy storage to cell signaling. The mode of action of many signaling lipids has remained elusive due to their low abundance, high lipophilicity, and inherent instability. Various chemical biology approaches, such as photoaffinity or activity-based protein profiling methods, have been employed to shed light on the biological role of lipids and the lipid-protein interaction profile. In this review, we will summarize the recent developments in the field of chemical probes to study lipid biology, especially in immunology, and indicate potential avenues for future research.